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Lesson 9 Cell Signaling:  Curations and Articles of reference as supplemental information for lecture section on WNTs: #TUBiol3373

Stephen J. Wiilliams, Ph.D: Curator

UPDATED 4/16/2019

Please click on the following links for the Powerpoint presentation for lesson 9.  In addition click on the mp4 links to download the movies so you can view them in Powerpoint slide 22:

cell motility 9 lesson_SJW 2019

movie file 1:

Tumorigenic but noninvasive MCF-7 cells motility on an extracellular matrix derived from normal (3DCntrol) or tumor associated (TA) fibroblasts.  Note that TA ECM is “soft” and not organized and tumor cells appear to move randomly if  much at all.

Movie 2:

 

Note that these tumorigenic and invasive MDA-MB-231 breast cancer cells move in organized patterns on organized ECM derived from Tumor Associated (TA) fibroblasts than from the ‘soft’ or unorganized ECM derived from normal  (3DCntrl) fibroblasts

 

The following contain curations of scientific articles from the site https://pharmaceuticalintelligence.com  intended as additional reference material  to supplement material presented in the lecture.

Wnts are a family of lipid-modified secreted glycoproteins which are involved in:

Normal physiological processes including

A. Development:

– Osteogenesis and adipogenesis (Loss of wnt/β‐catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes)

  – embryogenesis including body axis patterning, cell fate specification, cell proliferation and cell migration

B. tissue regeneration in adult tissue

read: Wnt signaling in the intestinal epithelium: from endoderm to cancer

And in pathologic processes such as oncogenesis (refer to Wnt/β-catenin Signaling [7.10]) and to your Powerpoint presentation

 

The curation Wnt/β-catenin Signaling is a comprehensive review of canonical and noncanonical Wnt signaling pathways

 

To review:

 

 

 

 

 

 

 

 

 

 

 

Activating the canonical Wnt pathway frees B-catenin from the degradation complex, resulting in B-catenin translocating to the nucleus and resultant transcription of B-catenin/TCF/LEF target genes.

Fig. 1 Canonical Wnt/FZD signaling pathway. (A) In the absence of Wnt signaling, soluble β-catenin is phosphorylated by a degradation complex consisting of the kinases GSK3β and CK1α and the scaffolding proteins APC and Axin1. Phosphorylated β-catenin is targeted for proteasomal degradation after ubiquitination by the SCF protein complex. In the nucleus and in the absence of β-catenin, TCF/LEF transcription factor activity is repressed by TLE-1; (B) activation of the canonical Wnt/FZD signaling leads to phosphorylation of Dvl/Dsh, which in turn recruits Axin1 and GSK3β adjacent to the plasma membrane, thus preventing the formation of the degradation complex. As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it promotes the expression of target genes via interaction with TCF/LEF transcription factors and other proteins such as CBP, Bcl9, and Pygo.

NOTE: In the canonical signaling, the Wnt signal is transmitted via the Frizzled/LRP5/6 activated receptor to INACTIVATE the degradation complex thus allowing free B-catenin to act as the ultimate transducer of the signal.

Remember, as we discussed, the most frequent cancer-related mutations of WNT pathway constituents is in APC.

This shows how important the degradation complex is in controlling canonical WNT signaling.

Other cell signaling systems are controlled by protein degradation:

A.  The Forkhead family of transcription factors

Read: Regulation of FoxO protein stability via ubiquitination and proteasome degradation

B. Tumor necrosis factor α/NF κB signaling

Read: NF-κB, the first quarter-century: remarkable progress and outstanding questions

1.            Question: In cell involving G-proteins, the signal can be terminated by desensitization mechanisms.  How is both the canonical and noncanonical Wnt signal eventually terminated/desensitized?

We also discussed the noncanonical Wnt signaling pathway (independent of B-catenin induced transcriptional activity).  Note that the canonical and noncanonical involve different transducers of the signal.

Noncanonical WNT Signaling

Note: In noncanonical signaling the transducer is a G-protein and second messenger system is IP3/DAG/Ca++ and/or kinases such as MAPK, JNK.

Depending on the different combinations of WNT ligands and the receptors, WNT signaling activates several different intracellular pathways  (i.e. canonical versus noncanonical)

 

In addition different Wnt ligands are expressed at different times (temporally) and different cell types in development and in the process of oncogenesis. 

The following paper on Wnt signaling in ovarian oncogenesis shows how certain Wnt ligands are expressed in normal epithelial cells but the Wnt expression pattern changes upon transformation and ovarian oncogenesis. In addition, differential expression of canonical versus noncanonical WNT ligands occur during the process of oncogenesis (for example below the authors describe the noncanonical WNT5a is expressed in normal ovarian  epithelia yet WNT5a expression in ovarian cancer is lower than the underlying normal epithelium. However the canonical WNT10a, overexpressed in ovarian cancer cells, serves as an oncogene, promoting oncogenesis and tumor growth.

Wnt5a Suppresses Epithelial Ovarian Cancer by Promoting Cellular Senescence

Benjamin G. Bitler,1 Jasmine P. Nicodemus,1 Hua Li,1 Qi Cai,2 Hong Wu,3 Xiang Hua,4 Tianyu Li,5 Michael J. Birrer,6Andrew K. Godwin,7 Paul Cairns,8 and Rugang Zhang1,*

A.           Abstract

Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy in the US. Thus, there is an urgent need to develop novel therapeutics for this disease. Cellular senescence is an important tumor suppression mechanism that has recently been suggested as a novel mechanism to target for developing cancer therapeutics. Wnt5a is a non-canonical Wnt ligand that plays a context-dependent role in human cancers. Here, we investigate the role of Wnt5a in regulating senescence of EOC cells. We demonstrate that Wnt5a is expressed at significantly lower levels in human EOC cell lines and in primary human EOCs (n = 130) compared with either normal ovarian surface epithelium (n = 31; p = 0.039) or fallopian tube epithelium (n = 28; p < 0.001). Notably, a lower level of Wnt5a expression correlates with tumor stage (p = 0.003) and predicts shorter overall survival in EOC patients (p = 0.003). Significantly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model. Mechanistically, Wnt5a antagonizes canonical Wnt/β-catenin signaling and induces cellular senescence by activating the histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway. In summary, we show that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence. We suggest that strategies to drive senescence in EOC cells by reconstituting Wnt5a signaling may offer an effective new strategy for EOC therapy.

Oncol Lett. 2017 Dec;14(6):6611-6617. doi: 10.3892/ol.2017.7062. Epub 2017 Sep 26.

Clinical significance and biological role of Wnt10a in ovarian cancer. 

Li P1Liu W1Xu Q1Wang C1.

Ovarian cancer is one of the five most malignant types of cancer in females, and the only currently effective therapy is surgical resection combined with chemotherapy. Wnt family member 10A (Wnt10a) has previously been identified to serve an oncogenic function in several tumor types, and was revealed to have clinical significance in renal cell carcinoma; however, there is still only limited information regarding the function of Wnt10a in the carcinogenesis of ovarian cancer. The present study identified increased expression levels of Wnt10a in two cell lines, SKOV3 and A2780, using reverse transcription-polymerase chain reaction. Functional analysis indicated that the viability rate and migratory ability of SKOV3 cells was significantly inhibited following Wnt10a knockdown using short interfering RNA (siRNA) technology. The viability rate of SKOV3 cells decreased by ~60% compared with the control and the migratory ability was only ~30% of that in the control. Furthermore, the expression levels of β-catenin, transcription factor 4, lymphoid enhancer binding factor 1 and cyclin D1 were significantly downregulated in SKOV3 cells treated with Wnt10a-siRNA3 or LGK-974, a specific inhibitor of the canonical Wnt signaling pathway. However, there were no synergistic effects observed between Wnt10a siRNA3 and LGK-974, which indicated that Wnt10a activated the Wnt/β-catenin signaling pathway in SKOV3 cells. In addition, using quantitative PCR, Wnt10a was overexpressed in the tumor tissue samples obtained from 86 patients with ovarian cancer when compared with matching paratumoral tissues. Clinicopathological association analysis revealed that Wnt10a was significantly associated with high-grade (grade III, P=0.031) and late-stage (T4, P=0.008) ovarian cancer. Furthermore, the estimated 5-year survival rate was 18.4% for patients with low Wnt10a expression levels (n=38), whereas for patients with high Wnt10a expression (n=48) the rate was 6.3%. The results of the present study suggested that Wnt10a serves an oncogenic role during the carcinogenesis and progression of ovarian cancer via the Wnt/β-catenin signaling pathway.

Targeting the Wnt Pathway includes curations of articles related to the clinical development of Wnt signaling inhibitors as a therapeutic target in various cancers including hepatocellular carcinoma, colon, breast and potentially ovarian cancer.

 

2.         Question: Given that different Wnt ligands and receptors activate different signaling pathways, AND  WNT ligands  can be deferentially and temporally expressed  in various tumor types and the process of oncogenesis, how would you approach a personalized therapy targeting the WNT signaling pathway?

3.         Question: What are the potential mechanisms of either intrinsic or acquired resistance to Wnt ligand antagonists being developed?

 

Other related articles published in this Open Access Online Scientific Journal include the following:

Targeting the Wnt Pathway [7.11]

Wnt/β-catenin Signaling [7.10]

Cancer Signaling Pathways and Tumor Progression: Images of Biological Processes in the Voice of a Pathologist Cancer Expert

e-Scientific Publishing: The Competitive Advantage of a Powerhouse for Curation of Scientific Findings and Methodology Development for e-Scientific Publishing – LPBI Group, A Case in Point 

Electronic Scientific AGORA: Comment Exchanges by Global Scientists on Articles published in the Open Access Journal @pharmaceuticalintelligence.com – Four Case Studies

 

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Targeting the Wnt Pathway

Writer and Curator: Larry H Bernstein, MD, FCAP 

 

7.11 Targeting the Wnt Pathway

7.11.1 Targeting the Wnt pathway in human cancers. Therapeutic targeting with a focus on OMP-54F28

7.11.2 Wnt signaling and hepatocarcinogenesis – Molecular targets

 

7.11.4 SALL4 is directly activated by TCF.LEF in the canonical Wnt signaling pathway

7.11.5 SALL4. An emerging cancer biomarker and target

7.11.6 Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer

7.11.7 The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma

7.11.8 Overexpression of the novel oncogene SALL4 and activation of the Wnt.β-catenin pathway in myelodysplastic syndromes

 

 

7.11.1 Targeting the Wnt pathway in human cancers. Therapeutic targeting with a focus on OMP-54F28

Le PN, McDermott JD, Jimeno A.
Pharmacol Ther. 2015 Feb; 146:1-11
http://dx.doi.org/10.1016/j.pharmthera.2014.08.005

The Wnt signaling pathways are a group of signal transduction pathways that play an important role in cell fate specification, cell proliferation and cell migration. Aberrant signaling in these pathways has been implicated in the development and progression of multiple cancers by allowing increased proliferation, angiogenesis, survival and metastasis. Activation of the Wnt pathway also contributes to the tumorigenicity of cancer stem cells (CSCs). Therefore, inhibiting this pathway has been a recent focus of cancer research with multiple targetable candidates in development. OMP-54F28 is a fusion protein that combines the cysteine-rich domain of frizzled family receptor 8 (Fzd8) with the immunoglobulin Fc domain that competes with the native Fzd8 receptor for its ligands and antagonizes Wnt signaling. Preclinical models with OMP-54F28 have shown reduced tumor growth and decreased CSC frequency as a single agent and in combination with other chemotherapeutic agents. Due to these findings, a phase 1a study is nearing completion with OMP-54F28 in advanced solid tumors and 3 phase 1b studies have been opened with OMP-54F28 in combination with standard-of-care chemotherapy backbones in ovarian, pancreatic and hepatocellular cancers. This article will review the Wnt signaling pathway, preclinical data on OMP-54F28 and other Wnt pathway inhibitors and ongoing clinical trials.

OMP-54F28

OMP-54F28

OMP-54F28
http://ars.els-cdn.com/content/image/1-s2.0-S0163725814001624-gr1.sml

Wnt signaling pathway

Three Wnt signaling pathways have been defined, including the canonical, non-canonical planar cell polarity pathway and the noncanonical Wnt/Ca2+ pathway.Of the three,the canonicalWnt pathway is the best described. Here, a cysteine-rich Wnt ligand binds the extracellular cysteine-rich domain (CRD) at the amino terminus of a seven pass transmembrane receptor termed Frizzled (FZ/Fzd [Vinson et al., 1989; Bhanot et al., 1996]) and low-density lipoprotein (LDL) receptor-related protein 5/6 (LRP5/6) that acts as a co-receptor (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000) to start the activation of the canonical Wnt signaling pathway. Nineteen Wnt ligands have been identified along with 10 Fzd receptors (Huang & Klein,2004).Various Wnt ligands have been shown to bind to particular Fzd receptors, but this interaction is promiscuous wherein oneWnt can bind multiple Fzd receptors (Bhanot et al., 1996). Wnt glycoproteins are relatively hydrophobic and insoluble possibly due to cysteine palmitoylation by Porcupine(PORC [Willertetal., 2003; Zhai et al., 2004]). However, PORC is required for Wnt signaling, suggesting that palmitoylation is essential in Wnt ligand secretion and pathway activation. Wnt ligands can activate signaling by both autocrine and paracrine signaling (Bafico et al., 2004). Wnt signaling can be inhibited through the binding of soluble Dickkopf (DKK) to LRP5/6 (Glinkaetal.,1998) or secreted Frizzled-related protein (SFRP) binding to Wnt ligands due to their sequence homology to the CRD domain of Fzd (Hoang et al., 1996). Wnt inhibitor factor (WIF) proteins, due to their similarity to the extracellular domain of derailed/RYK Wnt transmembrane receptors, can also regulate Wnt signaling by interacting with Wnt ligands (Hsieh et al., 1999a). When there is no Wnt ligand present, β-catenin levels are limited by the destruction complex that includes Adenomatous Polyposis Coli (APC) and AXIN. With Wnt signaling “off,” AXIN facilitates the phosphorylation of β-catenin by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK-3 [Peifer et al., 1994; Yost et al., 1996; Sakanaka etal.,1999;Liuetal.,2002]). These phosphorylated ser/thr sites are recognized by an E3ubiquitin ligase complex, and β-catenin is subsequently targeted for proteasomal degradation (Aberleetal.,1997). Therefore, β-catenin is maintained at low cytoplasmic and nuclear levels. In the “on” state, Wnt ligand binds the extracellular CRD of the amino terminus of Fzd and the LRP5/6 co-receptor (Dannet al., 2001; Pinson et al., 2000;Tamaietal.,2000). Dishevelled (Dsh/Dvl) is activated and recruited along with the destruction complex to the plasma membrane (Lee et al., 1999;Rothbacher et al., 2000).AXIN alsointeracts with the plasma membrane, possibly by binding the cytoplasmic tail of LRP5/6 (Mao et al., 2001). This binding is promoted by phosphorylation of LRP5/6 by GSK-3 and CK1 (Davidson et al., 2005; Zeng et al., 2005). AXIN is degraded, and GSK-3 is thus prevented from phosphorylating β-catenin. This leads to the accumulation of β-catenin in the nucleus and its interaction with T-cell factor (TCF) and lymphoid enhancerbinding protein (LEF) transcription factors to activated downstream targets (Behrenset al., 1996;Huber et al., 1996).

Wnt pathway and cancer

Aberrant Wnt signaling was first implicated in cancer in mouse studies, where mouse mammary tumor virus (MMTV) was found to be virally inserted into the promoter region of Int-1, promoting mammary tumors (Nusse & Varmus, 1982; Tsukamoto et al., 1988). It was later found that Int-1 was a homologue to Wg, and thus renamed Wnt (Nusse et al., 1991; Rijsewijk et al., 1987). Since this time, the Wnt pathway has been shown to be aberrantly regulated in many cancers. Abnormal β-catenin activation has been well characterized in colon cancer, where mutations in APC, or less frequently in β-catenin, results in constitutively active β-catenin and consequently active downstream effectors (Morin et al., 1997). While APC and β-catenin mutations are rare in lung cancer, overexpression of Dvl, Wnt-1 and Wnt-2 have all been correlated with non-small cell lung cancer (NSCLC) (He et al., 2004; Pongracz & Stockley, 2006; Ueda et al., 2001; Uematsu et al., 2003; You et al., 2004c). Moreover, increased tumor relapse was associated with a TCF4 Wnt gene signature in lung adenocarcinomas (Nguyen et al., 2009b). Together, these data provide strong evidence for the role of Wnt signaling in lung cancers. Wnt-5a has also been shown to be increased in breast cancer (Lejeune et al., 1995). Several of Fzds that have been shown to be overexpressed in cancers and/or cancer cell migration include Fzd4, Fzd7, Fzd8 and Fzd10 (Fukukawa et al., 2009; Jin et al., 2011; Ueno et al., 2009; Wang et al., 2012b; Yang et al., 2011). These have been shown to activate the canonical and/or non-canonical Wnt pathway. However, these are just a few of the studies linking Fzd overexpression with cancer, and an extensive list was previously covered by Ueno et al. (2013). Wnt expression has also beenassociated with metastasis and tumor microenvironment. Inhibition of Wnt signaling byRNAi targeting LEF1 and HOXB9 reduced brain and bone metastasis using a mouse model of lung adenocarcinoma (Nguyen et al., 2009b). The mechanism of LEF1 and HOXB9 metastasis promotion was not elucidated in this study although Wnt signaling, specifically Wnt-1 and Wnt-5a, has been shown to increase proliferation and survival of endothelial cells (Masckauchan et al., 2005,2006). β-catenin was also shown to correlate with VEGF expression in colon cancer (Easwaran et al., 2003; Zhang et al., 2001), suggesting ar ole for Wnt signaling in angiogenesis.Moreover, Wnt-5a expression has recently been shown to be increased in NSCLC; its expression in patient tissue was correlated with expression of angiogenesis related proteins such as vascular endothelial cadherin and matrix metalloprotease 2, microvessel density and vasculogenic mimicry, all of which suggest a role for Wnt-5a in promoting angiogenesis (Yao et al., 2014). Decreased expression of Wnt pathway inhibitors (WIF-1,DKKs, and SFRPs) “allows” for the activation of Wnt signaling and has also been observed in various cancers.For example, the down-regulation of associated Wnt antagonist, WIF-1, has been implicated in the breast, prostate, lung and bladder cancer (Wissmann et al., 2003). Furthermore, WIF-1 has been shown to be epigenetically silenced in lung and bladder cancer (Mazieres et al., 2004; Urakami et al., 2006). Epigenetic silencing of DKK-1 has been shown in colorectal cancer (Aguilera et al., 2006) and SFRP in NSCLC, hepatocellular carcinoma and colorectal cancer (Fukui etal., 2005; Shihetal., 2006; Suzukietal., 2004). Recent studies suggest that Wnt inhibitors may also play a pro-apoptotic role, where reduced apoptosis and p53 expression were observed in mammary glands isolated from SFRP-/- mice following induction of DNA damage by -irradiation (Gauger & Schneider, 2014). In addition, another study suggests that WIF- 1 may inhibit angiogenesis. DKK-1 and WIF-1 directly interact and together may act as co-regulators in promoting apoptosis in the human umbilical vein endothelial cell (HUVEC) system(Koetal.,2014). Although Wnt signaling is not as well correlated with head and neck squamous cell carcinoma (HNSCC) as with other cancers, such as colon cancer, recent studies provide evidence that Wnt signaling is an attractive target in HNSCC. Wnt pathway activation has been shown in HPV positive HNSCC, possibly driven by E6 and E7 (Rampias et al., 2010). β-catenin nuclear accumulation was also observed in the majority of patient HNSCC tumor samples (Wend et al., 2013). Up-regulation of several Fzd receptors was observed in HNSCC, including Fzd1, Fzd7a, Fzd10b, Fzd2 and Fzd13 (Rhee et al., 2002). Furthermore, Wnt expression may affect radio sensitivity in HNSCC cell lines, where β-catenin nuclear accumulation was correlated with radiation-resistance (Chang et al.,2008). Similarly, radiation-resistant mouse mammary progenitor cells were associated with active Wnt signaling (Chen et al., 2007; Woodward et al., 2007). Wnt expression has been correlated with therapy resistance in prostate cancer, where Wnt16B increased following therapy and lessened DNA damage following treatment with a topoisomerase inhibitor (Sun et al., 2012). In this study, Wnt16B increased growth and proliferation. Taken together, these studies suggest that Wnt expression not only promotes cancer cell proliferation, but may also affect treatment efficacy. Furthermore, the up-regulation of Wnt16B originating specifically in the stroma compartment, and through tumor-stroma interactions promoting therapy resistance in the tumor compartment, suggests that the stroma is a favorable target for therapy. Consistent with this,human ovarian fibroblasts released Wnt16B in to the stroma compartment following DNA damage by radiation or chemotherapy (Shen et al., 2014). Interestingly stromal Wnt16B activated the Wnt signaling pathway in dendritic cells (DCs), causing the release of interleukin-10 (IL-10) and tumor growth factor-β (TGF-β) and regulatory T-cell differentiation. Thus, Wnt16B may not only confer therapy resistance, but also alter the tumor microenvironment and as the authors suggest, possibly promote immune evasion.

Wnt signaling and cancer stem cells (CSCs)

Wnt signaling is important in stem cell homeostasis.In the intestinal villi Wnt signaling is particularly important in stem cell maintenance as well as in determining stem cell fate (Batlle et al., 2002; Korinek et al., 1998). Wnt signaling has also been shown to be essential in stem cell proliferation and hair follicle development and may function to activate stem cells in the bulge to more proliferative progenitor cells, as well as determining cell fate (Andl et al., 2002; Choi et al., 2013; Lien et al., 2014; Lowry et al., 2005).Similarly,Wnt overexpression in hematopoietic stem cells leads to the expansion of progenitor cells, suggesting that Wnt signaling is also important in hematopoiesis (Austin et al., 1997). Aberrant Wnt signaling in the stem cell compartment has been shown to contribute to tumorigenesis. Here, it is important to note thatwhile some authors appropriately choose conservative terminology in the definition of CSCs, for the purpose of coherency in this review, we loosely combine tumor-initiating, tumor propagating and CSCs into one term, as CSCs. Loss of APC, consequently leading to the accumulationof β-catenin, in colorectal cells resulted in cells maintaining a phenotype similar to progenitor cells of the crypt (Sansom et al., 2004). In another approach, high levels of Wnt expression were observed in CSCs from colon cancer grown as spheroids (Vermeulen et al., 2010). Similarly, Wnt-1, -3 and -5a all promoted murine mammosphere growth, a method that enriches for stem cells, and results suggested both canonical and non-canonical Wnt signaling could promote growth (Many & Brown, 2014). Furthermore, hair follicle tumors were observed to have stable expression of β-catenin in mice (Gat et al.,1998). A recent study found hair follicle stem cells (HFSC) treated with dimethylbenzanthracene (DMBA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced sebaceous neoplasms in C57BL/6 mice, as well as increased Wnt10b expression in basal cells via immunostaining (Qiu et al., 2014). Here the authors propose a model wherein increased Wnt10b results in proliferation and differentiation of HFSCs and thus promoting sebaceous neoplasms. High levels of Wnt expression were also observed in granulocyte-macrophage progenitors isolated from chronic myeloid leukemia (CML) patients and correlated with increased self-renewal (Jamieson et al., 2004). Fzd4 was suggested to regulate “stemness” of cancer cells and promote invasiveness in glioma cells (Jin et al., 2011). In HNSCC cell lines, side populations sorted by Hoechst efflux, a functional assay for enriching stem cells, were more invasive and tumorigenic in nude mice, and importantly these populations exhibited higher Wnt signaling (Song et al., 2010). Together, the data suggest that the same Wnt signaling mechanisms that regulate stem cells, when abnormal, may contribute to the tumorigenic potential of CSCs.

Targeting the Wnt pathway
Wnt pathway components are often difficult to target due to their redundancy in other functions. β-catenin, for example, also interacts with E-cadherin, an interaction that is essential for cell adhesion, as well as interacting with APC and TCF competitively within the same armadillo repeat domain (Behrens et al., 1996; Hulsken et al., 1994; Ozawa et al., 1989). In order to circumvent this, specific inhibitors that disrupt the β-catenin and TCF interaction have been widely explored, as well as RNAi approaches. However, even with utmost specificity, due to the essential role of the Wnt pathway in stem cell maintenance, tissue homeostasis and cell fate determination, targeting this signaling pathway has potential pitfalls. A potential concern is that toxicity, specifically to the GI tract, as well as anemia and immune suppression, might be too great for obtaining an adequate therapeutic index. In spite of these potential hurdles, research toward identifying potent Wnt pathway antagonists for cancer treatment has been promising.

Natural compounds

Non-steroidal anti-inflammatory drugs (NSAIDS), vitamins A and D, and polyphenols, such as curcumin and resveratrol, have all been shown to inhibit the Wnt pathway, and these have been elegantly reviewed (Table 1 and Fig. 1 [Barker & Clevers, 2006; Takahashi-Yanaga & Sasaguri, 2007; Takahashi-Yanaga & Kahn, 2010]). These compounds, although promising, have shown insufficient efficacy and thus may prove ineffectual as single-agent treatments. For example, the use of NSAIDS, specifically sulindac, in patients diagnosed with Familial Adenomatous Polyposis (FAP) reduced the number of polyps by only ~44% (Giardiello et al., 1993). Quercetin, a polyphenol and dietary flavonoid, has also been shown to decrease β-catenin and TCF protein levels (Fig.1) and inhibit colon cancer cell growth in vitro via decreased cyclin D1 and survivin levels (Park et al., 2005; Shan et al., 2009). Quercetin was also shown to inhibit murine mammary cancer cell growth and target theWnt pathway through DKK1,2,3 and 4 up-regulation (Kimetal., 2013). Salinomycin, an antibacterial potassium ionophore, was first identified by high throughput screening and was shown to inhibit breast CSCs (Gupta et al., 2009). Its mechanism was later elucidated and was shown to inhibit LRP5/6 phosphorylation, causing its degradation (Fig. 1 [Lu et al., 2011a]). Salinomycin has recently been shown to inhibit breastand prostate cancer cell proliferation and induce apoptosis, targeting Wnt signaling by decreased LRP5/6 expression, but also by targeting mTORC (Lu & Li, 2014), suggesting it may function in targeting multiple pathways. Salinomycin has also been shown to have antitumorigenic effects in hepatocellular carcinoma, osteosarcoma, gastric cancer, NSCLC and nasopharygeal carcinoma; studies suggest that it specifically targets CSCs by inhibiting cell proliferation, inducing apoptosis and limiting cell migration (Arafat et al., 2013; Mao et al., 2014; Tang et al., 2011; Wang et al., 2012a; Wu et al., 2014). COX-2 inhibitors may target the Wnt pathway by inhibiting prostaglandin E2 (PGE2), the product of COX-2, which acts to phosphorylate GSK-3 (Fig. 1 [Fujino et al., 2002]). Celecoxib, a NSAID and a COX-2 inhibitor, has been shown to decrease CD133 expression, a surface marker of prostate CSCs, by targeting the Wnt pathway, and this effect was observed to be independent of its COX-2 inhibiting activity (Deng et al., 2013). In order to circumvent the toxicities associated with long term COX-2 inhibition, one group suggests using synthetic derivatives of sulindac, another NSAID that was previously mentioned, that do not target COX-2 and were successful in limiting colon cancer cell growth and promoting apoptosis in vitro(Li et al., 2013;Whitt e tal., 2012). Resveratrol has recently been shown to inhibit the growth of breast CSCs both in invitroandwhenimplantedinNOD/SCIDmicebytargetingthecanonicalWntpathwayandinducingautophagy(Fuetal.,2014).Resveratrol also limited growth of cervical cancer cells by causing cell cycle arrest and inducing apoptosis (Zhang et al., 2014b). This study found resveratrol not only disrupted Wnt signaling, but also abrogated STAT3 signaling.

Fig.1.Mechanisms of inhibitors within the Wnt pathway.Wnt inhibitors act at various points within the active Wnt pathway.Common targets include Wnt ligands, including sequestration by OMP-54F28, and the β-catenin/TCF interaction. LGK974 is unique in that it inhibits pathway activation by preventingWnt ligand secretion by inhibiting palmitoylation by PORC. COX inhibition by NSAIDS prevents PGE2 from blocking the function of GSK-3 and Axin. Other targets are theWnt receptor, Fzd, and co-receptor LRP5/6. Several inhibitors act to stabilize the destruction complex, thus preventing the accumulation of β-catenin and transcription of downstream effectors. Alternatively, others prevent transcription by inhibiting transcriptional co-factors.

Small molecule inhibitors

There are many inhibitors that specifically disrupt the interaction of β-catenin with other key components of the Wnt pathway. PNU74654 was discovered by high-throughput screening, and was shown to inhibit the interaction of β-catenin and TCF (Fig. 1 [Trosset et al., 2006]). Treatment with certain 2,4-diamino-quinazoline derivatives, another compound that disrupts the β-catenin/TCF interaction, resulted in 20– 35% tumor growth inhibition when colorectal cells were implanted in nude mice(Chen et al., 2009c). Another approach is to disrupt a different β-catenin/activator interaction. Emami et al. identified a small molecule inhibitor using a cellbased screen that specifically bound to CREB binding protein (CBP), a TCF co-activator, termed ICG-001 (Fig. 1 [Emami et al., 2004]). This inhibitor has been experimentally explored in other diseases with aberrant Wnt signaling including kidney disease and pulmonary fibrosis with promising success (Hao et al., 2011; Henderson et al., 2010; Sasakiet al., 2013). ICG-001, at higher doses,was found to induce apoptosis in colon cancer cells with minimal effects on normal colon cells in vitro (Emami et al., 2004). Using ICG-001 in combination with a Met inhibitor and a CXCR4 inhibitor delayed tumor onset in a breast cancer mouse model (Holland et al., 2013). Tumors that arose from CSCs (CD24+ CD29+) isolated from salivary gland tumors grown in NOD/SCID mice and passed into NOD/SCID mice had decreased tumor volume when treatedwith ICG-001(Wend et al., 2013).These salivary gland tumors were originally grown in mice that were double-mutants, wherein mice had a gain of function mutation in β-catenin and a loss of function mutation in BMPR1A, a receptor in bone morphogenetic proteins (BMP) signaling which has been shown to inhibit CSCs proliferation in glioblastomas (Piccirillo et al., 2006). These were subsequently implanted into NOD/SCID mice, suggesting that Wnt inhibition with ICG-001 is effective in inhibiting tumor growth where Wnt activation is one of the key drivers. ICG-001 has also been shown to inhibit cell proliferation in pancreatic ductal adenocarcinoma(PDAC) by causing a G1 cell cycle arrest; however this effect appeared to be independent of Wnt signaling inhibition, suggesting ICG-001 may also target other pathways (Arensman et al., 2014). Several small molecule inhibitors, including XAV939, JW55 and IWR-1 promote β-catenin degradation by inhibiting PARsylation by Tankyrase 1 and Tankyrase 2 and thereby stabilizing axin (Fig. 1 [Chen etal.,2009a;Huangetal.,2009;Waaleretal.,2012]). XAV939 promoted Axin apoptosis in neuroblastoma cells and inhibited proliferation under serum-deprivation in breast and colorectal cancer cells (Bao et al., 2012; Tian et al., 2013). JW55 inhibited in vivo tumor growth in APC mutant mice using colorectal carcinoma cells (Waaler et al., 2012). Similarly, IWR-1 inhibited colon and prostate cancer cell growth (Chen et al., 2009a). Other small molecule inhibitors target Dvl or PORC (Fig. 1).LGK974 inhibits PORC, an O-acyltransferase that is required for the palmitoylation of Wnt ligands and ligand secretion (Zhai et al., 2004) and induced tumor regression in vivo using a mouse model for Wntdriven breast cancer and HNSCC (Liu et al., 2013). Interestingly exome sequencing a panel of 40 HNSCC cell lines showed a strong correlation between LGK974 sensitivity and Notch1 mutations although the significance of this has yet to be elucidated. Several small molecule inhibitors target the Wnt pathway by interacting with Dvl and thereby the destruction complex, ultimately leading to decreased β-catenin. NSC668036, FJ9 and 3289–8625 were shown to inhibit Wnt signaling by directly binding the PDZ domain of Dvl (Fujii et al., 2007; Grandy et al., 2009; Shan et al., 2005). 3289–8625 was shown to inhibit PC3 prostate cancer cell growth (Grandy et al., 2009), and FJ9 was shown to induce apoptosis in both melanoma and NSCLC cell lines (Fujii et al., 2007). FJ9 also inhibited tumor growth using implanted NSCLC cells in a mouse xenograft model. Two FDA-approved anthelmintics effectively inhibit the pathway by targeting several factors. Using a high-throughput small molecule screen, Pyrivinium was identified as a Wnt antagonist (Thorne et al., 2010). Pyrivinium, classically used in the treatment for pinworm infection (Royer & Berdnikoff, 1962), inhibits Wnt signaling at multiple points in the pathway (Fig. 1). It binds and induces a conformational change in CK1, promoting its kinase activity, and thus stabilizing axin and retaining β-catenin in the cytoplasm. Furthermore, it promoted the degradation of pygopus, a nuclear factor that is required by β-catenin for transcription of downstream Wnt targets (Thorne et al., 2010). Pyrivinium has recently been shown to target Wnt signaling in colon cancer cells, resulting in increased cell death, inhibition of cell migration and delaying liver metastasis growth in vivo (Wiegering et al., 2014). Another FDA-approved drug termed niclosamide, routinely used in the treatment of tapeworm, inhibited Wnt signaling by causing Fzd1 receptor internalization and decreased Dvl2 protein levels in human osteosarcoma cells (Fig. 1 [Chen et al., 2009b]). In contrast, another study suggests that niclosamide acts through targeting LRP6, both decreasing its phosphorylation and overall protein expression. In this study, Dvl2 was unperturbed, and decreased cell proliferation and apoptosis induction were observed in prostate and breast cancer cells (Lu et al., 2011b). These findings suggest the mechanisms are dependent on cell type, warranting more studies on this compound. Niclosamide was found to decrease spheroid growth, increase apoptosis and inhibit tumor growth in NOD/SCID mice when using the side population sorted from breast cancer cells (Wang et al., 2013). Both spheroid growth and side population highly enrich for CSCs, indicating that niclosamide may function in target CSCs. Ye et al. used breast cancer cells and observed decreased proliferation, migration and invasion, as well as increased apoptosis and decreased tumor growth in an in vivo mouse model (Ye et al., 2014). Niclosamide has also been used to target basal-like breast,liver,brain and ovarian cancer (Arend et al., 2014; Londono-Joshi et al., 2014; Tomizawa et al., 2013; Wieland et al., 2013; Yo et al., 2012). It is important to note that these in vivo studies, as well as the ones stated earlier, have shown little to limited levels of toxicity, providing hopeful optimism for Wnt inhibition in human cancer therapy.

Viral-based inhibitors

Numerous studies have used viral-based targeting with recombinant adenoviruses (Barker & Clevers, 2006). This is accomplished by integrating TCF binding sites into robust promoters and thus achieving
cell killing specific to cells with active Wnt signaling. Cancer cell killing was attained through manipulation of adenoviruses with E1 and E2 promoters, and these effectively targeted cancers with aberrant Wnt signaling (Brunori et al.,2001; Fuerer & Iggo, 2002). Surprisingly, Brunori et al. observed cell killing in lung cancer cells, and little effect in colon cancer cells. However, the reason for this was largely unknown. Variations of this have been done, and in one study, the addition of ADP cytosolic protein boosted the ability of the virus to spread from cell to cell(Toth et al., 2004). In addition, viral expression and effectiveness in tumor growth inhibition using mouse xenograft models were specific to colon cancer cells and non-effective in lung cancer cells. This suggests specificity to those cancers with greater levels of Wnt activity. In another approach, using TCF-driven E1 and E4 promoters, the Na/I symporter (hNIS) gene was included in the recombinant adenovirus (Peerlinck et al., 2009). This resulted in the enhancement of 131I− radiotherapy and allowed for imaging and tracking the spread of the adenovirus using computed tomography(CT)imagingwhen injected with 99mTcO4 −. Similarly, other studies combine cytotoxic gene expression with specificity to the Wnt pathway by the integration of promoters that are under the control of TCF. Using this technique and various promoters, apoptosis promoting Fas-associated via death domain (fadd), diphtheria toxin A (DTA) and herpes simplex virus thymidine kinase (HSV TK) genes have all been expressed and shown to be effective in targeting cancer cells with active Wnt signaling (Chen & McCormick, 2001; Kwong et al., 2002; Lipinski et al., 2004). Alternatively, others have added a gene that enhances cytotoxicity of prodrugs in order to increase therapeutic efficacy (Fuerer  & Iggo, 2004; Lukashev et al.,2005).

Antibody-based inhibitors

As stated earlier, because of the overexpression of Wnt ligands and/or receptors in many cancers, antibody-based inhibitors have been developed to bind and sequester either free ligand or Fzd receptors (Fig. 1). Several antibodies toward Wnt ligands have been produced. A monoclonal Wnt-1 antibody was shown to induce apoptosis in NSCLC and breast cancer cells by Wnt inhibition and activating cytochrome c and caspase 3, as well as decreasing survivin expression (He et al., 2004). Furthermore, the Wnt-1 antibody inhibited tumor growth in nude mice with NSCLC cells implanted subcutaneously, independent of whether the antibody was administered at implantation or once tumors were established, suggesting the timing of antibody administration was irrelevant for tumor control. Similar results were observed in colon cancer and sarcoma using theWnt-1antibody(Heetal.,2005;Mikamietal.,2005). The Wnt-1 antibody also induced apoptosis in mesothelioma cells that weredeficientin β-catenin, suggestingnon-canonical Wnt signaling inhibition was possible as well (You et al., 2004a). By the same token, a monoclonalWnt-2 antibody induced apoptosis in NSCLC and melanoma, as well as inhibited tumor growth in a melanoma xenograft model (You et al., 2004b, 2004c). In Wnt-activated HNSCC cells, both Wnt-1 and Wnt-10b antibodies effectively blocked Wnt signaling, induced apoptosis and inhibited cell proliferation (Rhee et al., 2002). OMP-18R5 is a monoclonal antibodythat was initially identified for its ability to bind Fzd7. Since then, OMP-18R5 has been found to bind Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8 and block β-catenin signaling in responseto Wnt3a ligand (Gurney et al., 2012). In the same study, using humantumorxenografts,OMP-18R5inhibitedtumorgrowthinseveral tumor types, including colon, breast, pancreatic and lung cancers. Tumor recurrence was also delayed. Furthermore, the addition of OMP-18R5 to standard-of-care chemotherapies, such as paclitaxel, increased efficacy in tumor growth inhibition in a synergistic manner. Although off-target effects were suggested in that several Wnt genes were inhibited in the mouse liver, at effective doses there was little toxicity observedin the GI tract. In another approach, peptides with complementary sequences interacting with either the Wnt ligand or Fzd receptor are fused with the immunoglobulin Fc domain. Using this approach, for example, WIF1-Fc and SFRP-Fc were expressed in cancer cells using recombinant adenoviruses (Hu et al.,2009).Wnt signaling inhibition by these antagonists inhibited tumor growth and prolonged survival in hepatocellular xenografts.

OMP-54F28:preclinical data

Effective Wnt targeting has been accomplished using an immunoglobin Fc fused to Fzd8, Fzd8(1–173)hFc (Fig. 1 [Hsieh et al., 1999b; Reya et al., 2003]). Others improved upon this, and constructed a minimal Fzd8 protein (residues1–155), wherein possible protease cleavage sites were removed (DeAlmeida et al., 2007). The fusion of the CRD domain of Fzd8 with Fc (F8CRDhFc) exhibited an extended half-life in vivo in comparison to Fzd8(1–173)hFc and successfully inhibited growth in human teratoma tumor xenografts with very limited toxicity to regenerating tissues. OMP-54F28 is a truncated Fzd8 receptor fused to the IgG1Fc region. This inhibitor has been shown to block Wnt signaling and block tumor growth using a MMTV-Wnt1 induced tumor model (Hoey, 2013). Furthermore,OMP-54F28 was shown to synergize with chemotherapeutic agents.When a patient-derived pancreatic cancer xenograft model was treated with gemcitabine and OMP-54F28, OMP-54F28 alone reduced tumor growth to a greater extent than gemcitabine alone and a combination of the two gave a slight advantage over single-agen tOMP-54F28. OMP-54F28 also reduced the frequency of CSCs as quantitated by the number of tumors that regrew when serially passaged (30, 90 or 270 cells) into NOD/SCID for 82 days. Similar to tumor growth inhibition, the greatest reduction in CSC frequency occurred in combination, and this was slightly greater than OMP-54F28 alone. However, with gemcitabine alone the frequency of CSCs increased when compared to control. The percent of cells expressing CD44+, a marker for CSCs, decreased from ~12.7% to 1.9%with OMP-54F28 treatment alone as compared to an increase from ~12.7% to 13.9% when treated with gemcitabine alone and from ~12.7% to 1.7% when a combination of OMP-54F28 and gemcitabine was used. Using luciferase-labeled pancreatic tumor cells implanted orthotopically, tumors were grown for 30 days, treated with OMP-54F28 and imaged in vivo for metastases. A decrease in both liver and lung metastases was observed (Hoey, 2013). Althoughcancers,including pancreatic cancers, are initially sensitive to gemcitabine, they can become resistant to treatment. A gemcitabine resistant pancreatic tumor model was created by continuously passing cells inincreasing concentration of gemcitabine. Using this gemcitabine resistant model, tumor growth was inhibited with a combination of 5FU and irinotecanor OMP-54F28 alone in comparison to control. However when all three are combined, there is a greater effect in tumor growth inhibition. Epithelial specific antigen (ESA)+CD201+, a marker of pancreatic CSCs, was assessed and a substantial decrease was observed with a combination of the three compounds, while treatment with OMP-54F28 alone showed the greatest decrease in ESA + CD201+. Treatment of gemcitabine resistant xenografts with OMP-54F28, gemcitabine and Abraxane resulted in tumor growth inhibition, and this growth inhibition was greater than that with the combination of gemcitabine and Abraxane (Hoey, 2013). Together the data suggest that OMP54F28 inhibits tumor growth, limits CSC frequency and tumor recurrence and is active in gemcitabine resistant tumors. It is effective as a single agent, but also in combination with chemotherapeutic agents.

OMP-54F28: first-in-human clinical data

With the efficacy seen in preclinical solid tumor models, OMP-54F28 has been recently investigated in a first-in-human phase1a study with advanced solid tumors (Jimeno, 2014). The primary objective of this study was to determine the safety and toxicity profile of the drug in patients with advanced solid tumors. Secondary objectives included pharmacokinetics, immunogenicity, and preliminary efficacy of OMP-54F28. The study was designed as a 3+3 dose escalation trial with dose levels between 0.5 and 20 mg/kg given intravenously every 3 weeks. Dose limiting toxicities (DLTs) were assessed every 28 days, and tumor assessment was done every eight weeks. At the time of submission of this review, only preliminary data from the phase1 study has been reported. The main adverse effects seen with OMP-54F28 included dysgeusia, fatigue, muscle spasms, decreased appetite, nausea and vomiting. Modulation of the WNT pathway has been shown to have effects in the bone including bone remodeling (Goldring & Goldring, 2007). In the present study, β-C-terminal telopeptide (β-CTX), a marker of increased bone turnover, was closely assessed, and it was recommended that zoledronic acid should be given to patients with doubling of their β-CTX levels.

Ongoing studies with OMP-54F28

There are 3 ongoing phase 1b studies combining OMP-54F28 with other drugs in solid tumors based on preclinical data and the safety and tolerability found in the phase 1a trial (Table 2 [OncoMed Pharmaceuticals Inc., 2014]). The first trial is combining OMP-54F28 with sorafenibin patients with hepatocellular cancer. Patients included must have locally advanced or metastatic hepatocellular cancer with no prior systemic therapies. Patients will receive sorafenib 400 mg orally twice daily with OMP-54F28 intravenously on day 1 of a 21-day cycle. Initiallydosesof5 mg/kgor10 mg/kgwillbeused,andbasedonsafety data higher or lower doses may be evaluated. The primary objectives are to evaluate the safety and tolerability of OMP-54F28 in combination with sorafenib, to identify dose limiting toxicities (DLTs) and maximum tolerated dose (MTD) and to determine the recommended phase 2 dose. Secondary objectives include characterization of the pharmacokinetics of OMP-54F28 in combination with sorafenib, characterization of the immunogenicity of OMP-54F28 and preliminary assessment of efficacyof the two drugs combined. Another phase1b study will be evaluating the combination of OMP54F28, nab-paclitaxel,and gemcitabine in patients with pancreatic cancer. Patients enrolled must have previously untreated stage IV ductal adenocarcinoma of the pancreas. They will receive nab-paclitaxel 125 mg/m2 and gemcitabine 1000 mg/m2 intravenously on days 1, 8 and 15 of a 28-daycycle. OMP-54F28 will be given at either 3.5 mg/kg or 7.0 mg/kg intravenously on days 1 and 15. Depending on emerging safety data, higher doses may also be evaluated. The primary objectives of this study will include evaluation of the safety and tolerability of the drug combinations, identification of the DLTs and MTD and identification of the recommended phase 2 dose for OMP-54F28 in combination with nab-paclitaxel and gemcitabine. Secondary objectives will be characterization of the pharmacokinetics and immunogenicity of the drug combinations and preliminary assessment of the efficacy of these drugs for metastatic pancreatic cancer. The third phase1b trial open is studying OMP-54F28 in combination with paclitaxel and carboplatin in ovarian cancer. The patients included should have recurrent, platinum-sensitive ovarian cancer, defined as disease progression greater than 6 months after completing a minimum of 4 cycles of a platinum-containing chemotherapy regimen. Patients who have received prior treatment with paclitaxel and carboplatin for recurrent disease will be excluded. Paclitaxel 175 mg/m2 and carboplatin AUC 5 will be given intravenously on day 1 of a 21-day cycle. OMP-54F28 will be given at 5 mg/kg or 10 mg/kg intravenously on day 1 with potential further dose escalation based on safety data. The paclitaxel and carboplatin will be given for a maximum total of 6 cycles with OMP-54F28 continuing until disease progression.The primary objectives are safety and tolerability of OMP-54F28 in combination with paclitaxel and carboplatin, determination of any DLTs and the MTD and planned phase2 dose of OMP-54F28. Secondary objectives will include pharmacokinetics, pharmacodynamics and efficacy of the drug combination.

 

7.11.2 Wnt signaling and hepatocarcinogenesis – Molecular targets

Pez F1Lopez AKim MWands JRCaron de Fromentel CMerle P.
J Hepatol. 2013 Nov; 59(5):1107-17.
http://dx.doi.org/10.1016/j.jhep.2013.07.001

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide. HCC can be cured by radical therapies if early diagnosis is done while the tumor has remained of small size. Unfortunately, diagnosis is commonly late when the tumor has grown and spread. Thus, palliative approaches are usually applied such as transarterial intrahepatic chemoembolization and sorafenib, an anti-angiogenic agent and MAP kinase inhibitor. This latter is the only targeted therapy that has shown significant, although moderate, efficiency in some individuals with advanced HCC. This highlights the need to develop other targeted therapies, and to this goal, to identify more and more pathways as potential targets. The Wnt pathway is a key component of a physiological process involved in embryonic development and tissue homeostasis. Activation of this pathway occurs when a Wnt ligand binds to a Frizzled (FZD) receptor at the cell membrane. Two different Wnt signaling cascades have been identified, called non-canonical and canonical pathways, the latter involving the β-catenin protein. Deregulation of the Wnt pathway is an early event in hepatocarcinogenesis and has been associated with an aggressive HCC phenotype, since it is implicated both in cell survival, proliferation, migration and invasion. Thus, component proteins identified in this pathway are potential candidates of pharmacological intervention. This review focuses on the characteristics and functions of the molecular targets of the Wnt signaling cascade and how they may be manipulated to achieve anti-tumor effects.

HCC represents a major public health problem with a high impact on society. HCC is the sixth most common tumor worldwide in terms of incidence (about one million per year). Projections are that this incidence will substantially increase during the next decades due to persistent infection with the hepatitis C virus as well as the emergence of non-alcoholic steatohepatitis as a major health problem. HCC portends a poor prognosis since ranking third in terms of “cause of death” by cancer, and often presents as a major complication of cirrhosis related to chronic hepatitis B and C infections, or non-virus related [[1], [2], [3]]. The dismal prognosis is generally related to a late diagnosis after HCC cells have infiltrated the liver parenchyma, have spread through the portal venous system and/or have formed distant metastases. However, if HCC is diagnosed early (<20% of patients), these smaller tumors may be cured by surgical resection, liver transplantation or radiofrequency ablation. In more advanced tumors (>80% of patients at diagnosis), only palliative approaches can be applied. In this regard, transarterial intrahepatic chemoembolization has been shown to be somewhat effective in increasing overall survival of individuals with tumors that have spread only into the liver parenchyma without extrahepatic metastasis (median overall survival is increased from 15 to 20 months compared to the best supportive care). In HCC with extrahepatic spread, only sorafenib, an anti-angiogenic and MAP kinase inhibitor, has been shown to increase overall survival of patients (from 8 to 11 months) [4]. All other systemic approaches such as cytotoxic chemotherapy have not been shown to be effective; thus, to date, no targeted therapy except sorafenib has been proven to prolong life in patients with HCC. However, there are ongoing or ended clinical trials with agents that target FGF, VEGF, PDGF, EGF, IGF, mTOR, and TGFβ signaling pathways but none has been shown yet to have a significant impact on patient survival [5].

Recently, cancer stem cells (CSC) have been hypothesized to play a key role in tumor maintenance as well as relapse after surgical resection. There is accumulating information that supports a role for CSC in hepatocarcinogenesis to maintain the tumor size and to initiate tumor recurrence following therapy [6]. The pool of CSC is maintained by self-renewal capabilities that are largely driven by reactivation of embryonic signaling programs mediated by Wnt, Notch, Bmi, and Hedgehog pathways, similar to what has been previously demonstrated during breast carcinogenesis [7]. Preclinical studies further underline the potential value of inhibiting activation of these signaling programs in some tumor types [[8],[9], [10], [11]].

In this review, we describe the features of a therapeutic target, i.e., the Wnt pathway, for potential therapy of HCC. We will discuss experimental and preclinical studies regarding the use of Wnt inhibitors as a therapeutic approach for HCC.

The Wnt-mediated signaling

The first member of the Wnt family of ligands was identified from the int-1 gene found in a mammary adenocarcinoma, located at the integration site of the mouse mammary tumor virus (MMTV); subsequently, it was demonstrated to have oncogenic properties [12]. More important, int-1 homolog genes have been found in human tumors as well [13]. In addition, a highly conserved int-1 homolog was also discovered in Drosophila and designated Wingless “Wg” [14]. The combination of int-1 and Wingless led to the common Wnt1 terminology and recently has been used to designate the Wnt family of ligands [15].

Wnt proteins are secreted extracellular auto-paracrine glycoproteins that interact with Frizzled receptors (FZD), a seven transmembrane domain protein, resembling the G-protein-coupled receptor (GPCR) family. Vinson and colleagues revealed that FZD contains an extracellular cysteine-rich domain (CRD) which is the putative binding site for the Wnt ligands. These investigators demonstrated the functional role of the frizzled locus to coordinate development of the cytoskeleton in Drosophila epidermal cells [16]. Subsequently, Wnt/FZD-mediated signaling has been extensively studied, and although it has been widely implicated in cellular homeostasis, these ligand/receptor interactions have now been appreciated as key factors during the oncogenesis process and therefore, could serve as new therapeutic targets.

Thus, Wnt proteins represent members of a highly conserved family that is involved in several processes including embryonic development, cell fate determination, proliferation, polarity, migration, and stem cell maintenance. In addition, Wnt/beta-catenin signaling has been found to play key roles in metabolic zonation of adult liver, regeneration [17]. In adult organisms, deregulation of Wnt signaling may lead to tumor development [[18], [19]]. The Wnt-mediated pathway is activated through the binding of one Wnt ligand to a FZD receptor. Ten different FZD receptors and 19 Wnt ligands have been identified in humans. The binding of Wnt to an FZD receptor can trigger activation of at least three different pathways. The first is the Wnt/β-catenin cascade, also called the Wnt-canonical pathway; the remaining two are the planar cell polarity (PCP) and the Wnt/calcium pathways, respectively. The two latter are β-catenin independent and represent examples of the non-canonical cascades. In this regard, a multitude of combinations between the 19 Wnt ligands and the 10 FZD receptors, such as co-receptors and other molecules, are theoretically possible. Classically, Wnt1/2/3/3a/8a/8b/10a/10b and FZD1/5/7/9 are classified as the canonical elements, whereas Wnt4/5a/5b/6/7a/7b/11 and FZD2/3/4/6 are designated as non-canonical components. The remaining Wnt2b/9a/9b/16 and FZD8/10 proteins remain unclassified [[19], [20]]. However, it remains elusive how selectivity between Wnt/FZD as well as specificity of downstream signaling is achieved. Some Wnt/FZD elements can share dual canonical and non-canonical functions. For instance, it has been shown that in absence of Ror2 co-receptors, Wnt5a can activate β-catenin signaling with FZD4 and Lrp5 [21]. FZD3 has been described to act likely through canonical pathways in mice neurogenesis [21]. Zhang et al. demonstrated that in Xenopus foregut, FZD7 can activate low level of β-catenin and non-canonical JNK signaling in which both pathways contributed to foregut fate and proliferation while JNK pathway regulated cell morphology [22]. It is of interest that canonical and non-canonical pathways can not only be driven by specific Wnt/FZD combinations, but also by cell type, differentiation status, localization and composition of the microenvironment [23].

The canonical Wnt/FZD pathway

The β-catenin protein, encoded by the CTNNB1 gene, is a key component of Wnt-canonical pathway signaling. β-catenin has a central region which presents armadillo domain repeats important for the binding of partners, such as Axin1 and adenomatous polyposis coli protein (APC) as well as transcription factors [24]. The C- and N-terminal regions are important. C-terminus of β-catenin serves as a binding factor for a multitude of complexes promoting β-catenin-mediated transcription, whereas phosphorylation of the N-terminus promotes degradation of β-catenin. Indeed, β-catenin may be present in several cellular compartments, such as the inner plasma membrane having a role in cell-cell junctions, the cytoplasm and the nucleus where it forms an active complex containing TCF/LEF transcription factors (T-cell factor/lymphoid enhancer factor) [25]. In the absence of nuclear β-catenin, TCF/LEF interact with the transcriptional co-repressor transducin like enhancer-1 (TLE-1) (Drosophila homolog Groucho), thus preventing β-catenin target gene expression [26]. Following translocation into the nucleus, β-catenin binds to TCF/LEF and replaces the TLE-1 repressor to form a transcriptional complex that activates the expression of its target genes (Fig. 1).

Canonical Wnt-FZD signaling pathway gr1_lrg

Canonical Wnt-FZD signaling pathway gr1_lrg

Canonical Wnt/FZD signaling pathway

http://www.journal-of-hepatology.eu/cms/attachment/2009077506/2031094357/gr1.sml

Fig. 1 Canonical Wnt/FZD signaling pathway. (A) In the absence of Wnt signaling, soluble β-catenin is phosphorylated by a degradation complex consisting of the kinases GSK3β and CK1α and the scaffolding proteins APC and Axin1. Phosphorylated β-catenin is targeted for proteasomal degradation after ubiquitination by the SCF protein complex. In the nucleus and in the absence of β-catenin, TCF/LEF transcription factor activity is repressed by TLE-1; (B) activation of the canonical Wnt/FZD signaling leads to phosphorylation of Dvl/Dsh, which in turn recruits Axin1 and GSK3β adjacent to the plasma membrane, thus preventing the formation of the degradation complex. As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it promotes the expression of target genes via interaction with TCF/LEF transcription factors and other proteins such as CBP, Bcl9, and Pygo.

In absence of the canonical Wnt signaling, cytosolic β-catenin is targeted for degradation by a complex composed of a scaffold of proteins named axin1, APC, and two serine/threonine kinases: the glycogen synthase kinase 3β (GSK3β) and the casein kinase 1 (CK1) [27] (Fig. 1A). Axin1 and APC act together as scaffolding proteins through binding of β-catenin, and enhance its N-terminal phosphorylation by GSK3β and CK1. The first phosphorylation event is generated by CK1 at Ser45 which allows the GSK3β-mediated sequential phosphorylation of Thr41, Ser37, and Ser33 [[28], [29]]. Ser37 and Ser33 phosphorylations provide a binding site for the E3 ubiquitin ligase β-TRCP (β-transducin repeat containing protein), leading to β-catenin ubiquitination in a β-TRCP/Skp1/cullin F-box complex (SCF) dependent manner followed by proteasomal degradation [[30], [31]].

Activation of the canonical Wnt signaling cascade leads to disruption of the β-catenin degradation complex, resulting in β-catenin accumulation in the cytoplasm followed by translocation into the nucleus where it serves as a transcription factor to activate downstream target genes (Fig. 1B). In brief, this process is as follows: Wnt ligand binds to the extracellular domain of an FZD receptor and Lrp5/6 co-receptors. This ternary complex (Wnt/FZD/Lrp) recruits the scaffolding phosphoprotein dishevelled (Dvl/Dsh) at the plasma membrane which in turn traps the axin-bound-GSK3β complex, thus preventing proteasomal degradation of cytosolic β-catenin. When stabilized, β-catenin is able to translocate into the nucleus, where it binds to TCF/LEF transcription factors and then forms a transcriptionally active complex with pygopus (Pygo), CBP (CREB-Binding Protein) and Bcl9 proteins [32]. In mammals, four TCF genes have been described, which adds further complexity to the mechanism(s) of activation of the Wnt canonical cascade [33]. Of notice is the β-catenin pool localized at the plasma membrane that plays a key role in cell-cell junctions. To this aim, a complex including either p120 catenin/γ-catenin(plakoglobin)/α-catenin or p120 catenin/β-catenin/α-catenin [25] binds to the cytoplasmic carboxyl terminus domain of E-cadherin adhesion molecule, in order to join cadherins to the actin cytoskeleton. More precisely, p120 catenin binds to the juxtamembrane and then β-catenin or γ-catenin binds to the cytoplasmic domain of E-cadherin. The remaining α-catenin serves as a link between actin and β/γ-catenin which leads to the stabilization of cell adhesion [34]. The possible consequences of inhibiting β-catenin at adherent junctions have to be discuss in respect of their role in epithelio-mesenchymal transition (EMT). Disruption of E-cadherin-mediated adherent junctions is a major event in EMT [35] and because of the interplay between cadherin-mediated cell adhesion and canonical/β-catenin signaling [36], targeting β-catenin could also promote the disruption of these junctions leading to enhance EMT. However, Wickline et al.have shown that in hepatocyte-specific β-catenin-conditional null mice, γ-catenin is upregulated and associated with E-cadherin and actin to maintain adherent junctions. In addition, no nuclear γ-catenin was detected in liver of KO mice, leading to the conclusion that despite armadillo domains on γ-catenin, there is no compensation at nuclear level. Nevertheless, authors warn us about preventing concurrent γ-catenin suppression that may increase tumor cell invasion [37]. More recent study confirmed these results in in vitro experiments with HCC cell lines and identified the mechanism of γ-catenin stabilization as serine/threonine phosphorylation induced by protein kinase A [38]. With regard to this recent data, targeting β-catenin in HCC therapies may not disturb cell junctions since the design of Wnt inhibitors for therapeutic intervention, specifically designates soluble active β-catenin as preferential target.

The non-canonical Wnt/FZD pathways

In contrast to the canonical Wnt pathway, non-canonical signaling does not depend on β-catenin and requires Ror2/Ryk co-receptors instead of Lrp5/6 (Fig. 2). In the Wnt/PCP pathway, Wnt/FZD interaction promotes the recruitment of Dvl/Dsh, which in turn binds to the small GTPase protein called Rac, leading to both the induction of ROCK (Rho-associated protein kinase) pathway and the activation of the MAP kinase cascade and subsequently to the activation of AP1-mediated target gene expression [[39], [40]]. In the Wnt/calcium pathway, the complex formation between FZD, Dvl/Dsh and G proteins results in PLC (Phospho Lipase C) activation which cleaves PIP2 (Phosphatidyl Inositol 4,5 biphosphate) into DAG (DiAcylGlycerol) and IP3 (Inositol 1,4,5-triphosphate). This process results in the activation of PKC (Protein Kinase C) through DAG while IP3 promotes calcium release from the endoplasmic reticulum. Increased intracellular concentration of calcium enhances phosphorylation and activation of PKCs. This also triggers the activation of Ca2+-calmodulin-dependent calcineurin and CAMKII (Ca2+-calmodulin dependent kinase II), leading to NFAT (Nuclear Factor of Activated T-cell) and NLK (Nemo Like Kinase) translocation, respectively. NLK acts as a β-catenin pathway inhibitor through phosphorylation and degradation of TCF/LEF transcription factors [41].

Non-canonical Wnt-FZD signaling pathway gr2_lrg

Non-canonical Wnt-FZD signaling pathway gr2_lrg

Non-canonical Wnt/FZD signaling pathway

http://www.journal-of-hepatology.eu/cms/attachment/2009077506/2031094361/gr2.sml

Fig. 2 Non-canonical Wnt/FZD signaling pathways. Interaction of Wnt, FZD, and ROR2/RYK co-receptors leads to either (1) JNK activation, (2) PKCs activation, (3) NFAT transactivation, or (4) inhibition of β-catenin activity through binding of NLK to TCF/LEF.

Antagonists and agonists of Wnt/FZD-mediated signaling

Several secreted proteins are known to negatively or positively regulate the Wnt/FZD complex. Four classes of antagonistic molecules have been described. Wnt inhibitory protein-1 (Wif1) and secreted FZD-related proteins (sFRP1, 2, 3, 4, 5) bind to and sequester the soluble Wnt ligands, thus inhibiting their interaction and binding to FZD receptors [[42],[43], [44], [45]]. The Dickkopf family is composed of four members (Dkk1, 2, 3, 4) that can interact with both Lrp5/6 and Krm1,2 (Kremen1,2) co-receptors [46]. The ternary complex Lrp-Dkk-Krm prevents β-catenin stabilization by promoting Lrp5/6 endocytosis [47]. Wise and Sost proteins form the other class of secreted antagonists. They bind to Lrp5/6 and thus disrupt the Wnt-induced FZD-Lrp5/6 interaction [[48], [49]].

Three agonistic molecules have recently been identified; the R-spondins (Rspo1, 2, 3, 4), norrin and glypican-3 (Gpc3). The Gpc3 is a heparan sulfate proteoglycan bound to the cell membrane through a glycosyl-phosphatidylinositol anchor. Gpc3 increases autocrine/paracrine canonical Wnt signaling by binding to Wnt ligands, thus facilitating the interaction between Wnt ligands and FZD receptors [50]. Mechanisms by which Rspo and Norrin activate the canonical Wnt pathway have not been clarified. Rspo1 is able to bind to both Lrps and FZDs but it has also been proposed that Rspo prevents Lrp6 internalization through binding to Krm instead of Dkk [[51], [52], [53]].

Wnt signaling deregulation in human hepatocarcinogenesis

Similar to other tumor tissue types, the canonical Wnt/FZD signaling is a critical contributor to HCC pathogenesis. Indeed, 40–70% of HCCs harbor nuclear accumulation of the β-catenin protein, one of the hallmarks of the Wnt/β-catenin pathway activation [[54], [55], [56]]. Activating mutations of the β-catenin gene (CTNNB1) occur in 8–30% of tumors, while loss-of-function/mutations in APC and Axin genes occur in 1–3% and 8–15%, respectively and are mutually exclusive to CTNNB1mutations [[54], [57], [58], [59], [60], [61], [62]]. Some observations suggest that the CTNNB1 mutation could be a late event during hepatocarcinogenesis. However, accumulation of β-catenin was detected in the early stage of HCC development, suggesting that other mechanisms could contribute to β-catenin stabilization (Table 1) [[60], [63]]. Strikingly, extrinsic activation of Wnt/β-catenin pathway and CTNNB1 mutation do not lead to the same molecular expression pattern, supporting different roles for wild type and mutated β-catenin. The Wnt/β-catenin activated HCC subclass with a CTNNB1mutation is characterized by upregulation of liver-specific Wnt-targets, low grade and well-differentiated tumors, with chromosome stability and a favorable prognosis. The Wnt/β-catenin activated HCC subclass without CTNNB1 mutation is characterized by dysregulation of classical Wnt targets, high chromosomal instability, aggressive phenotype, and is preferentially associated with chronic HBV infection [[54], [63], [64]].

Table 1Most prevalent potential mechanisms involved in activation of beta-catenin found so far in HCCs.

Modulation of Wnt ligands or FZD receptor expression could account for Wnt/β-catenin pathway activation without any other mutations in CTNNB1APC, or Axin genes. Indeed, upregulation of activators, such as ligands (Wnt1/3/4/5a/10b) or receptors/co-receptors (FZD3/6/7, Lrp6), and downregulation of inhibitors (sFRP1/4/5, Wif1, Dkk3, Dkk4) have been reported both in HCC tumors and surrounding precancerous liver tissues, which emphasizes that their over and/or underexpression may be early molecular events during hepatocarcinogenesis [[65], [66], [67], [68], [69], [70], [71],
[72],[73]].

Modulation of Wnt ligands or FZD receptor expression could account for Wnt/β-catenin pathway activation without any other mutations in CTNNB1APC, or Axin genes. Indeed, upregulation of activators, such as ligands (Wnt1/3/4/5a/10b) or receptors/co-receptors (FZD3/6/7, Lrp6), and downregulation of inhibitors (sFRP1/4/5, Wif1, Dkk3, Dkk4) have been reported both in HCC tumors and surrounding precancerous liver tissues, which emphasizes that their over and/or underexpression may be early molecular events during hepatocarcinogenesis [[65], [66], [67], [68], [69], [70], [71], [72],[73]].

Although β-catenin activation is crucial for liver development and regeneration, it is not sufficient per se for initiation of hepatocarcinogenesis. Indeed, animal models overexpressing an active β-catenin protein do not spontaneously form HCC [[74], [75], [76]]. However, β-catenin activation may cooperate with other oncogenic pathways such as insulin/IGF-1/IRS-1/MAPK, H-RAS, MET, AKT and chemicals to induce HCC formation in mice [[75], [77], [78], [79]]. It is described that beta-catenin mutation is a late event in hepatocarcinogenesis since present in some HCC tumors whereas absent in preneoplastic lesions, thus prompting us to speculate that only non-mutated beta-catenin could play a role in very early steps of hepatocarcinogenesis such as initiation and promotion. However, mutated forms of beta-catenin are used in experimental models to assess the role of activated beta-catenin in hepatocarcinogenesis. In these experimental mouse models, it is well shown that mutated beta-catenin is insufficient alone and per se for initiation of HCC but only enhance tumor promotion either in a context of chromosomal instability and increase of susceptibility to DEN-induced HCC formation [[78], [80]], or in a context of Lkb1+/− mice that spontaneously develop multiple hepatic nodular foci (NdFc) followed by HCC [81], or in a context of H-Ras transgenic mice where mutated beta-catenin appears as a strong carcinogenic co-factor collaborating with the mutated Ras oncogene [82]. In contrast and apparently paradoxically, invalidation of beta-catenin in hepatic beta-catenin conditional knockout mice has been found as enhancing DEN-induced tumorigenesis [83]. Of interest is another model of HCC developing in mice under exposure to phenobarbital (PB, potent tumor promoter in mouse liver) and DEN as tumor initiator. A tumor initiation–promotion study was conducted in mice with conditional hepatocyte-specific knockout (KO) of Ctnnb1 and in Ctnnb1 wild type controls. As expected, DEN + PB strongly enhanced liver tumor formation in Ctnnb1 wild type mice. Amazingly, the prevalence of tumors in Ctnnb1 KO mice was 7-fold higher than in wild type mice, suggesting an enhancing effect of the gene KO on liver tumor development [84]. Thus there is a paradox where the absence of wild type beta-catenin or presence of the mutated form, both lead to enhanced DEN-induced hepatocarcinogenesis. The issue is that the discussion is speculative since the mechanism of increased HCC in conditional beta-catenin KO is unknown. In the design of Wnt inhibitors for therapeutic intervention, these agents do target the Wnt pathway through the soluble beta-catenin cascade, but do not impact on invalidation of the beta-catenin pool involved in the membrane catenin/cadherin complexes involved in cell homeostasis. The beta-catenin therapeutic targeting may need to be personalized, based on the unexpected findings of enhanced tumorigenesis after chemical exposure in hepatocyte-specific beta-catenin conditional knockout mice.

Although the role of Wnt/β-catenin pathway is debated with respect to the initiation of hepatocarcinogenesis, it is definitively implicated in determining HCC aggressiveness, due to its promotion of increased cell proliferation, migration and invasion. This finding has been further substantiated by ectopic expression of Wnt3 and FZD7, Lrp6 or downregulation of sFRP1, Dkk1 and Dkk4 in HCC cell lines [[66], [69], [73], [85], [86]]. Moreover, recent studies have revealed that the Wnt/β-catenin pathway is also involved in the self-renewal and expansion of HCC initiating cells (i.e., the so-called liver CSC) which also influences tumor aggressiveness and resistance to chemo- radio-therapeutic agents [[87],[88]]. Furthermore, Wnt/FZD-mediated signaling could influence tumor microenvironment that supports tumor survival, growth, and size. Recent investigations emphasize the role of sFRP1 in the induction of senescence of tumor-associated fibroblasts after chemotherapeutic treatment [[89], [90], [91]].

It is noteworthy that the canonical and non-canonical Wnt/FZD pathways may have complementary roles in the pathogenesis of HCC. Indeed, β-catenin activation appears to be involved in the tumor initiation phase of hepatic oncogenesis, whereas subsequent activation of non-canonical pathways associated with inactivation of β-catenin may enhance tumor promotion and progression [88]. However, non-canonical pathways can also exhibit opposite effects on tumor behavior, since specific Wnt/FZD combinations are able to function as tumor suppressors [92]. Although little is known about the role of Wnt/PKC pathway in HCC, it has been demonstrated that inhibition of PKCβ activity reduces motility and invasion properties of HCC cells [93]. Finally, activation of the Wnt/JNK pathway during HCC progression would presumably support tumor growth, since enhanced JNK activity appears to be involved in HCC cell proliferation both in vitro and in vivo [94].

Identification of molecular targets for therapeutic interventions

There is some evidence to link the Wnt pathway activation to tumor cell properties characteristic of the malignant phenotype, such as enhanced cell proliferation, migration and invasion, which raises the possibility to target members of this signaling cascade as an attractive therapeutic approach for treatment of HCC [[95], [96]] (Fig. 3).

Potential Wnt-component targets  gr3_lrg

Potential Wnt-component targets gr3_lrg

Potential Wnt-component targets

http://www.journal-of-hepatology.eu/cms/attachment/2009077506/2031094360/gr3.sml

Fig. 3 Potential Wnt-component targets for therapeutic intervention on tumor development and growth. Inactivation of Wnt signaling pathway could be achieved by: (1) targeting extracellular signaling molecules with monoclonal antibodies, soluble factors or small molecules; (2) preventing the FZD/Dvl interaction; (3) stabilizing the destruction complex or (4) increasing β-catenin proteasomal degradation and (5) preventing the interaction between β-catenin and its co-factors for transactivity in the nucleus. The relationship between therapeutic molecules and their protein targets is indicated by a color code. Molecules in bold have been tested in HCC model, those in italics in other models of tumor growth.

Targeting extracellular molecules of the Wnt pathway

Antibody-based therapies directed against the overexpressed Wnt ligands and FZD proteins could provide a therapeutic approach. For instance, preclinical experiments have shown that an anti-Wnt1 monoclonal antibody inhibits the Wnt signaling pathway resulting in enhanced apoptosis and inhibiting cell proliferation, both in vitro and in vivo in a xenograft model of HCC [67]. These findings have been experimentally validated for several other types of tumors, such as sarcomas, colon, breast, non-small-cell lung cancer, and head-neck squamous cell carcinomas [[97], [98], [99], [100]]. Interestingly, as demonstrated with a colon cancer cell line, this anti-Wnt antibody was able to induce apoptosis even in the presence of downstream mutations in APC or CTNNB1 genes and appeared to be synergistic with docetaxel chemotherapy with respect to therapeutic response [97]. Although not tested in HCC tumors thus far, anti-Wnt2 antibodies may be useful to inhibit the Wnt/β-catenin cascade. Such antibodies induce apoptosis and inhibit tumor growth in vivo in several tumor types, including melanoma, mesothelioma, and non-small-cell lung cancer [[101], [102], [103]]. Since non-canonical pathways seem to be implied in tumor progression, the inhibition of Wnt-related ligand could be considered for therapy. For instance, WNT5A, which seems to be involved in the non-canonical pathway in HCC [88], could be antagonized by the use of anti-WNT5a antibodies. Indeed, in gastric cancer cells where WNT5A activates the non-canonical pathway, its inhibition reduces migration and invasion activities in vitro and in vivo [104]. Nevertheless, since the non-canonical pathway could antagonize the canonical one, it might be deleterious to inhibit the former. Anti-FZD7 antibodies that induce apoptosis and decrease cell proliferation both in vitro and in vivo of FZD7 positive Wilms’ tumor cells are also available [105]. More recently, a multispecific antibody that targets both FZD 1, 2, 5, 7, and 8 and mainly affects the canonical signaling pathway has been developed. It triggers a therapeutic reduction of breast, colon, lung and pancreas tumor growth and synergizes with other chemotherapeutic agents as well [106]. Strikingly, this antibody remains effective even in tumor cells with APC or CTNNB1 gene mutations. In addition, FZD co-receptors could also be attractive targets for monoclonal antibody therapy since, in a retinal pigment epithelial cell line, anti-Lrp6 antibody has been shown to inhibit Wnt signaling [107].

Another therapeutic strategy would be to trap the endogenous Wnt ligands with the exogenous soluble form of FZD receptors. This approach was reported for FZD7 by Tanaka and colleagues in esophagus carcinoma cells and confirmed later in HCC cells [[86], [108]]. More recently, Wei and co-workers have developed the same approach using an FZD7 extracellular domain peptide (sFZD7) that can bind to and sequester the soluble Wnt3 ligand. This peptide decreased the viability of HCC cell lines with high specificity, since normal hepatocytes were not sensitive to sFZD7. Moreover, sFZD7 cooperates with doxorubicin to reduce HCC cell proliferation in vitro and in a xenograft murine model as well. Interestingly, it has been shown to be highly efficient and independent of the β-catenin mutational status [109]. Inhibition of Wnt secretion by the small molecules, IWP2 and Wnt-C59 may also prevent autocrine Wnt signaling activation, as observed in colon cancer cell lines. These small molecules are also able to inhibit the progression of mammary tumors in Wnt1 transgenic mice [[110], [111]]. Addition of Wnt antagonist, such as sFRP1 or Wif1, has shown encouraging therapeutic results in HCC cell lines by blocking the Wnt/β-catenin signaling. These soluble molecules induce apoptosis, reduce angiogenesis and cell proliferation both in vitro and in vivo and are not influenced by the CTNNB1 mutation status [112]. Other Wnt antagonists such as sFRP2 and sFRP5 should also be considered, since they show similar treatment effects in colon cancer as sFRP1 exhibits in HCC [113]. Interestingly, Dkk1 and sFRP1 addition cooperates with anti-FZD7 antibodies to increase apoptosis in Wilm’s tumor demonstrating the importance of combinatorial therapies [105]. Therapeutic small molecules, such as niclosamide and silibinin, display anti-tumor activity in vitro and in vivo by suppressing Lrp6 expression, leading to inhibition of Wnt/β-catenin signaling in human prostate and breast tumor cells, as well as by promoting induction of apoptosis [[114], [115]].

Targeting the Wnt-mediated pathway in the cytosol

The straight-in approach to inhibit Wnt/β-catenin pathway is to directly target β-catenin by small interfering-RNA or antisense based therapy, which can reduce cell proliferation and survival of HCC cell line, providing a proof of principle for this approach [[116], [117], [118]]. However, its potential use as a therapeutic tool remains unlikely since β-catenin protein is essential for cell junction. Thus, targeting the soluble active pool of β-catenin seems more appropriate.

The interaction between the cytosolic tail of FZD and its adaptor Dvl protein is of importance in mediating Wnt signaling. A proof-of-principle has clearly been established in HCC cells, by using small interfering peptides capable of entering the tumor cells and disrupting the interaction between a specific motif on the FZD7 cytosolic tail and the PDZ domain of Dvl [11]. Similar results have been obtained in melanoma and non-small-cell lung cancer cells with small molecules using this same strategy [119].

Targeting the β-catenin destruction complex (APC, Axin, CK1, and GSK3β) as a therapeutic target has not been assessed in HCC so far. However, using other tumor model systems, such a strategy has demonstrated some potential. Since Axin1 overexpression induces apoptosis in HCC harboring APCAxin1 or CTNNB1 mutations, stabilization of Axin1 would be an attractive approach to trigger β-catenin degradation [120]. This may be achieved by using inhibitors of the Axin1 or/and 2 degradation, such as the smalls peptides IWR2, JW55 or XAV939 that inhibit the Wnt/β-catenin pathway, leading to a decreased proliferation of colon and breast cancer cell lines. Nevertheless, recent findings support the idea that this decrease may be restricted to low nutriment conditions, and emphasizes that stabilization of Axin needs to be combined with other therapeutic approaches [[110], [121], [122], [123]]. Preventing β-catenin stabilization through GSK3β activation would also be possible due to the discovery of differentiation-inducing factors (DIFs), which are natural metabolites expressed by Dictyostelium discoideum. Although the mechanisms of action of DIFs activity remain poorly understood, it is well known that DIFs induce β-catenin degradation and subsequently reduce cyclin D1 expression and function [124]. CK1α, another component of the destruction complex, may be stabilized by pyrvinium that inhibits both Wnt signaling and cell proliferation, even in the presence of APC or CTNNB1 mutations, as observed in colon cancer cell lines [125]. Another therapeutic approach would be to enhance β-catenin proteasomal degradation. In HCC, colon and prostate cancer cell lines, the small molecule antagonist CGK062 has been shown to exert such an effect, via the induction of β-catenin phosphorylation in the N-terminal domain which promotes its degradation [126]. Two chemicals agents, hexachlorophene and isoreserpine, upregulate Siah-1, an ubiquitin ligase that induces β-catenin degradation, independent of its phosphorylation status, thereby inhibiting Wnt signaling and subsequently has been shown to reduce colon cancer cell proliferation [127].

Targeting the Wnt pathway in the nucleus

Finally, an alternative way to block Wnt-mediated signaling is to target the nuclear β-catenin per se and/or the co-factors responsible for transcription of downstream Wnt-responsive genes. To accomplish this aim, several small molecules have been identified. The FH535 agent prevents both Wnt- and PPAR- (Peroxisome Proliferator-Activated Receptors) mediated signaling by suppressing the recruitment of β-catenin co-activators to target gene promoters and has been shown to be active in HCC, colon, and lung tumor cell lines [128]. PKF115-584, PKF118-310, and CGP049090 are inhibitors of TCF/β-catenin binding to DNA target sequences. They induce apoptosis in vitro and in vivo, as well as cell cycle arrest at the G1/S phase and suppress tumor growth in vivo independently of the mutated status of CTNNB1 [129]. Furthermore, inhibition of β-catenin/CBP interaction by ICG-001 both selectively induces apoptosis in transformed, but not in normal colonic cells and reduces growth of colon carcinoma cells in vitro as well as in vivo [130]. A second generation ICG-001 (PRI-724) is also available and in phase-I clinical trial (http://clinicaltrials.gov/show/NCT01302405). Other β-catenin binding proteins such as TBP, Bcl9, and Pygo also represent attractive approaches for inactivating Wnt signaling. Finally, interferon can inhibit β-catenin signaling through upregulation of RanBP3 that is a nuclear export factor, serving as extruding β-catenin outside the nucleus [131].

Conclusions and perspectives

Developmental regulated signaling pathways, such as Notch, Hedgehog and Wnt, have become important targets for new cancer drug development. While Notch and Hedgehog inhibitors are already in clinical trials, the Wnt inhibitors are still under preclinical assessment and only a few compounds have started to reach the phase-I clinical trials, since only recently has this pathway been recognized as playing a key role in tumor development. However, many studies have established proof-of-principle that specific targeting of molecules in this pathway can partially or fully switch off canonical as well as non-canonical Wnt signaling and lead to substantial anti-tumor activity. Thus, biotechnology and pharmaceutical organizations are currently developing Wnt signaling inhibitors. These inhibitors can target upstream or downstream proteins in this pathway. Targeting the Wnt cascade upstream of APC is controversial because downstream activating mutations in APC would, in theory, still drive tumor development. To cover the broadest number of activating mutations that occur in tumors, it seems that the ideal antagonist would be one that exerts its anti-tumor effect in the nucleus. Nevertheless, several experiments show that upstream targeting can also be very effective. Of importance is the potential toxicity of Wnt inhibitors on normal cells. Indeed, the Wnt pathway is critical for tissue and liver regeneration and for the ability of stem cells to self-renewal. Wnt pathway inhibitors could therefore have substantial and long-term side effects including anemia, immune suppression, as well as damage of the gastrointestinal tract. It is unknown what may occur in an adult mammal when this pathway is shut down or reduced in normal activity. Despite these known and unknown pitfalls, drug development is moving steadily forward to generate and characterize Wnt pathway inhibitors bothin vitro and in vivo. Indeed, agents that inhibit Wnt/β-catenin signaling as a means to produce anti-tumor effects are currently being assessed in clinical trials.

7.11.4 SALL4 is directly activated by TCF.LEF in the canonical Wnt signaling pathway

Böhm J1Sustmann CWilhelm CKohlhase J.
Biochem Biophys Res Commun. 2006 Sep 29; 348(3):898-907
http://dx.doi.org/10.1016/j.bbrc.2006.07.124

The SALL4 promoter has not yet been characterized. Animal studies showed that SALL4 is downstream of and interacts with TBX5 during limb and heart development, but a direct regulation of SALL4 by TBX5 has not been demonstrated. For other SAL genes, regulation within the Shh, Wnt, and Fgf pathways has been reported. Chicken csal1 expression can be activated by a combination of Fgf4 and Wnt3a or Wnt7a. Murine Sall1 enhances, but Xenopus Xsal2 represses, the canonical Wnt signaling. Here we describe the cloning and functional analysis of the SALL4 promoter. Within a minimal promoter region of 31 bp, we identified a consensus TCF/LEF-binding site.The SALL4 promoter was strongly activated not only by LEF1 but also by TCF4E. Mutation of the TCF/LEF-binding site resulted in decreased promoter activation. Our results demonstrate for the first time the direct regulation of a SALL gene by the canonical Wnt signaling pathway.

http://ars.els-cdn.com/content/image/1-s2.0-S0006291X06016615-gr1.sml

http://ars.els-cdn.com/content/image/1-s2.0-S0006291X06016615-gr2.sml

SALL4 is one out of five (four functional (SALL1-4) and one pseudogene (SALL1P)) human genes related to spalt (sal) of Drosophila melanogaster [1–5]. Mutations of SALL4 cause Okihiro/Duane-Radial Ray syndrome (DRRS, OMIM 607323), a rare autosomal dominant condition characterized by radial ray defects and Duane anomaly (a form of strabismus). Other features of Okihiro/ DRRS patients are anal, renal, cardiac, ear, and foot malformations, hearing loss, postnatal growth retardation, and facial asymmetry. The SALL4 gene product is a zinc finger protein thought to act as a transcription factor. It contains three highly conserved C2H2 double zinc finger domains, which are evenly distributed. A single C2H2 motif is attached to the second domain, and at the amino terminus SALL4 contains a C2HC motif. All but two reported SALL4 mutations lead to preterminal stop codons and are thought to cause the phenotype via haploinsufficiency.

The detection of larger deletions involving the whole SALL4 gene or single exons in patients with Okihiro or acro-renal-ocular syndrome provided proof for haploinsufficiency as the cause of the malformations. SALL4 is the only gene currently known to be mutated in patients with Okihiro/DRR syndrome. Mutations of SALL4 have been found in >90% of patients with classical Okihiro syndrome [6]. Sall4 is regulated by Tbx5 in mouse and zebrafish [7,8]. In the mouse, it controls Fgf10 expression in a synergistic manner together with Tbx5 in the forelimbs or with Tbx4 in the hindlimbs via direct effects on the Fgf10 promoter, whereas the effect of Sall4 and Tbx1 coexpression on the Fgf10 promoter is only additive [8]. The cooperative action of Sall4 with Tbx5 or Tbx4 can be counteracted by Tbx2 and Tbx3. In the heart, mouse Sall4 interacts with Tbx5 and activates Gja5 but interferes with Tbx5-dependent activation of Nppa. In zebrafish, fgf10 expression also depends on both tbx5 and sall4 [7]. Here, fgf10 expression is activated by fgf24 via fgfr2. While tbx5 controls expression of fgf24, sall1a (SALL1 orthologue), and sall4, fgf10 expression is indirectly activated by sall1a and sall4 via activation of fgfr2 expression. Although these data provide information on one pathway for SALL4 regulation, the SALL4 promoter and its regulation has neither been described in the mouse nor in human or zebrafish. Here we report on the cloning and functional analysis of the human SALL4 promoter.

Identification of SALL4 expressing cell lines

SALL4 expression had previously been detected in human teratocarcinoma cell lines H12.1 and 2102 EP by Northern blotting [4]. Since faint SALL4 expression had also been observed in human ovary tissue by RT-PCR, we analyzed SALL4 mRNA expression in human OVCAR-3 and OV-MZ-9 epithelial ovarian cancer cell lines by quantitative real-time RT-PCR. Only the 2102 EP cells as positive control and the epithelial ovarian cancer cells OVCAR-3 expressed SALL4 mRNA but not OVMZ-9 cells (Fig. 1). Interestingly, 2102 EP cells express SALL4 at an 18.7-fold higher level than OVCAR-3 cells and at levels similar to GAPDH.

Fig. 1. Real-time RT-PCR of human SALL4 mRNA from 2102 EP, OVCAR-3, and OV-MZ-9 cells. 2102 EP embryonal carcinoma cells show a 18.7-fold higher SALL4 expression level than epithelial ovarian cancer cells OVCAR-3, whereas human ovarian cancer cells OV-MZ-9 appeared to be consistently negative. Values are shown in comparison to endogenous GAPDH expression levels in the top left-hand corner of the diagram. Negative controls lacking template were always below threshold. The dissociation curve confirmed the amplification of only a single amplicon and verified the absence of secondary PCR products. Such single peak dissociation curves were found for all positive mRNAs in our samples.

Cloning of the 50 end of the SALL4 cDNA and identification of additional SALL4 transcripts

In order to identify the transcriptional startpoint of SALL4,5 0 RACE was used. Two amplification products of approximately 500 bp and 750 bp were obtained and sequenced. The 500 bp fragment contained parts of exon 2 (initial 130 base pairs) as well as exon 1 including the ATG and additional 28 base pairs in comparison to the SALL4 mRNA database sequence (NM020436), placing a transcriptional start site at 95 bp 50 of the ATG. This finding is supported by several ESTs (DA666635, DB066881, andCN308408) carrying similar 50 ends. No sequence identifying an additional upstream exon preceding exon 1 could be identified. The 750 bp fragment contained sequences of two putative alternative exons, positioned within intron 1 and following the AG-GT rule. One putative exon starts at position IVS1406 and ends at position IVS1574, covering 169 base pairs. The other putative exon starts at position IVS1+4577 and ends at position IVS1+4853, spanning 277 base pairs. These putative exons do not contain ATG start codons followed by an open reading frame, indicating the presence of an alternative transcript of SALL4 starting with a downstream ATG start codon within exon 2 (Fig. 2).

Fig. 2. Schematic representation of the SALL4 gene and the two detected transcript variants including alternative first exons. (above) The most common variant represented by database sequence NM020436 contains exons 1, 2, 3, and 4. Translation starts with the ATG in exon 1. (below) Alternative transcript variant detected by 50 RACE. Transcription starts with exon 1a and includes exon 1b, both located within the large 50 intron. Zinc finger domains are represented by rhombuses within the boxes, grey color indicates untranslated regions. Since neither contains an in-frame ATG, translation is likely to start at the first ATG 30 of the region coding for the first (C2HC) zinc finger domain. Similar variants were detected in the SALL3 gene [2].

Cloning and transcriptional characterization of the SALL4 promoter

In order to clone the SALL4 promoter, 5 initial constructs overlapping at their 30 ends were generated by PCR amplification of up to 1971 bp upstream of the SALL4 ATG start codon and cloning into the promoterless pGL3-Basic luciferase reporter plasmid. These constructs were transiently transfected into 2102 EP and OVCAR-3 cells, and co-transfected with pRL-SV40 vector in order to normalize luciferase activity in reference to the Renilla reniformis luciferase activity. Reporter constructs starting at 1971, 1446, 1003, 514, and 358 bp 50 of the ATG start codon demonstrated on average a 28-fold expression of luciferase (with no significant difference among the different constructs) in comparison to the empty pGL3-Basic vector in 2102 EP cells (Fig. 3A). Reporter gene activation was significantly lower (on average 10-fold as compared to pGL3-Basic) in OVCAR3 cells than in 2102 EP cells (Fig. 3B). Murine and human putative promoter regions were aligned and showed 88% homology over 375 bp upstream of ATG (Fig. 4). More 50 sequences did not reveal any significant homology. Further constructs of 278, 249, 218, 188, and 160 bp were generated and transfected to map the sequences responsible for transcriptional activation. The smallest construct showing full promoter activity included 249 bp 50 of ATG, while the constructs containing 218, 188, or 160 bp 50 of ATG did not show any significant higher luciferase activity than the promoterless pGL3-Basic vector, indicating that important DNA sequences for driving SALL4 expression through binding of activating proteins reside within only 31 base pairs. Analysis of those 31 base pairs with the ‘‘genomatix Matinspector’’ (http://www.genomatix.de) revealed two highly conserved recognition sequences for HepG2-specific P450 2C factor-1 (ZNF83) and TCF/LEF, involved in the Wnt
signal transduction pathway. Additionally, a classical CAAT-box is positioned at 165 and two putative GATA-binding sites are positioned in direct proximity at 144 and 123 from ATG.

Fig. 3. Analysis of luciferase reporter constructs for minimal DNA sequence required for human SALL4 promoter activity. (A) Constructs were transiently transfected into 2102 EP cells and normalized to Renilla reniformis luciferase activity. Constructs containing at least 249 bp 50 upstream of ATG demonstrated functional activity, whereas a construct containing 218 bp 50 of ATG did not show any significant luciferase activity. Reporter constructs comprising 1971, 1446, 1003, 514, and 358 bp did not show any significant difference in activating luciferase expression. In average, a 28-fold increase in luciferase activity was observed compared to the empty pGL3-Basic vector. Each transfection was performed at least in triplicate using two different DNA preparations of each construct. (B) Luciferase assay of transiently transfected constructs in OVCAR3 cells. As in 2102 EP cells, the minimal sequence stretch required for complete promoter activity contains 249 bp upstream of ATG. Smaller constructs do not promote any significant reporter gene expression. Compared to the empty pGL3-Basic vector, a 7.9-fold uprating luciferase activity was ascertained in average.

The SALL4 promoter region is highly conserved in mammals

Comparison of 367 bp upstream of the ATG of human SALL4 with corresponding sequences of Pan trogloydes, Mus musculus, Canis familialis or Bos taurus (http://www.ebi.ac.uk/clustalw/index.html) revealed homologies between 89% (Mus musculus) and 99% (Pan trogloydes)(Fig. 4). For 2000 bp 50 of the translational start point (not shown), the homology between human and chimpanzee is 98%, but this ratio decreases to 75% in Canis familialis and Bos taurus, and mouse and human sequences correspond only weakly (58%), with highly conserved regions residing adjacent to exon 1. The TCF/LEF-binding motiv TACAAAG is fully conserved with the exception of the putative mouse Sall4 promoter. Here, the last adenine is altered to a guanine, but this does not change the binding specificity for LEF1 [9]. The CAAT-box (165 from ATG) is identical between the analyzed species (Fig. 4).

Fig. 4. Cross-species sequence comparison between Homo sapiens, Pan trogloydes, Mus musculus, Canis familialis, and Bos taurus of the 50 upstream noncoding region of SALL4 and its orthologues. (A) Sequence identity to SALL4 varies between 89% (Mus musculus) and 99% (Pan trogloydes) within 367 base pairs upstream of ATG. (B) Sequence motifs putatively moderating SALL4 expression, accentuated by rectangles, as the CAAT-Box (165 from ATG) or GATA binding sites (144 and 123 from ATG) are highly conserved throughout the analyzed species. The TCF/LEF-binding motiv TACAAAG is invariable except in case of the putative mouse Sall4 promoter, where the third adenine is altered to a guanine. Asterisks indicate identical nucleotides at selected position across species. The position 1 denominates the first nucleotide 50 of ATG.

Mutation of the TCF/LEF-binding site

Since the closely related Sall1 protein was found to enhance the canonical Wnt signaling pathway, we sought to test if LEF1 binding was crucial for SALL4 expression. The core-binding sequence of LEF1 was altered from TACAAAG to GCACCCT by site-directed mutagenesis in the 1997Luc construct. The luciferase activity of the mutated construct was strongly reduced to approximately 36% as compared with the activity of the wild type (Fig. 5). In comparison to the empty pGL3-Basic vector, the mutated construct showed a 11.2-fold luciferase expression, while the wild-type construct had a 31.0-fold increase of promoter activity.

Fig. 5. Transcriptional activity of the SALL4 promoter with an altered TCF/LEF-binding motif compared to wild type. Constructs were transiently transfected at least in triplicate into 2102 EP cells using standard procedures and incubated for 24 h. Values were calculated by normalizing against Renilla reniformis luciferase activity and compared to the empty pGL3-Basic vector. Luciferase activitiy of the mutated construct appeared to be reduced to 36% of the wild-type construct, showing a 11.2- and 31-fold increase of promoter activity, respectively.

pGL3 empty vector                           + – – – – – –

SALL4 promoter-Luc                       – + + + – – –

SALL4 promoter mut-Luc               – – – – + + +

β-catenin                                            + – + + + + +

LEF1                                                    + – + – – + –

TCF4E                                                 – – – + – – +

Empty vector                                     – + – – + – –
Rel. luciferase activity
Co-transfection of SALL4-luciferase constructs and TCF/ LEF

Since human embryonal kidney cells HEK293 are known to exhibit a low level of endogenous Wnt signaling, we selected those cells for transfections and subsequent reporter gene assays. SALL4-Luc constructs, either wild type or bearing a mutated binding motif for TCF/LEF, were co-transfected with expression constructs for b-catenin and LEF1 or TCF4E. As the expression vectors for LEF1 and TCF4E were not identical, expression levels of both proteins were analyzed by immunoblot using HA-antibody (BD Clontech, Heidelberg, Germany) in HEK293  of the SALL4 promoter and members of the TCF/LEF family in HEK293 cells. Induction is depicted relative to Renilla reniformis luciferase activity. Cotransfections were performed using equal amounts of DNA and adjusted concentrations of expression vectors previously analyzed by immunoblot. Promoter activity of SALL4 is enhanced by overexpressing LEF1 or TCF4E in concert with b-catenin. Transfection of the mutated construct resulted in a comparatively 68% lower induction of promoter activity. Overexpression of LEF/TCF4E promotes rescue of the mutated construct resulting in luciferase values comparable to single transfection of the wild-type SALL4 promoter in HEK293 cells transfected with these expression plasmids (data not shown). Band intensities were compared and adjusted amounts of expression plasmids were subsequently used for reporter gene assays (Fig. 6). In comparison to the promoterless pGL3-Basic vector co-transfected with b-catenin and LEF1 expression vectors, transfection of SALL4-Luc plasmid resulted in a 4.8-fold increase in reporter gene activity. 13.2-fold luciferase activity was achieved by co-transfection of SALL4-Luc with bcatenin and LEF1 expression vectors. Co-transfection of SALL4-Luc with b-catenin and TCF4E expression plasmids revealed 10.6-fold expression of luciferase. Co-transfection of SALL4 mut-Luc and the LEF1 or TCF4E and b-catenin expression vectors in a 1:30/1:6 ratio resulted in a weaker induction of promoter activity as compared to the wild-type construct (4.2-fold as compared to 10.6-fold normalized luciferase activity). No statistically significant difference was seen between LEF1 and TCF4E. However, overexpression of these constructs in concert with b-catenin promotes rescue of the mutated SALL4 promoter leading to luciferase values comparable to transfection of the wild-type SALL4 promoter without LEF1/ TCF4E, indicating that altering the binding motif from TACAAAG to GCACCCT is not sufficient to abrogate affinity under the experimental conditions of cell culture mediated luciferase assays.

Fig. 6. Co-transfection of the SALL4 promoter and members of the TCF/LEF family in HEK293 cells. Induction is depicted relative to Renilla reniformis luciferase activity. Cotransfections were performed using equal amounts of DNA and adjusted concentrations of expression vectors previously analyzed by immunoblot. Promoter activity of SALL4 is enhanced by overexpressing LEF1 or TCF4E in concert with b-catenin. Transfection of the mutated construct resulted in a comparatively 68% lower induction of promoter activity. Overexpression of LEF/TCF4E promotes rescue of the mutated construct resulting in luciferase values comparable to single transfection of the wild-type SALL4 promoter in HEK293 cells.

LEF1 binds to the SALL4 promoter in vitro

LEF 1 (Lef1) is expressed in high levels in pre-B and Tcells in adult mice, and in the neural crest, mesencephalon, tooth germs, whisker follicles, and other sites during mouse embryonic development [10]. By Western blot analysis, we confirmed that LEF1 is also expressed in human 2102 EP cells (data not shown). To determine whether LEF1 protein interacts with the TCF/LEF-binding motif within the SALL4 promoter in vitro, we performed gel retardation assays. Nuclear extracts from 2102 EP cells as well as recombinant LEF1 and TCF4E proteins were incubated with a [c-32P]dATP-labeled synthetic double-strand DNA probe of the SALL4 promoter with the normal and a mutated TCF/LEF-binding site in the middle. A single complex was observed when the SALL4 probe was incubated with rising amounts of recombinant LEF1 protein (Fig. 7).

Fig. 7. Electrophoretic mobility shift assay confirms in vitro binding of LEF1 to a LEF/TCF consensus site within the SALL4 promoter region. (A) Recombinant LEF1 (100 and 300 ng) binds to a LEF/TCF consensus site in the SALL4 wild-type (WT) probe (lanes 2 and 3) and can be supershifted by the addition of a-LEF1 antibody (lane 4). It does not bind to a mutated (MUT) LEF/TCF site (lanes 6 and 7). As negative controls, no labeled probe was added in lanes 1 and 5. (B) LEF1 in nuclear extract derived from 2102 EP (2 and 6 lg) binds to SALL4 wild-type probe (lanes 2 and 3) and TCRa wt positive control probe (lanes 8 and 9) but not to a TCRa mutant probe (lanes 5 and 6). In lanes 1, 4, and 7 no radioactively labeled probe was added.

The application of a specific polyclonal rabbit a-LEF1 antibody resulted in a supershift of the complex. No retardation could be detected in case of the mutant probe. Adding recombinant TCF4E instead of LEF1 in a further assay also resulted in retardation of the wild-type probe (data not shown). However, members of the TCF family share highly conserved sequence homology and demonstrate similar affinity to canonical-binding sites. Nuclear extract proteins from 2102 EP cells bound to labeled LEF1 probe but not to the mutated probe in presence of unspecific competitor. Mutant and wild-type probes derived from the promoter of nuclear T-cell receptor (TCR)a [11] served as a control since it is known to bind LEF1 protein.

Discussion

In this report, we describe the first characterization of the SALL4 promoter and the activation of SALL4 by members of the TCF/LEF family in the canonical WNT signaling pathway. In order to identify the most suitable cell line for reporter gene studies, SALL4 mRNA expression was analyzed by quantitative real-time RT-PCR. SALL4 was found to be expressed in OVCAR-3 cells, but not as strong as in 2102 EP cells. In OV-MZ-9 cells, no expression was detected. By 50 RACE, the transcriptional startpoint was mapped to 95 bp 50 of the ATG start codon, and two additional, yet undescribed, exons 50 to exon 2 were identified. Reporter constructs were generated containing up to 1977 bp 50 of the ATG. Transfection of those constructs into 2102 EP cells identified a region responsible for reporter gene activation under the conditions tested within 249 bp upstream of the ATG. Transfection of other constructs containing 218, 188, and 160 bp 50 of the ATG revealed no reporter gene activation, rendering a region of 31 bp being responsible for activation of reporter gene expression. The region contains a TCF/LEF-binding site, and mutation of this site resulted in decreased but not abolished activation of the SALL4 promoter. Co-transfection of SALL4-reporter gene constructs and LEF1/b-catenin or TCF4E/ b-catenin confirmed SALL4 activation by LEF1 but also by TCF4E. EMSA studies provided evidence of direct binding of both members of the TCF/ LEF family to the presumed binding site, which was further substantiated by a supershift observed upon incubation with a LEF1 antibody.

Identification of alternative transcripts

By 50 RACE, we could identify a previously unknown SALL4 transcript containing exons 1a and 1b instead of exon 1. No transcript was detected containing exon 1 in addition to exons 1a or 1b, suggesting that the two detected variants are the only existing mRNAs with different 50 ends. These exons are conserved in the genomic SALL4 sequence of Pan trogloydes but not in mouse Sall4 (data not shown). Interestingly, translation of the exon 1a–1b transcript would likely result in a SALL4 protein not containing the conserved amino terminal region including the C2HC zinc finger domain and the region which contains the amino terminal repressor domain in SALL1 [12,13]. A similar transcript had previously been detected for the gene SALL3, which appeared to be the most closely related SALL gene [2,3]. The resulting protein would also miss a conserved stretch of 12 amino acids encoded by exon 1 important for recruitment of the nucleosome remodeling and deacetylase corepressor complex (NuRD) involved in global transcriptional repression and regulation of specific developmental processes [14]. Although SALL4 and SALL3 are not (yet) known to be transcriptional repressors as shown for SALL1, the existence of a transcript lacking essential repressor domains might indicate that the alternative transcripts in SALL4 and SALL3 reflect different functional properties.

Structure of the SALL4 promoter

By 50 RACE, the transcriptional start-point was mapped to 95 from ATG. No consensus TATA box was found within 2 kb upstream of this site, suggesting that SALL4 has a TATA-less promoter as shown for SALL1 and 2 [15,16]. Only one putative, but non-canonical, TATA box in proximity to the transcriptional start site was detected, TAATAATAATTA, 41 nt from the predicted initiator region (INR). The consensus for the INR in mammalian cells is Py-Py(C)-A+1-N-T/A-Py-Py, fitting quite well with the predicted INR element for SALL4 (CCCAACTCC, Fig. 4). INR regions are important for binding the basal transcription factor TFIID in a sequence specific manner and cooperatively with a DPE motif. The DPE is found most commonly in TATA-less promoters and is located at +28 to +32 relative to the A+1 position in the INR with a consensus sequence A/G+28-G-A/T-C/T-G/A/C (for review, see Ref. [33–35]. Such a sequence cannot be found within the SALL4 promoter, indicating that this promoter may be TATA-box dependent despite the lack of a classical TATA box. On the other hand, TATA-less promoters are often associated with multiple transcription start sites.

The results of the 50 RACE in comparison with the sequences of several ESTs suggest multiple transcription start sites for SALL4, since the transcription start site obviously varies in a few nucleotides. By reporter gene analysis, we could demonstrate that a minimal region of 31 bp is important for SALL4 expression in the 2102 EP cell line. The region contained a TCF/LEF-binding site highly conserved throughout mammals, which we could show to be functional by reporter studies and mobility shift assays. Earlier reports described activation of Sall4 in mouse and sall4 in zebrafish by Tbx5 [7], but no Tbx5-binding site was detected within 2 kb upstream of the ATG in exon 1 of SALL4. This does not exclude a direct interaction of SALL4 by TBX5 but indicates that TBX5 might rather bind to a limb-specific enhancer. Within the 31 bp region crucial for SALL4 expression, only one other binding site for ZNF83 was predicted, which however overlaps with the TCF/LEF-binding site. Mutation of the TCF/LEF-binding site in the 1997 bp construct resulted in a decreased but not abolished activation of the SALL4 promoter, which might be explained by other activating factors binding upstream of the TCF/LEF site.

SALL4 is a target of the canonical WNT signaling

Our results provide a new and important aspect on the relation of SALL genes and WNT signaling. Studies in chicken have shown that ectopic expression of the SALL1 homologue csal1 is depending upon interaction of wnt3a or wnt7a with Fgf4 [17]. Studies in mouse and Xenopus laevis have shown that SALL genes can also actively act on the Wnt signaling pathway. While Sall1 was found to interact with β-catenin and to enhance the canonical Wnt signaling by binding to heterochromatin [18], the Xsal2 protein acts by repressing canonical Wnt signaling [19].

Lymphoid enhancer-binding factor-1 (LEF-1) belongs to the high mobility group (HMG) regulating, e.g., genes involved in T-cell development through binding the core consensus sequence 50-CTTTGA/TA/T-3 in the minor groove of the DNA [20, 21]. Binding induces formation of a sharp bend in the double helix, thus facilitating the binding of transcription factors to the adjacent DNA sequences required for driving transcription [9]. TCF/LEF apparently operates as a repressor when not linked to b-catenin by interacting with Groucho, in turn recruiting histone deacetylases. These HDAC remove an acetyl group from histones, which allows histones to bind DNA and inhibit gene transcription [22–24]. Binding of β-catenin possibly replaces Groucho by the histone acetylase CBP/p300 (cyclic AMP response element-binding protein), inducing chromatin remodeling resulting in transcriptional activation of the target gene [25,26].

As in the cytoplasm and the extracellular compartment, negative regulators of the Wnt induced pathway also exist in the nucleus. Chibby and ICAT can bind β-catenin and thereby prevent its interaction with LEF1 [26,27]. Phosphorylation by MAPK related protein kinase also serves as a mechanism for regulating LEF1 activity, leading to a reduced capability of the LEF1/b-catenin complex to form a ternary complex with DNA [28, 29]. A key protein within the signaling pathway initiated by WNTs is β-catenin. The primary structure of the β-catenin protein comprises an N-terminal domain that is subject to phosphorylation events leading to its proteolytic degradation, a 42 amino acid arm repeat interacting with TCF/ LEF, and a C-terminal domain important for transcriptional activation.

Phosphorylation, and hence targeting of β-catenin for ubiquitination and degradation, through glycogen synthase kinase-3 β (GSK3) is faciliated by Axin and Adenomatous Polyposis Coli (APC), constituting a kind of scaffold. Cytoplamic β-catenin levels are consequently kept low. Binding of the signal molecule Wnt to its transmembrane receptor Frizzled induces diverse processes leading to an inhibition of the GSK3 activity and thus to a cytoplasmic accumulation of β-catenin. Free cytoplasmic β-catenin enters the nucleus and induces gene expression by complexing TCF/LEF and forming a ternary complex with DNA (for review, see Ref. [36]).

In addition to enhancement or repression of the canonical WNT pathway by SALL genes [18,19], we report here a further interaction of a SALL gene with the canonical WNT pathway by demonstrating that the canonical WNT signaling can directly regulate expression of SALL4, and that this regulation is achieved by direct interaction of LEF1 and the SALL4 promoter. A large number of Wnt targets have been identified, including also members of the Wnt signaling pathway resulting in feedback regulation and underlying the complex machinery working in concert for fine-tuning signal transduction. One of the key roles of Wnt signaling concerns cell proliferation, and lack of Wnt5a inhibits outgrowth of the limbs [30], a major phenotype seen in patients with Okihiro syndrome. Mutation of human WNT3 results in tetraamelia [31], and mutation of WNT7A in Fuhrmann and Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndromes [32], supporting an important role of WNT signaling in the pathogenesis of human limb malformations. The finding of a direct regulation of SALL4 by TCF/LEF supports now the assumption that SALL4 may be activated by WNT3 and/ or WNT7A by means of TCF/ LEF during limb development.

References

[1] R. Al-Baradie, K. Yamada, C. St Hilaire, W.M. Chan, C. Andrews, N. McIntosh, M. Nakano, E.J. Martonyi, W.R. Raymond, S. Okumura, M.M. Okihiro, E.C. Engle, Duane Radial Ray Syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family, Am. J. Hum. Genet. 71 (2002) 1195–1199. [2] J. Kohlhase, S. Hausmann, G. Stojmenovic, C. Dixkens, K. Bink, W. Schulz-Schaeffer, M. Altmann, W. Engel, SALL3, a new member of the human spalt-like gene family, maps to 18q23, Genomics 62 (1999) 216–222. [3] J. Kohlhase, M. Heinrich, L. Schubert, M. Liebers, A. Kispert, F. Laccone, P. Turnpenny, R.M. Winter, W. Reardon, Okihiro syndrome is caused by SALL4 mutations, Hum. Mol. Genet. 11 (2002) 2979–2987. [4] J. Kohlhase, A. Ko ¨hler, H. Ja ¨ckle, W. Engel, R. Stick, Molecular cloning of a SALL1-related pseudogene and mapping to chromosome Xp11.2, Cytogenet. Cell Genet. 84 (1999) 31–34. [5] J. Kohlhase, R. Schuh, G. Dowe, R.P. Ku ¨hnlein, H. Ja ¨ckle, B. Schroeder, W. Schulz-Schaeffer, H.A. Kretzschmar, A. Ko ¨hler, U. Mu ¨ller, M. Raab-Vetter, E. Burkhardt, W. Engel, R. Stick, Isolation, characterization, and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene spalt, Genomics 38 (1996) 291–298. [6] J. Kohlhase, D. Chitayat, D. Kotzot, S. Ceylaner, U.G. Froster, S. Fuchs, T. Montgomery, B. Rosler, SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders, Hum. Mutat. 26 (2005) 176–183. [7] S.A. Harvey, M.P. Logan, sall4 acts downstream of tbx5 and is required for pectoral fin outgrowth, Development 133 (2006) 1165–1173. [8] K. Koshiba-Takeuchi, J.K. Takeuchi, E.P. Arruda, I.S. Kathiriya, R. Mo, C.C. Hui, D. Srivastava, B.G. Bruneau, Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart, Nat. Genet. 38 (2006) 175–183. [9] J.J. Love, X. Li, D.A. Case, K. Giese, R. Grosschedl, P.E. Wright, Structural basis for DNA bending by the architectural transcription factor LEF-1, Nature 376 (1995) 791–795. [10] M. Oosterwegel, M. van de Wetering, J. Timmerman, A. Kruisbeek, O. Destree, F. Meijlink, H. Clevers, Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis, Development 118 (1993) 439–448. [11] J. Galceran, S.C. Hsu, R. Grosschedl, Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression, Proc. Natl. Acad. Sci. USA 98 (2001) 8668–8673. [12] S. McLeskey Kiefer, K.K. Ohlemiller, J. Yang, B.W. McDill, J. Kohlhase, M. Rauchman, Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects, Hum. Mol. Genet. 12 (2003) 2221–2227. [13] C. Netzer, S. Bohlander, M. Hinzke, Y. Chen, J. Kohlhase, Defining the heterochromatin localization and repression domains of SALL1, Biochim. Biophys. Acta 1762 (2006) 386–391. [14] S.M. Lauberth, M. Rauchman, A conserved twelve amino acid motif in sall1 recruits nuRD, J. Biol. Chem. (2006). [15] L. Chai, J. Yang, C. Di, W. Cui, R. Lai, Y. Ma, Transcriptional activation of the sall1 by the human six1 homeodomain during kidney development, J. Biol. Chem. 281 (2006) 18918–18926. [16] Y. Ma, D. Li, L. Chai, A. Luciani, D. Ford, J. Morgan, A. Maizel, Cloning and characterization of two promoters for the human HSAL2 gene and their transcriptional repression by the Wilms tumor suppressor gene product, J. Biol. Chem. 276 (2001) 48223–48230. [17] E.R. Farrell, A.E. Munsterberg, csal1 is controlled by a combination of FGF and Wnt signals in developing limb buds, Dev. Biol. 225 (2000) 447–458. [18] A. Sato, S. Kishida, T. Tanaka, A. Kikuchi, T. Kodama, M. Asashima, R. Nishinakamura, Sall1, a causative gene for Townes-Brocks syndrome, enhances the canonical Wnt signaling by localizing to heterochromatin, Biochem. Biophys. Res. Commun. 319 (2004) 103–113. [19] T. Onai, N. Sasai, M. Matsui, Y. Sasai, Xenopus XsalF: anterior neuroectodermal specification by attenuating cellular responsiveness to Wnt signaling, Dev. Cell 7 (2004) 95–106.

 

7.11.5 SALL4. An emerging cancer biomarker and target

Zhang X1Yuan X2Zhu W2Qian H2Xu W3.
Cancer Lett. 2015 Feb 1; 357(1):55-62
http://dx.doi.org:/10.1016/j.canlet.2014.11.037

SALL4 is a transcription factor that plays essential roles in maintaining self-renewal and pluripotency of embryonic stem cells (ESCs). In fully differentiated cells, SALL4 expression is down-regulated or silenced. Accumulating evidence suggest that SALL4 expression is reactivated in cancer. Constitutive expression of SALL4 transgene readily induces acute myeloid leukemia (AML) development in mice. Gain- and loss-of-function studies reveal that SALL4 regulates proliferation, apoptosis, invasive migration, chemoresistance, and the maintenance of cancer stem cells (CSCs). SALL4 controls the expression of its downstream genes through both genetic and epigenetic mechanisms. High level of SALL4 expression is detected in cancer patients, which predicts adverse progression and poor outcome. Moreover, targeted inhibition of SALL4 has shown efficient therapeutic effects on cancer. We have summarized the recent advances in the biology of SALL4 with a focus on its role in cancer. Further study of the oncogenic functions of SALL4 and the underlying molecular mechanisms will shed light on cancer biology and provide new implications for cancer diagnostics and therapy.

SALL4 (sal-like4) is a member of the mammal homologs of Drosophila homeoticgene spalt (sal). In humans, SALL4 gene mutations are known to cause Okihiro syndrome (Duane radial ray syndrome), an autosomal dominant disease involving multiple organ defects [1–4]. In mice, SALL4 homozygous knockout is embryonic lethal and SALL4 heterozygous knockout causes dysplasia of multiple organs [5,6]. SALL4 is an essential factor for the maintenance of self-renewal and pluripotency of embryonic stem cells(ESCs)[7,8]. SALL4 expression gradually decreases with the maturation of tissues and organs. However, SALL4 expression is found to be restored in numerous human malignancies. High levels of SALL4 has been observed in both hematological diseases and solid tumors, including acute myeloid leukemia, chronic myeloid leukemia, breast cancer, lung cancer, gastric cancer, colorectal cancer, liver cancer, endometrial cancer and glioma. SALL4 acts as an oncogene that plays multifaceted roles in the processes of cancer initiation, development, and progression. Exploring the underlying mechanisms responsible for the oncogenic functions of SALL4 will allow the development of a novel target for cancer therapy. In this review, we focus on recent progress in understanding the roles of SALL4 in cancer and the molecular mechanisms, with an emphasis on the potential of SALL4 in cancer diagnostics and treatment.

Roles of SALL4 in stem cells

SALL4 has two isoforms,S ALL4A and SALL4B, which resulted from different internal splicing patterns in exon 2 (Fig.1).

Fig. 1. SALL4 gene and protein structure. Human SALL4 gene localizes on chromosome 20q13.13-q13.2 and consists of four exons. The human SALL4 protein has multiple zinc finger (ZF) domains of the SAL type, which is composed of one N terminal C2HC type zinc finger (NT ZF) and seven C2H2 type zinc fingers. A Q rich motif is responsible for interactions between members of SALL1-4. The SALL4 protein has two isoforms, SALL4A and SALL4B, which resulted from different internal splicing patterns in exon 2. SALL4B lacks the 386 to 822 amino acids of full-length SALL4 protein. A conserved “KRLR” motif at amino acid positions of 64–68 is identified in both SALL4A and SALL4B as nuclear localization signal (NLS).

SALL4A and SALL4B are able to form homodimers or heterodimers with distinct DNA binding sites and exhibit different roles during early embryogenesis [9]. In murine ESCs, depletion of both isoforms of SALL4 by shRNA leads to multilineage differentiation. SALL4A and SALL4B have overlapping, but not identical binding sites of epigenetic marks in target loci or their interactions with other pluripotent factors. In addition, SALL4B, but not SALL4A, alone can maintain the pluripotent state of mouse ESCs. SALL4 expression could be detected in embryos as early as at the 4-cell stage and is gradually restricted to inner cell mass from which ESCs are normally derived. Disruption of both SALL4 alleles cause embryonic lethality during peri-implantation and depletion of SALL4 results in early embryonic development defects, suggesting that SALL4 is critical for early embryonic development. SALL4 plays a vital role in stem cell self-renewal and pluripotency through multiple layers of mechanisms.

First, SALL4 regulates the activation of several important signaling pathways in stem cells. Activation of Wnt/β-catenin signaling maintains the pluripotency of human and mouse embryonic stem cells [10]. SALL4 binds to β-catenin and up-regulates the expression of the target genes of the Wnt/β-catenin pathway [11], suggesting that SALL4 may promote stem cell self-renewal and inhibit stem cell differentiation through the interaction with β-catenin. STAT3 activation mediates the self-renewal and pluripotency of embryonic stem cells [12]. SALL4 may interact with STAT3 to regulate the self-renewal and differentiation of stem cells. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development [13]. Genome-wide analysis reveals that SALL4 regulates Sonic Hedgehog (SHH) pathway [7]. SALL4 may modulate SHH signaling to prevent the differentiation of embryonic stem cells. SALL4 inhibits the expression of PTEN and induces the activation of the Akt pathway [14], which may enhance stem cell self-renewal and expansion and maintain stem cells at undifferentiated state.

Second, SALL4 modulates the transcription of key stemness factors including Oct4, Nanog, Sox2, and c-Myc [7,15–17]. Compared to wild type ESCs, the expression of these 4 genes is remarkably down-regulated in SALL4+/− ESCs [7], suggesting that SALL4 could be a key regulator for stem cell factors. SALL4 can activate the expression of Oct4, Nanog, Sox2, and c-Myc and form a transcriptional core network with these factors to maintain cell stemness. The down-regulation of core stemness factors may be responsible for SALL4 knockdown-induced spontaneous cell differentiation [18]. Due to its critical role in maintaining pluripotency, SALL4 has been used as an enhancer for induced pluripotent stem cell (iPS) generation from somatic cells [19].

Third, SALL4 may regulate the expression of key genes that are associated with stem cell self-renewal and differentiation through epigenetic modulation. SALL4 induces the activation of Bmi-1, an important factor for regulating stem cell self-renewal, by mediating H3K4 trimethylation and H3K79 dimethylation at thepromoter region [20]. In addition, SALL4 recruits MLL (mixed lineage leukemia), a histone methyltransferase, to prompt H3K4 and H3K79 methylation, resulting in HOXA9 up-regulation [21].

In summary, these findings indicate that SALL4 is involved in regulating self-renewal and pluripotency of stem cells through a variety of signaling pathways, transcription factors, and epigenetic modulators. SALL4 expression has also been found in adult stem/progenitor cells. In human bone marrow, SALL4 expression is strictly limited to the CD34+ hematopoietic stem/progenitor cells (HSCs/HPCs) and decreased following differentiation [22,23]. SALL4 is found to be a robust stimulator for both human and mouse HSC/HPC self-renewal [24,25]. Human HSCs transduced with SALL4 are able to expand rapidly and efficiently in vitro. On the contrary, depletion of endogenous SALL4 leads to reduced HSC proliferation and accelerated cell differentiation. SALL4 regulates the expression of genes that are critical in maintaining short-term and long-term HSC proliferation, including Bmi-1, HOXA9, and c-Myc [26]. SALL4 works with these factors to form a concerted network for normal hematopoiesis [27]. In addition to HSCs/HPCs, SALL4 is expressed in fetal hepatoblasts but silenced in adult hepatocytes [28]. The expression levels of SALL4 gradually fall during liver development. SALL4 overexpression enhances while SALL4 knockdown impairs induced differentiation of hepatoblasts to cholangiocytes and the formation of bile duct, suggesting that SALL4 regulates cell fate decision in fetal hepatic stem/progenitor cells.

Roles of SALL4 in cancer

SALL4 is overexpressed in cancer and affects multiple cellular processes which are involved in tumorigenesis, tumor growth and tumor progression. SALL4 regulates a variety of targets in distinct types of cancer cells. In this section, we review the targets of SALL4 and their functions in cancer (Fig.2).

Fig. 2. Proposed model for SALL4 regulatory network in cancer. SALL4 regulates cell proliferation, apoptosis, migration/invasion, drug resistance, and stemness by targeting a variety of genes. SALL4 regulates cell proliferation through the β-catenin/cyclin D1, Bmi-1, and PTEN pathways. SALL4 regulates cell migration/invasion through the ZEB1/ E-cadherin and the c-Myc pathways. SALL4 inhibits apoptosis through the Bmi-1, HOXA9, and FADD pathways. SALL4 regulates the resistance of cancer cells to chemotherapy by targeting the ABCA3, ABCG2, and c-Myc pathways. SALL4 regulates the self-renewal of cancer stem cells through the Oct4, Nanog, Sox-2, and Bmi-1 pathways. STAT3, CDX1, and TCF/LEF are upstream positive regulators of SALL4. SALL4 is a downstream target of microRNA-107. Natural compounds matrine and apigenin could inhibit the expression of SALL4.

SALL4 and cell transformation

During normal hematopoiesis, SALL4 is expressed in the CD34+ HSC/HPC population. SALL4 expression is down-regulated or silenced in mature blood cells. In contrast, SALL4 is constitutively expressed in human primary acute myeloid leukemia (AML) and myeloid leukemia cell lines. To test whether constitutive expression of SALL4 is sufficient to induce AML, Ma and colleagues generated a SALL4B transgenic mouse model with SALL4B expression in most organs. They demonstrated that all the monitored SALL4B transgenic mice exhibit dysregulated hematopoiesis and develop myelodysplastic syndrome (MDS)-like features at ages 6–8 months and half of the monitored mice eventually progressed to AML [11]. Mice injected with serially transplanted SALL4B-induced AML cells also develop aggressive AML, suggesting that SALL4B-induced AML is transplantable.

The potential signaling pathway that SALL4 may affect in leukemogenesis has been postulated, which includes SALL4 binding to β-catenin and activating the Wnt/β-catenin signaling pathway. The expression of Wnt/β-catenin downstream target genes, such as c-Myc and Cyclin D1, is upregulated in SALL4B transgenic mice. Thus, constitutive expression of SALL4 in AML may enable leukemic blasts to gain stem cell properties, such as self-renewal and/or lack of differentiation, and thus become leukemic stem cells (LSCs). In addition, Bmi-1 is identified as a target gene for SALL4 in both hematopoietic and leukemic cells [20].

Bmi-1 is a putative oncogene that modulates stem cell pluripotency and plays a role in leukemogenesis. SALL4 binds to Bmi-1 promoter and directly affects the levels of endogenous Bmi-1 expression. In vitro knockdown of SALL4 by siRNA in leukemic cells or in vivo deletion of one copy of SALL4 in mouse bone marrow significantly reduces Bmi-1 expression. Bmi-1 expression is up-regulated in transgenic mice that constitutively overexpress SALL4B, and the levels of Bmi-1 in these mice increase as they progress from normal to preleukemic (myelodysplastic syndrome) and leukemic (acute myeloid leukemia) stages. Furthermore, there is a strong positive correlation between SALL4 and Bmi-1 expression in human AML samples. SALL4 induces high levels of H3K4 trimethylation and H3K79 dimethylation in the binding region of the Bmi-1 promoter, suggesting that SALL4 provides epigenetic modifications at the Bmi-1 gene promoter. These findings indicate a link between SALL4 and leukemogenesis by regulating self-renewal of leukemic stem cells.

SALL4 and cell proliferation and apoptosis

SALL4 acts as a key regulator of cell proliferation and apoptosis in cancer cells. SALL4 knockdown induces massive apoptosis and significant growth arrest in human leukemic cells [29]. In addition, reduction of SALL4 markedly diminishes tumorigenicity of human leukemia cells in immunodeficient mice. ChIP-chip assay for the global gene target of SALL4 in human leukemic cells reveals that SALL4 binds to the promoter of genes that are critically involved in apoptosis. The expression levels of these genes change significantly after SALL4 knockdown, indicating that SALL4 is a key regulator of apoptosis-associated genes. In addition, SALL4 has an important role in the proliferation and survival of chronic myeloid leukemia (CML) cells, and its expression is associated with an advanced stage of CML disease. Downregulation of SALL4 leads to cell cycle arrest and apoptosis in CML cells [30].

In AML and CML cells, SALL4 knockdown-induced apoptosis and cell cycle arrest are rescued by forced expression of Bmi-1, suggesting that SALL4 regulation of Bmi-1 may at least be partially responsible for its effects on cell proliferation and apoptosis. Moreover, HOXA9 is identified as another downstream target of SALL4 [21]. SALL4 interacts with mixed lineage leukemia (MLL) and co-occupies the HOXA9 promoter region in AML leukemic cells. Compared with wild-type controls, HOXA9 is up-regulated in SALL4B transgenic mice. In primary human AML cells, downregulation of SALL4 also reduces HOXA9 expression and induces cell apoptosis. Furthermore, SALL4 knockdown leads to growth inhibition of lung cancer cells as a result of cell cycle arrest [31]. Similarly, reduction of SALL4 expression by siRNA completely also inhibits the proliferation of breast cancer cells as a result of cell cycle arrest [32]. Conversely, SALL4-overexpressing liver cancer cells exhibit enhanced cell proliferation with the characteristics of reduced cell population in the G1 phase through the up-regulation of cyclin D1 and D2 [33]. In contrast, down-regulation of SALL4 inhibits liver cancer cell proliferation in vitro as well as in tumor xenograft models. We have recently reported that SALL4 overexpression enhances while knockdown of SALL4 inhibits the proliferation of gastric cancer cells [34]. In consistent with this observation, SALL4 knockdown also inhibits endometrial cancer cell growth in vitro and tumorigenicity in vivo due to the inhibition of cell proliferation and increased apoptosis [35].

SALL4 and invasive migration

The research from our group has indicated that the SALL4 level is highly correlated with lymph node metastasis of gastric cancer [34]. Enforced expression of SALL4 enhances the migration of human gastric cancer cells, whereas knockdown of SALL4 by siRNA leads to the opposite effects. SALL4 overexpression up-regulates the expression of Twist1, N-cadherin while down-regulating E-cadherin expression, thus inducing epithelial–mesenchymal transition (EMT) in gastric cancer cells. In endometrial cancer, down-regulation of SALL4 significantly impedes the migration and invasion properties of cancer cells in vitro and their metastatic potential in vivo[35]. SALL4 specifically binds to the c-Myc promoter region in endometrial cancer cells. Reduction of SALL4 leads to a decreased expression of c-Myc and ectopic SALL4 overexpression causes increased c-Myc expression, indicating that c-Myc is one of the SALL4 downstream targets in endometrial cancer. In addition, SALL4 suppresses E-cadherin expression and maintains cell dispersion in basal-like breast cancer [36]. SALL4 inhibits intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. SALL4 knockdown leads to EMT in basal-like breast cancer cells. Further study showed that SALL4 positively regulates the EMT factor ZEB1, therefore suppressing E-cadherin transcription and leading to cell dispersion and mesenchymal gene expression.

SALL4 and cancer stem cells

SALL4 is essential for maintaining the properties of cancer stem cells (CSCs). The expression of SALL4 is significantly higher in side population (SP) cells than that in non-SP cells in leukemia cell lines, suggesting that SALL4 is more abundant in CSCs [37]. Knockdown of SALL4 leads to reduced frequency of SP cells, indicating that SALL4 is required for the self-renewal of cancer stem cells. Similarly, SALL4 is also enriched in SP of breast cancer cells and increased SALL4 expression leads to an expansion of SP subset in breast cancer cells.

We have recently demonstrated that SALL4 overexpression induces the acquirement of stemness in gastric cancer cells through increasing the levels of Sox2, Bmi-1, CD133, and Lin28B [34]. SALL4 overexpressing gastric cancer cells could be efficiently induced to differentiate into osteoblasts and adipocytes under the appropriate conditions, suggesting that SALL4 overexpression endows gastric cancer cells with stemness and pluripotency. SALL4 is suggested as a stem cell marker in liver cancer that regulates the stemness of liver cancer cells [33]. SALL4 overexpression down-regulates differentiation markers ALB, TTR, and UGT2B7, suggesting that SALL4 inhibits hepatocytic differentiation in human liver cancer cells. SALL4 is identified as one of the transcription factors that are potentially activated in hepatic stem cell-like HCC (HpSC-HCC)[38]. SALL4-positive HCCs are associated with expression of the hepatic stem cell markers including EpCAM.

EpCAM+ cells have higher expression of SALL4 and possess a stronger spheroid formation capacity than EpCAM− cells, indicating that SALL4 is activated in EpCAM+liver CSCs. Ectopic expression of SALL4 leads to up-regulation of the hepatic stem cell markers and down-regulation of the mature hepatocyte marker. Moreover, SALL4 overexpression results in the significant activation of spheroid formation while knockdown of SALL4 results in a compromised spheroid formation capacity with decreased expression of EpCAM, suggesting that SALL4 regulates stemness of HpSC-HCC.

SALL4 and chemoresistance

SALL4 expression is associated with therapy response in cancer. In acute myeloid leukemia (AML), SALL4 expression is higher in drug resistant patients than those from drug-responsive cases. AML cells with decreased SALL4 expression are more sensitive to drug treatments than their parental cells. SALL4 modulates drug sensitivity through the maintenance of SP cells in AML [37]. SALL4 directly binds to the promoter region of ABCA3, a resistance-mediating transporter, affecting the formation of SP cells in AML. SALL4 expression is positively correlated to that of ABCA3 in primary leukemic patient samples. In addition to AML patients, constitutive expression of SALL4 has also been observed in CML cells in the blast crisis or accelerated phase. Exposure to tyrosine kinase inhibitors (TKI) leads to increased expression of SALL4 in CML cells, which consequently upregulates ABCA3 [39]. High ABCA3 levels facilitate detoxification of TKI, protecting CML cells against TKI effects. The positive regulation of ABCA3 through SALL4 constitutes an auto-protective loop to protect CML cells from the lytic activity of TKI. Suppression of SALL4 by siRNA partially abrogates a TKI-associated increase in ABCA3 expression and increases susceptibility of CML cells to cytotoxicity of tyrosine kinase inhibition. Treatment with indomethacin interrupts the inducible SALL4/ABCA3 pathway in CML cells to restore TKI susceptibility.

Constitutive expression of SALL4 affects the sensitivity of endometrial cancer cells to carboplatin treatment [35]. Overexpression of SALL4 in carboplatin sensitive endometrial cancer cells could further promote carboplatin resistance in a dose-dependent manner. SALL4-transfected endometrial cancer cells show increased proliferationaftercarboplatintreatmentcomparedwithcontrolcellswhile the overexpression also protects endometrial cancer cells from carboplatin-inducedapoptosis.Incontrast,incarboplatin-resistant endometrial cancer cells, SALL4 knockdown significantly sensitizes the cells to carboplatin treatment. SALL4 directly regulates c-Myctranscriptionalactivityinendometrialcancercells,whichmay be partially responsible for the chemotherapy resistance induced by SALL4 upregulation. In addition, SALL4 expression is correlated with chemosensitivity in liver cancer cells. SALL4-overexpression induces survival and proliferation of liver cancer cells in response to 5-FU treatment, suggesting that SALL4 expression results in selection of chemoresistant cells [33].

SALL4 and epigenetic modulation

In addition to transcriptional control, SALL4 also regulates gene expression through epigenetic mechanisms. DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs are the four major molecular mechanisms responsible for epigenetic modification. Yang et al. suggest that SALL4 protein directly interacts with DNA methyltransferases (DNMTs), indicating that SALL4 is able to repress transcription through recruitment of DNA methyltransferases [40]. In addition, SALL4 has been shown to co-occupy target genes with polycomb repressive complex (PRC) [7].

SALL4 may repress gene transcription though the induction of PRC components (such as Bmi-1) or interaction with PRC complex members. Moreover, SALL4 interacts with histone lysine-specific demethylase1 (LSD1) to repress gene transcription in stem cells [41]. In addition to gene repression, SALL4 is capable of binding to the histone methyltransferase MLL to activate HOXA9 gene expression [21], suggesting that SALL4-mediated methylation and demethylation in DNA and histone may distinctly regulate gene expression in stem cells and cancer.

Second, SALL4 is associated with Mi-2/nucleosome remodeling and deacetylase (NuRD) complex and theSALL4-interacting protein complex exhibits histone deacetylase (HDAC)activity [42]. For instance, SALL4 co-occupies the same promoter regions of PTEN as HDAC2 and represses its expression in vitro, indicating that SALL4-repressed gene transcription could be mediated by histone deacetylation and nucleosome remodeling. However, there is a lack of studies about the regulatory effects of SALL4 on non-coding RNA in epigenetic modulation. Therefore, the existing data suggest that SALL4 may recruit multiple epigenetic modifiers to synergistically remodel local chromatin structure and coordinately regulate gene transcription (Fig.3).

 

Fig. 3. SALL4 and epigenetic machinery. SALL4 represses or activates gene transcription through the interaction with distinct epigenetic modifiers. SALL4 suppresses gene transcription through recruitment of DNA methyltransferases (DNMT), Mi-2/nucleosome remodeling and deacetylase (NuRD) complex, polycomb repressive complex (PRC), and histone demethylase (LSD1). SALL4 activates gene expression through recruitment of histone methyltransferase such as MLL (mixed-lineage leukemia).

SALL4 regulation in cancer

SALL4 is regulated at multiple levels in cancer. Aberrant hypomethylation of the promoter region of SALL4 gene is observed in MDS patients and SALL4 mRNA level is highly associated with the status of SALL4 hypomethylation, indicating that SALL4 gene expression is dysregulated in MDS patients by epigenetic mechanism [43]. The frequency of SALL4 hypomethylation is significantly increased in higher risk MDS patients, suggesting that hypomethylation of SALL4 gene is involved in the progression of MDS. In addition, SALL4 gene is aberrantly hypomethylated in acute myeloid leukemia patients and the status of SALL4 gene methylation is associated with intermediate and poor karyotypes of AML [44]. SALL4 is also regulated by a variety of transcription factors that are closely linked with tumor development and progression.

Multiple STAT3-binding sites have been identified in the SALL4 gene promoter region. Down-regulation of STAT3 activity remarkably decreased the expression of SALL4 [45]. STAT3-mediated SALL4 regulation is critical for the survival of breast cancer cells. SALL4 expression is also regulated by the canonical Wnt signaling pathway. SALL4 promoter contains a conserved TCF/LEF-binding site. Co-transfection of β-catenin with LEF1 (or TCF4E) greatly increases SALL4 luciferase activity while mutation of the TCF/LEF-binding site attenuates SALL4 luciferase activity, suggesting that SALL4 is a direct transcriptional target of canonical Wnt signaling [46]. Furthermore, SALL4 interacts with β-catenin to cooperatively activate its target genes. Therefore, the regulation of SALL4 by Wnt signaling may form a feedback loop to fine-tune the Wnt signaling pathway.

SALL4 is identified as a direct target of caudal-related homeobox 1 (CDX1) transcription factor [47]. SALL4 is aberrantly expressed in the CDX1-positive intestinal metaplasia of the stomach in both humans and mice. CDX1-induced SALL4 converts gastric epithelial cells into tissue stem-like progenitor cells, which then transdifferentiate into intestinal epithelial cells, suggesting that SALL4 is a critical component of CDX1-directed transcriptional circuitry that promotes intestinal metaplasia. Furthermore, SALL4 is found to be regulated by microRNA in glioma cells [48]. MiR-107 mimics reduce while miR-107 inhibitors increase the SALL4 mRNA level. MiR-107 overexpression inhibits cell proliferation and induces apoptosis in glioma cells, which are reversed by SALL4 reintroduction. An obvious inverse correlation between miR-107 expression and SALL4 level is observed in clinical glioma samples, indicating that upregulation of miR-107 inhibits glioma cell growth through direct targeting of SALL4.

SALL4B can be modified by both ubiquitination and sumoylation at the post-translational level. However, SALL4B sumoylation is independent of ubiquitination while lysine residues 156, 316, 374, and 401 are essential for sumoylation. It is known so far that only SALL4 sumoylation is functionally important [49]. A constitutive sumoylation of SALL4B is readily detectable in teratocarcinoma cells. SUMO-deficiency compromises the transactivational or trans-repressional activities of SALL4B, suggesting that sumoylation is an important post-translational modification for SALL4 activity.

SALL4 as cancer biomarker and target

SALL4 seems to be a sensitive and specific cancer biomarker. SALL4 expression is reported in numerous malignancies, such as precursor B-cell lymphoblastic lymphoma [50,51], myelodysplastic syndromes [52], acute myeloid leukemia [11], chronic myeloid leukemia [30], breast cancer [32], lung cancer [31,53], endometrial cancer [35], liver cancer [33,38], gastrointestinal carcinoma [34,54–56], glioma [48,57], germ cell tumors (GCTs), and yolk sac tumors [58–60]. SALL4 expression correlates with disease progression in human AML and its expression in AML patients is correlated with treatment status. Therefore, SALL4 may be used as a marker for diagnosis and prognosis for AML. SALL4 is expressed at a high level in the early clinical stages of breast cancer, indicating that SALL4 may be helpful for breast cancer screening.

SALL4 expression is reactivated in human HCC patients. HCC patients with high SALL4 expression are significantly associated with shorter survival. SALL4 is an independent prognostic factor for overall survival and early recurrence of HCC. In endometrial cancer patients, the level of SALL4 expression is positively correlated with worse patient survival and aggressive features such as metastasis. In colorectal carcinoma (CRC), significant increase in SALL4 expression is detected in 87 tissues and SALL4 expression is highly correlated with tumor metastasis. In esophageal carcinoma (ESCC), SALL4 expression has a significant correlation with invasion and metastasis of the disease [61]. We have shown that SALL4 expression is up-regulated in gastric cancer patients and high level of SALL4 predicts poor prognosis in these patients. Furthermore, a high level of SALL4 protein is detected in the serum of HCC patients [62]. HCC patients with high SALL4 serum levels have poor prognosis evidenced by both tumor recurrence and overall survival rate, suggesting that the high serum level of SALL4 is a novel prognosis biomarker for HCC patients.

In addition to being a biomarker for cancer diagnostics, SALL4 may also constitute a possible therapeutic target. The inhibition of SALL4 expression by siRNA causes reduced cell survival and impaired migration and invasion in distinct cancer cells in vitro.Stable knockdown of SALL4 by shRNA efficiently retards tumor growth and restrains tumor metastasis in animal models. Moreover, SALL4 silencing by miRNA inhibits glioma cell proliferation and induces apoptosis in vitro and in vivo. These findings suggest that depletion of SALL4 has potential anti-tumor effects. In addition, natural compounds such as matrine and apigenin have been shown to suppress SALL4 expression and inactivate the β-catenin signaling pathway in leukemic cells [63]. However, the specificity of these natural compounds to SALL4 still needs to be further investigated.

SALL4 also serves as an ideal target for combined therapy. SALL4 knockdown in combination with Bcl2 inhibitor treatment increases the apoptotic AML cells to 2–3 fold compared to cells treated alone [64], suggesting that the combination of Bcl2 inhibitor and down-regulation of SALL4 could be a novel therapeutic strategy in treating AML patients. In human hepatocellular carcinoma, HDAC inhibitor SBHA reduces SALL4 expression and inhibits the proliferation of SALL4-positive HCC cells, suggesting the therapeutic potential of these inhibitors in the treatment of SALL4-positive HpSC HCC through targeting SALL4 [38]. Furthermore, interfering with the interaction of SALL4 and its epigenetic partner complex has therapeutic effects in cancer.

Gao and colleagues have developed a peptide inhibitor that can compete with SALL4 in interacting with the HDAC complex [65]. They demonstrate that treating SALL4 expressing leukemic cells with this peptide leads to cell death through the reactivation of PTEN. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells by using siRNA. The biological activity of this peptide is further confirmed in hepatocellular carcinoma.TheSALL4 peptide inhibitor inhibits the viability of SALL4-overexpressing hepatocellular carcinoma cells in a PTEN-dependent manner, with minimaltoxiceffectsonSALL4-negativecells,as compared with the HDAC inhibitor trichostatin (TSA). To test the therapeutic effect of this peptide for in vivo treatment, the peptide is conjugated with the transactivator of transcription (TAT) protein transduction domain and intraperitoneally injected into NOD/SCID mice bearing subcutaneous xenograft tumor [66]. TAT fusion peptide greatly reduces the tumorigenicity of SALL4-overexpressing hepatocellular carcinoma cells, suggesting that the SALL4 peptide inhibitor is a potent anti-cancer agent for SALL4-overexpressing hepatocellular carcinoma.

Conclusions and future directions

Over the past decade, accumulating evidence indicates that SALL4 plays a key role in cancer biology in addition to its seminal function in stem cells and development. In distinct types of cancer,SALL4 has been shown to be crucial for cell survival and proliferation, invasive migration, and chemoresistance. Evidence from mouse models suggests that SALL4 is critically involved in leukemogenesis. SALL4 protein may either function as a transcription activator or suppressor to regulate the expression of downstream target genes. However, the pathological roles of SALL4 in cancer seem to be dependent on cell type and context. Therefore, it is necessary to establish mouse models in which SALL4 isoforms are conditionally activated or knocked down in certain cell types. In addition, the oncogenic functions of SALL4 have not been completely characterized. Much less is known about the roles of SALL4 in the other hallmarks of cancer such as sustained angiogenesis, immune evasion, and deregulated energy metabolism. The underlying molecular mechanisms responsible for the different functions of SALL4 in tumor development and progression have not been fully elucidated. Up to now only a few mediators have been identified.

It is conceivable that many other downstream targets of the SALL4 signaling pathway remain to be discovered. Thus, transcriptomic and proteomic analyses to reveal the global downstream target genes (including both coding and noncoding genes) and interacting proteins for SALL4 will help establish the integrated signaling network in cancer. Moreover, the mechanisms driving the re-activation of SALL4 in cancer remain largely unknown. The activation of SALL4 upstream regulators is frequently seen in human cancers. For instance, STAT3 is associated with constitutive SALL4 expression in breast cancer and inhibition of STAT3 activity disrupts SALL4 expression [58]. Thus, we need to understand if any other genetic or epigenetic modifications of SALL4 gene exist that contribute to tumor development and progression. In addition, a specific peptide inhibitor for SALL4 has shown promising anti-cancer activity and further efforts to develop small molecule peptide mimetics or combine with conventional anticancer drugs may help rediscover its therapeutic value. Targeted delivery of siRNA and other inhibitors to disrupt the expression and function of SALL4 in cancer cells by using advanced biotechnologies will provide a new strategy for cancer therapy. Finally, SALL4 is known to maintain the self-renewal and pluripotency of stem cells.

It is interesting to test whether targeting SALL4 will be able to eradicate CSCs given that CSCs are thought to cause metastasis, chemoresistance, and subsequently tumor recurrence. Answers to these important questions will shed light on the role of SALL4 in cancer biology and provide full potential for SALL4 as a valid cancer biomarker and target.

7.11.6 Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer

Itou J1Matsumoto YYoshikawa KToi M.
FEBS Lett. 2013 Sep 17; 587(18):3115-21
http://dx.doi.org/10.1016/j.febslet.2013.07.049

Highlights

  • SALL4 suppresses CDH1 transcription.
    • SALL4 positively regulates expression of ZEB1, a CDH1 suppressor.
    • SALL4 prevents intercellular adhesion in basal-like breast cancer.
    • SALL4 maintains cell motility in basal-like breast cancer.

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal-like 4 (SALL4) as a dispersion factor in basal-like breast cancer. Our shRNA-mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal-like breast cancer.

Cell migration is recognized in various fields, including cancer. A hallmark of migratory cells is the dispersed phenotype in in vitro condition, in which a cell located at the edge of a cluster loses intercellular adhesion, possesses membrane spikes and front–rear polarity, and moves away independently from the cluster. In contrast, plated non-migratory cells form compacted clusters, where adhesiveness is augmented, and single dispersed cell is not seen. One of the phenomena to induce the migratory ability is epithelial mesenchymal transition (EMT), by which epithelial properties, e.g., the compacted morphology and epithelial marker expression, are replaced by the dispersed phenotype and mesenchymal gene expression [1]. An advantageous model to study cell dispersion and EMT is basal-like breast cancer. Some of basal-like breast cancer cell lines, such as SUM159 and MDA-MB-231, have the dispersed phenotype and mesenchymal gene expression. These characteristics are convertible to epithelial properties by genetic manipulation, which allows us to digest what factor(s) functions to control cell dispersion and EMT. For instance, the zinc finger- and homeobox containing transcription factor ZEB1 (also known as deltaEF1 and TCF8) acts as an EMT activator. ZEB1 suppresses the transcription of the adhesion gene CDH1, and ZEB1 knockdown enhances cell–cell adhesion [2]. The miR200 family of microRNAs is known as a suppressor of the ZEB family [3]. Introduction of miR200-mediated ZEB1 silencing diminishes the dispersed phenotype and motility in MDA-MB-231 [4]CDH1 encodes the cell–cell adhesion protein E-cadherin. MDA-MB-231 having ectopic E-cadherin expression exhibits the compacted epithelial morphology and loss of the migratory ability [5]. These revealed that CDH1 suppression by ZEB1 plays a key role in the maintenance of cell dispersion in basal-like breast cancer.

Sal-like 4 (SALL4) is one of the mammalian homologs of the Drosophila region specific homeotic genespalt (sal), which encodes a multiple zinc finger transcription factor. SALL4 consists of four exons, and the second of which has an internal splicing donor site. SALL4A, one of two SALL4 variants, is translated from the mRNA having the entire exon2, whereas the mRNA for SALL4B has the short form of exon2 [6]. SALL4 has been identified as a causative factor in acute myeloid leukemia [6]. An increase in SALL4 expression has also been reported in breast- [7] and [8], lung- [9], colorectal- [10] and liver cancers [11] as well as germ cell tumors [12] and [13]. In addition to in cancerous tissues and cancer cell lines, SALL4 expression has been detected in circulating breast cancer cells [14]. In breast cancer cell lines, SALL4 transcription is positively regulated by STAT3 [7], and SALL4 suppression provides proliferative inhibition [7] and [8].

In this study, we identified SALL4 as a cell dispersion factor. We demonstrated that basal-like breast cancer cell lines undergo transition to a compacted epithelial state by SALL4 knockdown. In reciprocal experiments, the overexpression of SALL4 provided the dispersed phenotype and a reduction in CDH1expression to epithelial cells. The time-course observation revealed that SALL4 prevents cell–cell adhesion, and maintains cell motility in basal-like breast cancer.

Epithelial transition is induced by SALL4 knockdown in basal-like breast cancer

SALL4 involves in cell proliferation in breast cancer cell lines [7] and [8]. The functions of SALL4, however, remain elusive. To analyze the functions of SALL4 in breast cancer, we established a DOX inducible shRNA expression system with shGFP and shSALL4 constructs in the basal-like breast cancer cell line SUM159. To evaluate effects of our system, we analyzed the cell proliferative ability, a known function of SALL4. In our system, reduced cell number was observed in cells having shSALL4#3 and #5 expression, but not in shGFP expression (Fig. S1A–C). Target sites of shSALL4#3 and #5 were designed at the regions common to the mRNAs of SALL4 variants. Because the shSALL4#5 is more effective than the #3, we mainly used the #5 in this study. Quantitative RT-PCR and immunoblotting showed significant reductions in SALL4 mRNA and protein levels in DOX-induced cells (Fig. S1D and E). The ratio between the numbers of dead cells and total cells in shSALL4-expressing cells was identical to that in the no-DOX control (Fig. S1F), indicating that the reduced cell number observed by SALL4 knockdown is not due to decreased cell survival. To analyze changes in expression of the proliferation genes, we quantified the mRNA levels.BMI1, a polycomb group gene, is positively regulated by SALL4 [15]. BMI1 suppresses expression of the cyclin-dependent kinase inhibitors, such as p16p18 and p21 [16]. In our system, shSALL4 reduced theBMI1 level and increased the p16 and p18 levels ( Fig. S2A–D). We analyzed other proliferation markers,MYCCCNE1 and CCND1. It has been reported that SALL4 positively regulates CCND1 in transcription level [11]. We observed a reduction in expression of CCND1 in shSALL4-expressing cells ( Fig. S2E–G). In protein analysis, Cyclin D1, the product of CCND1, level was reduced ( Fig. S2H). These results indicate that SALL4 regulates cell proliferation in breast cancer, and our inducible shRNA expression system is useful to explore SALL4 functions.

In in vitro conditions, some of basal-like breast cancer cell lines, including SUM159, tend to be dispersed. Surprisingly, almost cells having shSALL4 expression lost membrane spikes, and formed compacted clusters (Fig. 1A–F). In order to examine this difference, we measured the lengths of perimeters and contacting areas of cells located at the edges of the clusters (Fig. S3). Polarized and spine-rich cells typically have a longer perimeter than spineless cells. We compared the lengths of perimeters of shSALL4-expressing cells to that of no-DOX and shGFP controls. Small values observed in shSALL4-expressing cells indicate that the cells became spineless (Fig. 1G). The ratio of the length of contacting area to that of perimeter reflects the degree of compaction. Cells having shSALL4 expression were more compacted than the controls (Fig. 1H).

Fig. 1. The compacted phenotype and epithelial gene expression observed by SALL4 knockdown in SUM159. (A–F) Cell compaction was observed in cells having shSALL4 expression.
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Since mammary cells possess a potential to shift between compacted epithelial and dispersed mesenchymal states [2] and [17], the compaction observed in shSALL4-expressing cells was suggestive of a transition to the epithelial state. Thus, we analyzed mRNA levels of the epithelial marker CDH1 and the mesenchymal markers VIM and CDH2 ( Fig. 1I–K). In shSALL4-expressing cells, the CDH1 level was increased and the VIM level was reduced. The CDH2 level was not significantly changed. We detected immunoreaction of E-cadherin, the product of CDH1, in shSALL4-expressing cells ( Fig. 1L–O). Our observations, the compacted phenotype ( Fig. 1D, F and H) and the up-regulation of epithelial markerCDH1 ( Fig. 1I and O), indicate that SALL4 knockdown induces the epithelial transition. The previous study has demonstrated that ectopic E-cadherin expression induces the compacted phenotype and reduction in the vimentin, the product of VIM, level in basal-like breast cancer MDA-MB-231 [5]. SALL4 might regulate cell dispersion and mesenchymal gene expression by suppressing CDH1 transcription.

SALL4 regulates the EMT factor ZEB1

We suspected that SALL4 regulates transcription factors involving in EMT, because SALL4 knockdown induces epithelial transition. In order to identify the factor(s), we used quantitative RT-PCR to screen the transcription factors, SNAI1SNAI2TWIST1TWIST2FOXC1FOXC2TGFB1TCF3GSCGRHL2,ZEB1 and ZEB2 [18][19] and [20]. In the result, we found reduction in the ZEB1 mRNA level in shSALL4-expressing cells ( Fig. 2A), while the others were not significantly changed ( Fig. 2B and Fig. S4). No detectable amplification was observed in the experiments for GSC and GRHL2. In addition to change in theZEB1 mRNA level, ZEB1 protein level was reduced in shSALL4-expressing cells ( Fig. S5).

Fig. 2.  The SALL4-ZEB1 network in SUM159.

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ZEB1 mRNA is one of known targets of the miR200 family-mediated gene silencing [4] and [21]. Thus we assessed the activities of miR200s by using a miR200 reporter ( Fig. S6), which has the ZEB1 3′ untranslated region, a target of miR200 family. To evaluate the miR200 reporter, we introduced expressions of two miR200 regions, miR200b-a-429 and miR200c-141. The expression of miR200 family decreased the luciferase activity ( Fig. 2C), indicating that using the miR200 reporter enables us to examine the activities of miR200s-mediated gene silencing. Comparing to the shGFP control, shSALL4-expressing cells showed no alteration of the luciferase activity ( Fig. 2D). This result indicates that the activities of miR200s are not changed by SALL4 knockdown. It is known that expressions of the ZEB family and the miR200 family are mutually exclusive [3]. ZEB1 and ZEB2 bind to the promoter regions of miR200 family, and suppress their transcription. The miR200s act as the silencer for ZEB1 and ZEB2 mRNAs by binding to their 3′ untranslated regions. A previous study has demonstrated that ZEB1 knockdown increases miR200s activities [22]. We showed reduction in ZEB1 level ( Fig. 2A). However the activities of miR200s were not increased ( Fig. 2D). No alteration of miR200s activity observed in shSALL4-expressing cells is likely due to the function of another miR200s suppressor ZEB2, the mRNA level of which was not changed by SALL4 knockdown ( Fig. 2B). A similar observation has been reported in a study in ovarian cancer, which showed that the miR200c-141 level was not altered by ZEB1 knockdown in cells expressing ZEB2 [23]. To analyze whether ZEB1 promoter activity was affected by SALL4 knockdown, we connected the 5 kbp upstream region of the ZEB1 initiation codon to the luciferase2 gene ( Fig. S6). Cells having thisZEB1 promoter construct showed a reduction in the luciferase activity when shSALL4 was expressed ( Fig. 2E), suggesting that SALL4 positively regulates ZEB1 transcription.

Our results demonstrated that SALL4 regulates two transcriptional regulators, BMI1 ( Fig. S2A) and ZEB1 (Fig. 2A). To analyze whether BMI1 regulates ZEB1 transcription, we performed shRNA-mediated BMI1knockdown assays. Due to severe proliferative inhibition of BMI1 knockdown [16], we could not obtain enough number of shBMI1 infectants to analyze the gene expression. We therefore established the DOX inducible shBMI1 expression system to obtain a sufficient number of cells with avoiding the proliferative inhibition. The ZEB1 mRNA level was not changed by shBMI1 induction ( Fig. 2F and G). In head and neck squamous cell carcinoma, CDH1 transcription is suppressed by BMI1, and BMI1 knockdown increases the E-cadherin level [24]. In basal-like breast cancer, however, the CDH1 mRNA level was not affected by shBMI1 expression ( Fig. 2H). This suggests that the mechanism of CDH1 regulation is different among cell types.

Besides analyses in shBMI1 expressing cells, we performed ZEB1 knockdown experiments. The BMI1mRNA level was not affected by ZEB1 knockdown ( Fig. 2I and J). The results of ZEB1 and BMI1 knockdown experiments suggest that these transcriptional regulators are independently regulated by SALL4. Since ZEB1 acts as the suppressor for CDH1 transcription [2], the CDH1 mRNA level was up-regulated by shZEB1 expression ( Fig. 2K). This suggests that the SALL4-ZEB1 network regulates CDH1transcription.

If CDH1 transcription is suppressed by the SALL4-ZEB1 network, a change in CDH1 level should be observed after a reduction in ZEB1 expression when shSALL4 is induced. To analyze the timing of changes in SALL4ZEB1 and CDH1 expressions, we performed quantitative RT-PCR in the DOX inducible shSALL4 expression system at time points 0.5, 1, 2 and 4 days post DOX administration. A reduction in theSALL4 mRNA level was observed from 0.5 day ( Fig. 3A). The ZEB1 level was significantly changed from 1 day ( Fig. 3B). An increase in the CDH1 level was observed from 2 days, suggesting that up-regulation ofCDH1 transcription occurs between 1 and 2 days ( Fig. 3C). These results support the suggestion thatCDH1 is suppressed by the SALL4-ZEB1 network in basal-like breast cancer.

Sequential gene expression changes after SALL4 knockdown in SUM159.

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Fig. 3. Sequential gene expression changes after SALL4 knockdown in SUM159. (A–C) Quantification of the mRNA levels for SALL4 (A,= 3), ZEB1 (B, = 3) and CDH1 (C, = 3) were performed at 0.5, 1, 2 and 4 days. The same values of no-DOX controls as for the analyses shown in Fig. S1 (SALL4), Fig. 2 (ZEB1) and Fig. 1 (CDH1) were used to calculate relative values. Error bars indicate standard deviations. Asterisks indicate statistical significance. Data between the no-DOX control and each time point were analyzed by the Student’s t-test.

SALL4 maintains cell dispersion and regulates gene expression in MDA-MB-231 as well as in SUM159

The previous study has reported that SALL4 knockdown impairs the proliferative ability in another basal-like breast cancer cell line MDA-MB-231 [7]. To assess the generality of our observation in SUM159, we analyzed the changes in the phenotype and gene expression in MDA-MB-231. Cells having shSALL4 expression lost spikes and exhibited an oval-shape (Fig. 4A and B). However, unlike SUM159, the cells were enlarged. The mean perimeter length of enlarged oval-shaped cells was comparable to that of spine-rich controls (Fig. 4C). SALL4 knockdown increased the degree of compaction in MDA-MB-231 (Fig. 4D). In quantitative RT-PCR analyses for the epithelial and mesenchymal genes, CDH1 expression was augmented, and VIM was reduced ( Fig. 4E and F). The CDH2 level was not significantly changed ( Fig. 4G). The levels of transcriptional regulators BMI1 and ZEB1 were reduced by SALL4 knockdown ( Fig. 4H and Fig. S7). These results, except for the effect on cell size, were similar to the observations in SUM159, suggesting that SALL4 maintains cell dispersion and regulates the expressions of epithelial and mesenchymal genes in basal-like breast cancer cell lines.

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Mammary epithelial cells exhibit the dispersed phenotype and express the mesenchymal genes by SALL4 overexpression

HMLE is utilized as an epithelial cell model in breast cancer studies [17]. We overexpressed SALL4 variants, SALL4A and SALL4B, in HMLE to analyze whether SALL4 induces cell dispersion in compacted epithelial cells. The EGFP control showed compacted clusters (Fig. 5A). The clusters of SALL4A and SALL4B overexpressing cells had more spaces than that of EGFP control (Fig. 5B and C arrowheads). These spaces were likely to be caused by a loss of adhesiveness. In SALL4 overexpressions, cells exhibited membrane spikes, and the mean lengths of perimeters were increased (Fig. 5D). The degrees of compaction were reduced (Fig. 5E). These results imply that SALL4 forces cell dispersion in HMLE. However, SALL4 itself is insufficient to induce complete cell dispersion as in basal-like breast cancers, suggesting that other supportive factor(s) is required to induce it.

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Fig. 5. The dispersed phenotype and mesenchymal gene expression induced by SALL4 overexpression in epithelial cells.

In basal-like breast cancers, SALL4 knockdown increases the expression of adhesion gene CDH1 and reduces the levels of mesenchymal genes VIM and ZEB1 ( Fig. 1Fig. 2 and Fig. 4). We analyzed the mRNA levels of CDH1VIM and ZEB1 in HMLE having SALL4 overexpression. SALL4A and SALL4B reduced the CDH1 level ( Fig. 5F), which might involve in a loss of cell–cell adhesion. Conversely, the VIMand ZEB1 expressions were up-regulated ( Fig. 5G and H). Our results suggest that in addition to the maintenance of dispersed phenotype in basal-like breast cancers, SALL4 is capable of inducing cell dispersion with a reduction in CDH1 expression and an increase in the transcription of mesenchymal genes in epithelial cells. Given that SALL4 up-regulates the ZEB1 transcription ( Figs. 2A and E, 4H, 5H), and that ZEB1 suppresses the CDH1 transcription ( Fig. 2K) [2], the down-regulation of CDH1 was likely to be caused by an ectopic activation of SALL4-ZEB1 network. Since loss of CDH1 function diminishes intercellular adhesiveness in HMLE [25]CDH1 suppression by the SALL4-ZEB1 network might result in a loss of adhesiveness. We observed identical effects between the SALL4A and SALL4B overexpressions in HMLE, indicating that the regulation of cell dispersion is a fundamental function of SALL4.

SALL4 suppresses cell–cell adhesion to maintain cell dispersion in basal-like breast cancer

Cells having shSALL4 expression exhibited compacted clusters (Fig. 1D and F), suggesting that SALL4 knockdown changes cell behavior. Time-lapse microscopy is utilized to explore cell movements. We performed the time-lapse analyses from 1 to 2 days after starting incubation with DOX in which compaction is initiated in our SALL4 knockdown system. The up-regulation of CDH1 transcription is also initiated between 1 and 2 days post DOX administration ( Fig. 3C). The no-DOX controls repeated contact and dispersion ( Movie S1). For instance, as shown in Fig. 6A top, one cell interacted with another cell at time point 20 min, and the contact was preserved until 120 min. At 140 min, the cells were uncoupled. Subsequently, one uncoupled cell collided with the other cell at 160 min, and dispersed immediately. In comparison to the control, the contacting period of the cell having shSALL4 expression was extended ( Fig. 6A bottom, Movie S2). Cells were interacted at 20 min, and adhered. This contact was persisted for longer than 200 min.

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Fig. 6. Attenuation of the dispersion ability in shSALL4-expressing SUM159

For further understanding of the behavior, we compared the frequencies of cells immediately dispersed, dispersed in 1, 3 and 5 h, and adhered longer than 5 h after cell–cell interaction (Fig. 6B). Most of the control cells were dispersed within 5 h after interaction (78.11%). In shSALL4-expressing cells, the frequencies of dispersion within 5 h were decreased (48.12%), and the rate of formation of intercellular adhesion was increased (21.89–51.88%). In addition, we analyzed the frequencies after cell division (Fig. 6C). Similarly to the results of after cell–cell interaction, the frequencies of dispersion within 5 h were reduced (24.57–5.31%). These suggest that SALL4 knockdown impairs the dispersion ability by enhancing intercellular adhesiveness.

We asked whether shSALL4-expressing cells do not disperse from highly compacted clusters, and whether the compacted clusters move around. We performed the wound healing assay in 80–90% confluent cultures with the proliferation inhibitor mitomycin C (Fig. 7). Because the proliferative ability is different between the control and shSALL4-expressing cells (Fig. S1), inhibition of proliferation was demanded to count the exact number of cells moved into the scratched areas, and to analyze the dispersion ability in the wound healing assay. To determine the concentration of mitomycin C, we performed a growth assay, and found that 0.5 μg/ml of it sufficiently inhibited cell proliferation (Fig. S8). In controls, cells were dispersed and filled the scratched areas (Fig. 7E–G). However the number of cells in the scratched areas was significantly reduced in shSALL4-expressing cells (Fig. 7H and I), indicating that shSALL4-expressing cells do not disperse from their cluster, and that the compacted cluster is immobile. We, moreover, analyzed the speeds of movement in the time-lapse movies used for the analyses shown inFig. 6. The mean and maximum speeds were not changed between single cells with and without shSALL4 expression (Fig. 8A and B, single). We also analyzed the motility of cells in contact with other cell(s). Although the no-DOX control had an identical moving speed to single cells, shSALL4-expressing cells showed reduced moving speeds (Fig. 8A and B contacting). The attenuation of the motility observed in contacting shSALL4-expressing cells is consistent with the results of the wound healing assay, and suggests that the trigger to lose cell motility is intercellular adhesion.

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Fig. 7. Loss of migratory ability in compacted shSALL4-expressing SUM159.

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Fig. 8. Attenuation of the motility in contacting shSALL4-expressing SUM159.

We showed that the ability of cell–cell adhesion after interaction and cell division was enhanced in shSALL4-expressing cells (Fig. 6A–C). In epithelial cells, the adhesion protein E-cadherin localizes in the contacting area to form cell–cell adhesion after interaction [26]. Our immunostaining for E-cadherin showed strong signals in the contacting areas (Fig. 1O), supporting the notion that cell–cell adhesion is enhanced by SALL4 knockdown. As exemplified by the wound healing assay and the analysis of moving speeds (Fig. 7 and Fig. 8), intercellular adhesion observed in shSALL4-expressing cells persists for more than 24 h, and adhered cells loses their motility. Accumulation of the low-motile adhered cells could develop to compacted clusters. Taken together, SALL4 functions to suppress the formation of cell–cell adhesion to preserve cell motility when cells interact, which contributes to the dispersed phenotype.

In this study, we identified SALL4 as the cell dispersion factor. SALL4 suppresses the adhesion geneCDH1, and positively regulates the CDH1 suppressor ZEB1. Consistent with the previous study [5], basal-like breast cancer having shSALL4-induced CDH1 expression lost the dispersed phenotype. The STAT3 inhibitor impairs the dispersion ability in glioma cells [27]. Given that STAT3 is a positive regulator forSALL4 transcription in breast cancer [7], our findings are in agreement with the report in glioma cells. Dispersion from an adhesive cluster is one of the characteristics of metastatic cancer [28]. Some of compacted cancer cells acquire the motility and migrate from a cluster to a distant site. Similar events are known in other research fields, such as migratory neural crest cells in development [29] and cardiomyocyte migration in regeneration [30]. Therefore, this study might not only contribute to therapies for cancer metastasis, but also facilitate understanding of the nature of cell migration.

7.11.7 The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma

Zeng SS1Yamashita T2Kondo M1Nio K1Hayashi T1Hara Y1, et al.
J Hepatol. 2014 Jan; 60(1):127-34
http://dx.doi.org:/10.1016/j.jhep.2013.08.024

Background & Aims: Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. We investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. Methods: Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. Results: The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells.  Conclusions: SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features.

7.11.8 Overexpression of the novel oncogene SALL4 and activation of the Wnt.β-catenin pathway in myelodysplastic syndromes

Shuai X1Zhou DShen TWu YZhang JWang XLi Q.
Cancer Genet Cytogenet. 2009 Oct 15; 194(2):119-24
http://dx.doi.org:/10.1016/j.cancergencyto.2009.06.006

Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell diseases with a tendency to progress to leukemic transformation. The cytogenetic and molecular pathogenesis of MDS has not been well understood. SALL4, a newly identified oncogene, modulates stem cell pluripotency and self-renewal capability in embryonic development and also plays a role in leukemogenesis. Overexpression of SALL4 induces MDS-like features and subsequent leukemic progression in transgenic mice. Here, we examined SALL4 expression levels in bone marrow mononuclear cells from MDS patients, acute myeloid leukemia (AML) patients, and normal control subjects using a semiquantitative reverse transcription polymerase chain reaction. Higher levels of SALL4 expression were seen in MDS and AML samples than in control samples. The expression level of SALL4 positively correlated with those of MYC and CCND1, both of which are downstream target genes in the Wnt/beta-catenin pathway. We therefore propose that SALL4 plays a critical role in the pathogenesis of MDS by causing the aberrant activation of the Wnt/beta-catenin pathway.

Comments:

  1. sjwilliamspa

    Would be good to talk about how different Wnt isoforms activate either the noncanonical or canonical pathways in different cancer types. Different Wnts have different specificities for different tissues and activate different pathways. For reference see work by Rugang Zhang in ovarian.

  2. In addition it would be good to find literature on why, after nearly a decade, drug development strategies against the Wnt pathway, have never come to fruition, much like the early days of the farnesylation inhibitors. I believe the early inhibitors were too toxic. In addition, in relation to the OMP trial, Wnt inhibitors target the cancer stem cell and results showing a few months benefit in survival.

    good review on current status of Wnt inhibitor development
    http://link.springer.com/chapter/10.1007/978-1-4419-8023-6_9

    also this is a reference which should be linked to great article by Emma Hill
    http://www.onclive.com/publications/oncology-live/2012/december-2012/wnt-signaling-inhibition-will-decades-of-effort-be-fruitful-at-last/2

    shows all the trials and tribulations of a decade worth of effort.

Date: 28 Dec 2010
Inhibiting the Wnt Signaling Pathway with Small Molecule
Wnt signaling plays important roles in embryonic development and in maintenance of adult tissues. Mutation, loss, or overexpression of key Wnt pathway components has been linked to various types of cancer. Therefore, inhibition of Wnt signaling is of interest for the development of novel anticancer agents. The results of recent structure-based screening, high-throughput screening (HTS), and chemical genomics studies demonstrate that small molecules, including synthetic and natural compounds, can inhibit Wnt signaling in various cancers by blocking specific protein–protein interactions or the activity of specific enzymes. In biological studies, these compounds appear promising as potential anticancer agents; however, their efficacy and toxicity have yet to be investigated. Small molecule inhibitors of Wnt signaling also have wide-ranging potential as tools for elucidating disease and basic biology. Indubitably, in the near future, these compounds will yield agents that are clinically useful against malignant diseases.

Wnt Signaling Inhibition: Will Decades of Effort Be Fruitful at Last?

Emma Hitt, PhD

Oncology Live  Published Online: Monday, January 7, 2013

The Wnt signaling pathway was first characterized in the 1970s in Drosophila melanogaster development. It was later recognized in mammalian systems for its importance in cancer. Specifically, core components of this pathway were shown to be dysregulated in colorectal disease. It has since been shown to play a role in other forms of cancer, where it promotes proliferation and survival. It also plays an important role in the maintenance of the pool of tumor-initiating cells, which promote the regrowth of tumors after an insult like surgery or chemotherapy. Tumor-initiating cells are thought to be the source of metastasis. For these reasons, the Wnt pathway is viewed as a strong candidate for therapeutic intervention.

Wnt Signaling in Cancer

The Wnt pathway takes on many forms that fall under the broad classifications of canonical and noncanonical. The canonical pathway deals with the regulation of β-catenin protein levels. Under normal conditions, a cytosolic scaffold known as the destruction complex binds and phosphorylates β-catenin, resulting in its ubiquitylation and degradation. The destruction complex includes the adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase-3β. When Wnt ligand binds to the Frizzled receptor, its coreceptor LRP5/6 is recruited and phosphorylated in the intracellular domain, promoting the binding of Dishevelled protein and the sequestration of axin. This disintegrates the destruction complex, resulting in accumulation of β-catenin in the cytosol and upregulated trafficking into the nucleus. β-catenin promotes transcription of genes related to proliferation and survival by acting as a coactivator for the Tcf/Lef family of transcription factors in the nucleus.

Aside from canonical Wnt signaling, two major noncanonical pathways have been studied. In the first of these two pathways, Wnt ligand binding to the Frizzled receptor induces recruitment of Dishevelled protein and the Dishevelled-associated activator of morphogenesis 1 (Daam1). This complex initiates a cascade that activates the Rac and Rho GTPases to mediate cell polarity. The other most widely studied noncanonical Wnt signaling pathway is related to calcium signaling. Wnt ligand binding to the Frizzled receptor promotes the recruitment of Dishevelled in complex with a G protein. This complex promotes intracellular calcium levels to mediate other signaling pathways.

Biological systems tightly regulate the Wnt signaling pathway to prevent aberrant cell growth. It has been known for decades that dysregulation of Wnt signaling leads to cancer, where it was first recognized in familial colorectal disease with mutations in the APC gene. Since then, Wnt signaling has been found to act prominently in breast, liver, skin, and prostate cancers.

Aberrations in canonical Wnt signaling can manifest in many ways. For example, proteins involved in the destruction complex can become nonfunctional through mutations or truncations, inhibiting β-catenin downregulation. The β-catenin protein itself can be mutated to inhibit its recognition by the destruction complex. In addition, the production of Wnt ligand or receptors can be upregulated, resulting in excessive signaling. These different routes of activation complicate the use of a single therapeutic against the Wnt pathway.

Wnt Signaling Pathways

This illustration depicts the best-elucidated cancer-promoting routes of Wnt cell signaling, which draws its name from the wingless mutation in the fruitfly Drosophila melanogaster.

Wnt-Signaling cancer-promoting routes

Wnt-Signaling cancer-promoting routes

Therapeutic Targeting of Wnt Pathway

Wnt signaling is of great interest to cancer researchers because it is linked to many different forms of the disease. Preclinical models throughout the last decade have established this pathway as an attractive drug target. However, to date, therapies meant to attenuate the Wnt pathway have remained largely theoretical and preclinical. Thankfully, compounds are now starting to enter clinical trials.

Vitamin D has been postulated as a suitable anti-Wnt therapy. The vitamin D receptor binds and sequesters β-catenin at the plasma membrane, inhibiting its nuclear translocation. Mice bearing an APC mutation that promotes spontaneous colon cancer developed more disease when the vitamin D receptor was knocked out. Additionally, the association between sunlight exposure and decreased risk of colon cancer implies that the inhibition of Wnt signaling by vitamin D may be conserved in humans. This phenomenon has yet to be tested in a systematic trial, however.

High-throughput screens have been used extensively to identify small-molecule targeted inhibitors of different Wnt pathway constituents. The binding of β-catenin to its nuclear targets T-cell factor (Tcf) or Creb-binding protein (CBP) is a prototypical example of study in this realm. Screens were performed to identify specific inhibitors of this interaction. This work has identified useful hits in cell-based assays, but translation into the clinic has remained difficult due to the potential off-target effects. First, the drugs identified so far fail to discriminate between the binding of β-catenin to Tcf or APC, so the drug may prevent degradation of β-catenin in addition to its intended effect on transcription. Disruption of the destruction complex binding to β-catenin could lead to severe side effects in normal tissue. Another deleterious effect identified with blockers of β-catenin binding is the inhibition of complex formation with E-cadherin at cell-cell junctions. The net effect of this phenomenon could be to impair cell adhesion on a grand scale. As a result of these challenges, only one compound that directly disrupts β-catenin function has moved into clinical trials.

Other drug candidates inhibit Wnt aberrations upstream of β-catenin function. Several of these compounds have recently begun clinical trials. They block either Wnt ligand secretion or recognition, and preclinical evidence has been encouraging so far. Targeting at this level can also lead to side effects, however. By blocking Wnt signaling at the level of the membrane, it is possible to inhibit noncanonical in addition to canonical signaling. The effect this will have on the body is unknown. In addition, blocking Wnt ligand-receptor interactions may not be sufficient to inhibit Wnt signaling since the activating event may be mutation within the destruction complex or β-catenin itself. In these settings, Wnt signaling can be rendered constitutive and independent of ligand, and the therapy would likely fail.

Moving Into the Clinic

No approved compounds exist for the treatment of Wnt signaling. Phase I trials for these inhibitors should be illustrative in the coming years. While many cancers are addicted to this signaling for long-term growth and renewal, high-turnover tissues like the gastric epithelium and hair follicles are similarly reliant. Therefore, the field is cautious when utilizing any blockade of Wnt signaling, as significant toxicity may result.

If Wnt pathway inhibitors can be proven safe, it may represent a milestone in cancer research given the strong preclinical evidence for cancer cell cytotoxicity. Since this pathway is also crucial in the maintenance of tumor-initiating cells, inhibition could represent a powerful tool in our arsenal to target cells that are resistant to traditional chemotherapy and promote metastasis.

Wnt-Targeting Compounds in Development

Phase I/II Trials
OMP-18R5
(OncoMed Pharmaceuticals/ Bayer)
This monoclonal antibody targets the Frizzled receptors to block association with Wnt ligands. It was recently shown to potently block the capabilities of pancreatic tumor-initiating cells in a serial dilution assay. In xenograft models of breast, lung, pancreatic, and colon cancer, OMP-18R5 demonstrated significant inhibition of tumor growth, and it synergized with standard-of-care treatment in these models (paclitaxel in breast cancer, for example). (NCT01345201)
OMP-54F28
(OncoMed Pharmaceuticals/ Bayer)
This agent is a fusion protein of the Frizzled8 ligand-binding domain with the Fc region of a human immunoglobulin. It binds and sequesters soluble Wnt ligand, impairing its recognition by receptors on tissues. (NCT01608867)
PRI-724
(Prism Pharma Co, Ltd/Eisai)
This is a small-molecule inhibitor of the interaction between β-catenin and CBP. Disrupting the interaction prevents activated transcription by aberrant Wnt signaling at many levels. It is being studied in both solid tumors and myeloid malignancies. (NCT01606579, NCT01302405)
LGK974
(Novartis Pharmaceuticals)
This small molecule inhibits acyltransferase porcupine. Preclinical work demonstrated this enzyme’s action is crucial in the secretion of Wnt ligands out of the cell; therefore, inhibiting porcupine can be a small-molecule–based method for inhibiting Wnt ligand-mediated activation. (NCT01351103)
Preclinical Studies
XAV939
(Novartis Pharmaceuticals)
This small-molecule poly(ADP ribose) polymerase (PARP) inhibitor has demonstrated efficacy in cellular models of cancer survival. In Wnt signaling, PARPs like the tankyrases promote the ribosylation and subsequent degradation of axin, a key scaffolding protein of the destruction complex. By inhibiting tankyrase, the axin protein is stabilized and can promote the degradation of β-catenin.
JW55
(Tocris Bioscience)
This selective tankyrase1/2 inhibitor has been shown to inhibit the growth of colon cancer cells in both cell and animal models.

Sources: ClinicalTrials.gov website, company websites.

Key Research

  • Chen B, Dodge ME, Tang W, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer [published online ahead of print January 4, 2009]. Nat Chem Biol. 2009;5(2):100–107. doi: 10.1038/nchembio.137.
  • Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192-1205.
  • Eckhardt SG. Targeting the WNT Pathway for Cancer Therapy. Presented at: 10th International Congress on Targeted Therapies in Cancer; August 17-18, 2012; Washington, DC.
  • He B, Reguart N, You L, et al. Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations. Oncogene. 2005;24(18):3054-3058.
  • Ichii S, Horii A, Nakatsuru S, et al. Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis (FAP). Hum Mol Genet. 1992;1(6):387-390.
  • Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science. 1997;275(5307):1784-1787.
  • Rubinfeld B, Souza B, Albert I, et al. Association of the APC gene product with beta-catenin. Science. 1993;262(5140):1731-1734.

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Pathway Specific Targeting in Anticancer Therapies

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

7.7 Pathway specific targeting in anticancer therapies

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

 

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Thangavelua, CQ Pana, …, BC Lowa, and J. Sivaramana
Proc Nat Acad Sci 2012; 109(20):7705–7710
http://dx.doi.org:/10.1073/pnas.1116573109

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA’s active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

The Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (12). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (23). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (25).

Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly, direct suppression of miR23a and miR23b (9) or activation of TGF-β (10) enhances KGA expression. Similarly, Rho GTPase that controls cytoskeleton and cell division also up-regulates KGA expression in an NF-κB–dependent manner (11). In addition, KGA is a substrate for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1, linking glutaminolysis to cell cycle progression (12). In comparison, function and regulation of LGA is not well studied, although it was recently shown to be linked to p53 pathway (1314). Although intense efforts are being made to develop a specific KGA inhibitor such as BPTES [bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] (15), its mechanism of inhibition and selectivity is not yet understood. Equally important is to understand how KGA function is regulated in normal and cancer cells so that a better treatment strategy can be considered.

The previous crystal structures of microbial (Mglu) and Escherichia coli glutaminases show a conserved catalytic domain of KGA (1617). However, detailed structural information and regulation are not available for human glutaminases especially the KGA, and this has hindered our strategies to develop inhibitors. Here we report the crystal structure of the catalytic domain of human apo KGA and its complexes with substrate (L-glutamine), product (L-glutamate), BPTES, and its derived inhibitors. Further, Raf-Mek-Erk module is identified as the regulator of KGA activity. Although BPTES is not recognized in the active site, its binding confers a drastic conformational change of a key loop (Glu312-Pro329), which is essential in stabilizing the catalytic pocket. Significantly, EGF activates KGA activity, which can be abolished by the kinase-dead, dominant negative mutants of Mek2 (Mek2-K101A) or its upstream activator Raf-1 (Raf-1-K375M), which are the kinase components of the growth-promoting Raf-Mek2-Erk signaling node. Furthermore, coexpression of phosphatase PP2A and treatment with Mek-specific inhibitor or alkaline phosphatase all abolished enhanced KGA activity inside the cells and in vitro, indicating that stimulation of KGA is phosphorylation dependent. Our results therefore provide mechanistic insights into KGA inhibition by BPTES and its regulation by EGF-mediated Raf-Mek-Erk module in cell growth and possibly cancer manifestation.

Structures of cKGA and Its Complexes with L-Glutamine and L-Glutamate.
The human KGA consists of 669 amino acids. We refer to Ile221-Leu533 as the catalytic domain of KGA (cKGA) (Fig. 1A). The crystal structures of the apo cKGA and in complex with L-glutamine or L-glutamate were determined (Table S1). The structure of cKGA has two domains with the active site located at the interface. Domain I comprises (Ile221-Pro281 and Cys424 -Leu533) of a five-stranded anti-parallel β-sheet (β2↓β1↑β5↓β4↑β3↓) surrounded by six α-helices and several loops. The domain II (Phe282-Thr423) mainly consists of seven α-helices. L-Glutamine/L-glutamate is bound in the active site cleft (Fig. 1B and Fig. S1B). Overall the active site is highly basic, and the bound ligand makes several hydrogen-bonding contacts to Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Fig. 1C and Fig. S1C), and these residues are highly conserved among KGA homologs (Fig. S1D). Notably, the putative serine-lysine catalytic dyad (286-SCVK-289), corresponding to the SXXK motif of class D β-lactamase (17), is located in close proximity to the bound ligand. In the apo structure, two water molecules were located in the active site, one of them being displaced by glutamine in the substrate complex. The substrate side chain is within hydrogen-bonding distance (2.9 Å) to the active site Ser286. Other key residues involved in catalysis, such as Lys289, Tyr414, and Tyr466, are in the vicinity of the active site. Lys289 is within hydrogen-bonding distance to Ser286 (3.1 Å) and acts as a general base for the nucleophilic attack by accepting the proton from Ser286. Tyr466, which is close to Ser286 and in hydrogen-bonding contact (3.2 Å) with glutamine, is involved in proton transfer during catalysis. Moreover, the carbonyl oxygen of the glutamine is hydrogen-bonded with the main chain amino groups of Ser286 and Val484, forming the oxyanion hole. Thus, we propose that in addition to the putative catalytic dyad (Ser286 XX Lys289), Tyr466 could play an important role in the catalysis (Fig. 1Cand Fig. S2).

structure of the cKGA-L-glutamine complex

structure of the cKGA-L-glutamine complex

http://www.pnas.org/content/109/20/7705/F1.medium.gif

Fig. 1.  Schematic view and structure of the cKGA-L-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (L-glutamine) is shown as a cyan stick. (C) Fourier 2Fo-Fc electron density map (contoured at 1 σ) for L-glutamine, that makes hydrogen bonds with active site residues are shown.

Allosteric Binding Pocket for BPTES. The chemical structure of BPTES has an internal symmetry, with two exactly equivalent parts including a thiadiazole, amide, and a phenyl group (Fig. S3A), and it equally interacts with each monomer. The thiadiazole group and the aliphatic linker are well buried in a hydrophobic cluster that consists of Leu321, Phe322, Leu323, and Tyr394 from both monomers, which forms the allosteric pocket (Fig. 2 B–E). The side chain of Phe322 is found at the bottom of the allosteric pocket. The phenyl-acetamido moiety of BPTES is partially exposed on the loop (Asn324-Glu325), where it interacts with Phe318, Asn324, and the aliphatic part of the Glu325 side chain. On the basis of our observations we synthesized a series of BPTES-derived inhibitors (compounds2–5) (Fig. S3 AF and SI Results) and solved their cocrystal structure of compounds 2–4. Similar to BPTES, compounds 24 all resides within the hydrophobic cluster of the allosteric pocket (Fig. S3 CF).

Fig. 2. Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES.

Allosteric Binding of BPTES Triggers Major Conformational Change in the Key Loop Near the Active Site.  The overall structure of these inhibitor complexes superimposes well with apo cKGA. However, a major conformational change at the Glu312 to Pro329 loop was observed in the BPTES complex (Fig. 2F). The most conformational changes of the backbone atoms that moved away from the active site region are found at the center of the loop (Leu316-Lys320). The backbone of the residues Phe318 and Asn319 is moved ≈9 Å and ≈7 Å, respectively, compared with the apo structure, whereas the side chain of these residues moved ≈14 Å and ≈12 Å, respectively. This loop rearrangement in turn brings Phe318 closer to the phenyl group of the inhibitor and forms the inhibitor binding pocket, whereas in the apo structure the same loop region (Leu316-Lys320) was found to be adjacent to the active site and forms a closed conformation of the active site.

Binding of BPTES Stabilizes the Inactive Tetramers of cKGA.  To understand the role of oligomerization in KGA function, dimers and tetramers of cKGA were generated using the symmetry-related monomers (Fig. 2 A–E and Fig. S4 D and E). The dimer interface in the cKGA: BPTES complex is formed by residues from the helix Asp386-Lys398 of both monomers and involves hydrogen bonding, salt bridges, and hydrophobic interactions (Phe389, Ala390, Tyr393, and Tyr394), besides two sulfate ions located in the interface (Fig. 2E). The dimers are further stabilized by binding of BPTES, where it binds to loop residues (Glu312-Pro329) and Tyr394 from both monomers (Fig. 2 D and E). Similarly, residues from Lys311-Asn319 loop and Arg454, His461, Gln471, and Asn529-Leu533 are involved in the interface with neighboring monomers to form the tetramer in the BPTES complex.

BPTES Induces Allosteric Conformational Changes That Destabilize Catalytic Function of KGA

Fig. 3A shows that 293T cells overexpressing KGA produced higher level of glutamate compared with the vector control cells. Most significantly, all of these mutants, except Phe322Ala, greatly diminished the KGA activity.

Fig. 3. Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity.

Raf-Mek-Erk Signaling Module Regulates KGA Activity. Because KGA supports cell growth and proliferation, we first validated that treatment of cells with BPTES indeed inhibits KGA activity and cell proliferation (Fig. S5 A–D and SI Results). Next, as cells respond to various physiological stimuli to regulate their metabolism, with many of the metabolic enzymes being the primary targets of modulation (18), we examined whether KGA activity can be regulated by physiological stimuli, in particular EGF, which is important for cell growth and proliferation. Cells overexpressing KGA were made quiescent and then stimulated with EGF for various time points. Fig. 4A shows that the basal KGA activity remained unchanged 30 min after EGF stimulation, but the activity was substantially enhanced after 1 h and then gradually returned to the basal level after 4 h. Because EGF activates the Raf-Mek-Erk signaling module (19), treatment of cells with Mek-specific inhibitor U0126 could block the enhanced KGA activity with parallel inhibition of Erk phosphorylation (Fig. 4A). Interestingly, such Mek-induced KGA activity is specific to EGF and lysophosphatidic acid (LPA) but not with other growth factors, such as PDGF, TGF-β, and basic FGF (bFGF), despite activation of Mek-Erk by bFGF (Fig. S6A).

The results show that KGA could interact equally well with the wild-type or mutant forms of Raf-1 and Mek2 (Fig. 4C). Importantly, endogenous Raf-1 or Erk1/2, including the phosphorylated Erk1/2 (Fig. 4 C and D), could be detected in the KGA complex. Taken together, these results indicate that the activity of KGA is directly regulated by Raf-Mek-Erk downstream of EGF receptor. To further show that Mek2-enhanced KGA activity requires both the kinase activity of Mek2 and the core residues for KGA catalysis, wild-type or triple mutant (Leu321Ala/Phe322Ala/Leu323Ala) of KGA was coexpressed with dominant negative Mek2-KA or the constitutive active Mek2-SD and their KGA activities measured. The result shows that the presence of Mek2-KA blocks KGA activity, whereas the triple mutant still remains inert even in the presence of the constitutively active Mek2 (Fig. 4E), and despite Mek2 binding to the KGA triple mutant (Fig. S7B). Consequently, expressing triple mutant did not support cell proliferation as well as the wild-type control (Fig. S7C).

Fig. 4. EGFR-Raf-Mek-Erk signaling stimulates KGA activity.

When cells expressing both KGA and Mek2-K101A were treated with subthreshold levels of BPTES, there was a synergistic reduction in cell proliferation (Fig. S6C and SI Results). Lastly, to determine whether regulation of KGA by Raf-Mek-Erk depends on its phosphorylation status, cells were transfected with KGA with or without the protein phosphatase PP2A and assayed for the KGA activity. PP2A is a ubiquitous and conserved serine/threonine phosphatase with broad substrate specificity. The results indicate that KGA activity was reduced down to the basal level in the presence of PP2A (Fig. 5A). Coimmunoprecipitation study also revealed that KGA interacts with PP2A (Fig. 5B), suggesting a negative feedback regulation by this protein phosphatase. Furthermore, treatment of immunoprecipitated and purified KGA with calf-intestine alkaline phosphatase (CIAP) almost completely abolished the KGA activity in vitro (Fig. S6D). Taken together, these results indicate that KGA activity is regulated by Raf-Mek2, and KGA activation by EGF could be part of the EGF-stimulated Raf-Mek-Erk signaling program in controlling cell growth and proliferation (Fig. 5C).

KGA activity is regulated by phosphorylation

KGA activity is regulated by phosphorylation

http://www.pnas.org/content/109/20/7705/F5.medium.gif

Fig. 5. KGA activity is regulated by phosphorylation. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation.

Small-molecule inhibitors that target glutaminase activity in cancer cells are under development. Earlier efforts targeting glutaminase using glutamine analogs have been unsuccessful owing to their toxicities (2). BPTES has attracted much attention as a selective, nontoxic inhibitor of KGA (15), and preclinical testing of BPTES toward human cancers has just begun (20). BPTES selectively suppresses the growth of glioma cells (21) and inhibits the growth of lymphoma tumor growth in animal model studies (22). Wang et al. (11) reported a small molecule that targets glutaminase activity and oncogenic transformation. Despite extensive studies, nothing is known about the structural and molecular basis for KGA inhibitory mechanisms and how their function is regulated during normal and cancer cell metabolism. Such limited information impedes our effort in producing better generations of inhibitors for better treatment regimens.

Comparison of the complex structures with apo cKGA structure, which has well-defined electron density for the key loop, we provide the atomic view of an allosteric binding pocket for BPTES and elucidate the inhibitory mechanism of KGA by BPTES. The key residues of the loop (Glu312-Pro329) undergo major conformational changes upon binding of BPTES. In addition, structure-based mutagenesis studies suggest that this loop is essential for stabilizing the active site. Therefore, by binding in an allosteric pocket, BPTES inhibits the enzymatic activity of KGA through (i) triggering a major conformational change on the key residues that would normally be involved in stabilizing the active sites and regulating its enzymatic activity; and (ii) forming a stable inactive tetrameric KGA form. Our findings are further supported by two very recent reports on KGA isoform (GAC) (2324), although these studies lack full details owing to limitation of their electron density maps. BPTES is specific to KGA but not to LGA (15). Sequence comparison of KGA with LGA (Fig. S8A) reveals two unique residues on KGA, Phe318 and Phe322, which upon mutation to LGA counterparts, become resistant to BPTES. Thus, our study provides the molecular basis of BPTES specificity.

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Islam SS, Mokhtari RB, Noman AS, …, van der Kwast T, Yeger H, Farhat WA.
Molec Carcinogenesis mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22300

shh sonic hedgehog signaling pathway nri2151-f1

shh sonic hedgehog signaling pathway nri2151-f1

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis

Sonic_hedgehog_pathway

Sonic_hedgehog_pathway

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Gaikwad SM, Thakur B, Sakpal A, Singh RK, Ray P.
Int J Biochem Cell Biol. 2015 Apr; 61:90-102
http://dx.doi.org:/10.1016/j.biocel.2015.02.001

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

Kuboki MIto ASimizu SUmezawa K.
Biochem Biophys Res Commun. 2015 Jan 16; 456(3):810-4
http://dx.doi.org:/10.1016/j.bbrc.2014.12.024

Activation of caspase 3 and caspase-dependent apoptosis  nrmicro2071-f1

Activation of caspase 3 and caspase-dependent apoptosis nrmicro2071-f1

Highlights

  • We have prepared RelB mutants that are resistant to caspase 3-induced scission.
  • Vinblastine induced caspase 3-dependent site-specific RelB cleavage in cancer cells.
  • Cancer cells expressing cleavage-resistant RelB showed less sensitivity to vinblastine.
  • Caspase 3-induced RelB cleavage may provide positive feedback mechanism in apoptosis.

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

apoptotic-caspases-control-microglia-activation-cdd2011107f3

apoptotic-caspases-control-microglia-activation-cdd2011107f3

 

 

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

He GDhar DNakagawa HFont-Burgada JOgata HJiang Y, et al.
Cell. 2013 Oct 10; 155(2):384-96
http://dx.doi.org/10.1016%2Fj.cell.2013.09.031

Il-6 signaling in cancer cells

Il-6 signaling in cancer cells

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from pre-malignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Clonal evolution and selective pressure may cause some descendants of the initial progenitor to cross the bridge of no return and form a premalignant lesion. Cancer genome sequencing indicates that most cancers require at least five genetic changes to evolve (Wood et al., 2007). It has been difficult to isolate and propagate cancer progenitors prior to detection of tumor masses. Further, it is not clear whether cancer progenitors are the precursors for the  cancer stem cells (CSCs)isolated from cancers. An answer to these critical questions depends on identification and isolation of cancer progenitors, which may also enable definition of molecular markers and signaling pathways suitable for early detection and treatment.

Hepatocellular carcinoma (HCC), the end product of chronic liver diseases, requires several decades to evolve (El-Serag, 2011). It is the third most deadly and fifth most common cancer worldwide, and in the United States its incidence has doubled in the past two decades. Furthermore, 8% of the world’s population are chronically infected with hepatitis B or C viruses (HBV and HCV) and are at a high risk of new HCC development (El-Serag, 2011). Up to 5% of HCV patients will develop HCC in their lifetime, and the yearly HCC incidence in patients with cirrhosis is 3%–5%. These tumors may arise from premalignant lesions, ranging from dysplastic foci to dysplastic hepatocyte nodules that are often seen in damaged and cirrhotic livers and are more proliferative than the surrounding parenchyma (Hytiroglou et al., 2007). There is no effective treatment for HCC and, upon diagnosis, most patients with advanced disease have a remaining lifespan of 4–6 months. Premalignant lesions, called foci of altered hepatocytes (FAH), were described in chemically induced HCC models (Pitot, 1990), but it was questioned whether these lesions harbor tumor progenitors or result from compensatory proliferation (Sell and Leffert, 2008). The aim of this study was to determine whether HCC progenitor cells (HcPCs) exist and if so, to isolate these cells and identify some of the signaling networks that are involved in their maintenance and progression.

We now describe HcPC isolation from mice treated with the procarcinogen diethyl nitrosamine (DEN), which induces poorly differentiated HCC nodules within 8 to 9 months (Verna et al., 1996). The use of a chemical carcinogen is justified because the finding of up to 121 mutations per HCC genome suggests that carcinogens may be responsible for human HCC induction (Guichard et al., 2012). Furthermore, 20%–30% of HCC, especially in HBV-infected individuals, evolve in noncirrhotic livers (El-Serag, 2011). Nonetheless, we also isolated HcPCs fromTak1Δhep mice, which develop spontaneous HCC as a result of progressive liver damage, inflammation, and fibrosis caused by ablation of TAK1 (Inokuchi et al., 2010). Although the etiology of each model is distinct, both contain HcPCs that express marker genes and signaling pathways previously identified in human HCC stem cells (Marquardt and Thorgeirsson, 2010) long before visible tumors are detected. Furthermore, DEN-induced premalignant lesions and HcPCs exhibit autocrine IL-6 production that is critical for tumorigenic progression. Circulating IL-6 is a risk indicator in several human pathologies and is strongly correlated with adverse prognosis in HCC and cholangiocarcinoma (Porta et al., 2008Soresi et al., 2006). IL-6 produced by in-vitro-induced CSCs was suggested to be important for their maintenance (Iliopoulos et al., 2009). Little is known about the source of IL-6 in HCC.

DEN-Induced Collagenase-Resistant Aggregates of HCC Progenitors

A single intraperitoneal (i.p.) injection of DEN into 15-day-old BL/6 mice induces HCC nodules first detected 8 to 9 months later. However, hepatocytes prepared from macroscopically normal livers 3 months after DEN administration already contain cells that progress to HCC when transplanted into the permissive liver environment of MUP-uPA mice (He et al., 2010), which express urokinase plasminogen activator (uPA) from a mouse liver-specific major urinary protein (MUP) promoter and undergo chronic liver damage and compensatory proliferation (Rhim et al., 1994). HCC markers such as α fetoprotein (AFP), glypican 3 (Gpc3), and Ly6D, whose expression in mouse liver cancer was reported (Meyer et al., 2003), were upregulated in aggregates from DEN-treated livers, but not in nonaggregated hepatocytes or aggregates from control livers (Figure S1A). Using 70 μm and 40 μm sieves, we separated aggregated from nonaggregated hepatocytes (Figure 1A) and tested their tumorigenic potential by transplantation into MUP-uPA mice (Figure 1B). To facilitate transplantation, the aggregates were mechanically dispersed and suspended in Dulbecco’s modified Eagle’s medium (DMEM). Five months after intrasplenic (i.s.) injection of 104 viable cells, mice receiving cells from aggregates developed about 18 liver tumors per mouse, whereas mice receiving nonaggregated hepatocytes developed less than 1 tumor each (Figure 1B). The tumors exhibited typical trabecular HCC morphology and contained cells that abundantly express AFP (Figure S1B).

Only liver tumors were formed by the transplanted cells. Other organs, including the spleen into which the cells were injected, remained tumor free (Figure 1B), suggesting that HcPCs progress to cancer only in the proper microenvironment. Indeed, no tumors appeared after HcPC transplantation into normal BL/6 mice. But, if BL/6 mice were first treated with retrorsine (a chemical that permanently inhibits hepatocyte proliferation [Laconi et al., 1998]), intrasplenically transplanted with HcPC-containing aggregates, and challenged with CCl4 to induce liver injury and compensatory proliferation (Guo et al., 2002), HCCs readily appeared (Figure 1C). CCl4 omission prevented tumor development. Notably, MUP-uPA or CCl4-treated livers are fragile, rendering direct intrahepatic transplantation difficult. CCl4-induced liver damage, especially within a male liver, generates a microenvironment that drives HcPC proliferation and malignant progression. To examine this point, we transplanted GFP-labeled HcPC-containing aggregates into retrorsine-treated BL/6 mice and examined their ability to proliferate with or without subsequent CCl4 treatment. Indeed, the GFP+ cells formed clusters that grew in size only in CCl4-treated host livers (Figure S1E). Omission of CC14 prevented their expansion.

Because CD44 is expressed by HCC stem cells (Yang et al., 2008Zhu et al., 2010), we dispersed the aggregates and separated CD44+ from CD44 cells and transplanted both into MUP-uPA mice. Whereas as few as 103 CD44+ cells gave rise to HCCs in 100% of recipients, no tumors were detected after transplantation of CD44 cells (Figure 1E). Remarkably, 50% of recipients developed at least one HCC after receiving as few as 102 CD44+ cells.

HcPC-Containing Aggregates in Tak1Δhep Mice

We applied the same HcPC isolation protocol to Tak1Δhep mice, which develop HCC of different etiology from DEN-induced HCC. Importantly, Tak1Δhep mice develop HCC as a consequence of chronic liver injury and fibrosis without carcinogen or toxicant exposure (Inokuchi et al., 2010). Indeed, whole-tumor exome sequencing revealed that DEN-induced HCC contained about 24 mutations per 106 bases (Mb) sequenced, with B-RafV637E being the most recurrent, whereas 1.4 mutations per Mb were detected inTak1Δhep HCC’s exome (Table S1). By contrast, Tak1Δhep HCC exhibited gene copy number changes. HCC developed in 75% of MUP-uPA mice that received dispersed Tak1Δhep aggregates, but no tumors appeared in mice receiving nonaggregated Tak1Δhep or totalTak1f/f hepatocytes (Figure 2B). bile duct ligation (BDL) or feeding with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), treatments that cause cholestatic liver injuries and oval cell expansion (Dorrell et al., 2011), did increase the number of small hepatocytic cell aggregates (Figure S2A). Nonetheless, no tumors were observed 5 months after injection of such aggregates into MUP-uPA mice (Figure S2B). Thus, not all hepatocytic aggregates contain HcPCs, and HcPCs only appear under tumorigenic conditions.

The HcPC Transcriptome Is Similar to that of HCC and Oval Cells

To determine the relationship between DEN-induced HcPCs, normal hepatocytes, and fully transformed HCC cells, we analyzed the transcriptomes of aggregated and nonaggregated hepatocytes from male littermates 5 months after DEN administration, HCC epithelial cells from DEN-induced tumors, and normal hepatocytes from age- and gender-matched littermate controls. Clustering analysis distinguished the HCC samples from other samples and revealed that the aggregated hepatocyte samples did not cluster with each other but rather with nonaggregated hepatocytes derived from the same mouse (Figure S3A). 57% (583/1,020) of genes differentially expressed in aggregated relative to nonaggregated hepatocytes are also differentially expressed in HCC relative to normal hepatocytes (Figure 3B, top), a value that is highly significant (p < 7.13 × 10−243). More specifically, 85% (494/583) of these genes are overexpressed in both HCC and HcPC-containing aggregates (Figure 3B, bottom table). Thus, hepatocyte aggregates isolated 5 months after DEN injection contain cells that are related in their gene expression profile to HCC cells isolated from fully developed tumor nodules.

Figure 3 Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells

We examined which biological processes or cellular compartments were significantly overrepresented in the induced or repressed genes in both pairwise comparisons (Gene Ontology Analysis). As expected, processes and compartments that were enriched in aggregated hepatocytes relative to nonaggregated hepatocytes were almost identical to those that were enriched in HCC relative to normal hepatocytes (Figure 3C). Several human HCC markers, including AFP, Gpc3 and H19, were upregulated in aggregated hepatocytes (Figures 3D and 3E). Aggregated hepatocytes also expressed more Tetraspanin 8 (Tspan8), a cell-surface glycoprotein that complexes with integrins and is overexpressed in human carcinomas (Zöller, 2009). Another cell-surface molecule highly expressed in aggregated cells is Ly6D (Figures 3D and 3E). Immunofluorescence (IF) analysis revealed that Ly6D was undetectable in normal liver but was elevated in FAH and ubiquitously expressed in most HCC cells (Figure S3C). A fluorescent-labeled Ly6D antibody injected into HCC-bearing mice specifically stained tumor nodules (Figure S3D). Other cell-surface molecules that were upregulated in aggregated cells included syndecan 3 (Sdc3), integrin α 9 (Itga9), claudin 5 (Cldn5), and cadherin 5 (Cdh5) (Figure 3D). Aggregated hepatocytes also exhibited elevated expression of extracellular matrix proteins (TIF3 and Reln1) and a serine protease inhibitor (Spink3). Elevated expression of such proteins may explain aggregate formation. Aggregated hepatocytes also expressed progenitor cell markers, including the epithelial cell adhesion molecule (EpCAM) (Figure 3E) and Dlk1 (Figure 3D). We compared the HcPC and HCC (Figure 3A) to the transcriptome of DDC-induced oval cells (Shin et al., 2011). This analysis revealed a striking similarity between the HCC, HcPC, and the oval cell transcriptomes (Figure S3B). Despite these similarities, some genes that were upregulated in HcPC-containing aggregates and HCC were not upregulated in oval cells. Such genes may account for the tumorigenic properties of HcPC and HCC.

Figure 4  DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells

We examined the aggregates for signaling pathways and transcription factors involved in hepatocarcinogenesis. Many aggregated cells were positive for phosphorylated c-Jun and STAT3 (Figure 4A), transcription factors involved in DEN-induced hepatocarcinogenesis (Eferl et al., 2003He et al., 2010). Sox9, a transcription factor that marks hepatobiliary progenitors (Dorrell et al., 2011), was also expressed by many of the aggregated cells, which were also positive for phosphorylated c-Met (Figure 4A), a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) and is essential for liver development (Bladt et al., 1995) and hepatocarcinogenesis (Wang et al., 2001). Few of the nonaggregated hepatocytes exhibited activation of these signaling pathways. Despite different etiology, HcPC-containing aggregates from Tak1Δhep mice exhibit upregulation of many of the same markers and pathways that are upregulated in DEN-induced HcPC-containing aggregates. Flow cytometry confirmed enrichment of CD44+ cells as well as CD44+/CD90+ and CD44+/EpCAM+ double-positive cells in the HcPC-containing aggregates from either DEN-treated or Tak1Δhep livers (Figure S4B).

HcPC-Containing Aggregates Originate from Premalignant Dysplastic Lesions

FAH are dysplastic lesions occurring in rodent livers exposed to hepatic carcinogens (Su et al., 1990). Similar lesions are present in premalignant human livers (Su et al., 1997). Yet, it is still debated whether FAH correspond to premalignant lesions or are a reaction to liver injury that does not lead to cancer (Sell and Leffert, 2008). In DEN-treated males, FAH were detected as early as 3 months after DEN administration (Figure 5A), concomitant with the time at which HcPC-containing aggregates were detected. In females, FAH development was delayed. FAH contained cells positive for the same progenitor cell markers and activated signaling pathways present in HcPC-containing aggregates, including AFP, CD44, and EpCAM (Figure 5C). FAH also contained cells positive for activated STAT3, c-Jun, and PCNA (Figure 5C).

HcPCs Exhibit Autocrine IL-6 Expression Necessary for HCC Progression

In situ hybridization (ISH) and immunohistochemistry (IHC) revealed that DEN-induced FAH contained IL-6-expressing cells (Figures 6A, 6B, and S5), and freshly isolated DEN-induced aggregates contained more IL-6 messenger RNA (mRNA) than nonaggregated hepatocytes (Figure 6C). We examined several factors that control IL-6 expression and found that LIN28A and B were significantly upregulated in HcPCs and HCC (Figures 6D and 6E). LIN28-expressing cells were also detected within FAH (Figure 6F). As reported (Iliopoulos et al., 2009), knockdown of LIN28B in cultured HcPC or HCC cell lines decreased IL-6 expression (Figure 6G). LIN28 exerts its effects through downregulation of the microRNA (miRNA) Let-7 (Iliopoulos et al., 2009).

Figure 6  Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression

Figure 7  HCC Growth Depends on Autocrine IL-6 Production

The isolation and characterization of cells that can give rise to HCC only after transplantation into an appropriate host liver undergoing chronic injury demonstrates that cancer arises from progenitor cells that are yet to become fully malignant. Importantly, unlike fully malignant HCC cells, the HcPCs we isolated cannot form s.c. tumors or even liver tumors when introduced into a nondamaged liver. Liver damage induced by uPA expression or CCl4 treatment provides HcPCs with the proper cytokine and growth factor milieu needed for their proliferation. Although HcPCs produce IL-6, they may also depend on other cytokines such as TNF, which is produced by macrophages that are recruited to the damaged liver. In addition, uPA expression and CCl4 treatment may enhance HcPC growth and progression through their fibrogenic effect on hepatic stellate cells. Although HCC and other cancers have been suspected to arise from premalignant/dysplastic lesions (Hruban et al., 2007Hytiroglou et al., 2007), a direct demonstration that such lesions progress into malignant tumors has been lacking. Based on expression of common markers—EpCAM, CD44, AFP, activated STAT3, and IL-6—that are not expressed in normal hepatocytes, we postulate that HcPCs originate from FAH or dysplastic foci, which are first observed in male mice within 3 months of DEN exposure.

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

Lin R1Tao RGao XLi TZhou XGuan KLXiong YLei QY.
Mol Cell. 2013 Aug 22; 51(4):506-18
http://dx.doi.org:/10.1016/j.molcel.2013.07.002

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

Fatty acid synthesis occurs at low rates in most nondividing cells of normal tissues that primarily uptake lipids from circulation. In contrast, increased lipogenesis, especially de novo lipid synthesis, is a key characteristic of cancer cells. Many studies have demonstrated that in cancer cells, fatty acids are preferred to be derived from de novo synthesis instead of extracellular lipid supply (Medes et al., 1953Menendez and Lupu, 2007;Ookhtens et al., 1984Sabine et al., 1967). Fatty acids are key building blocks for membrane biogenesis, and glucose serves as a major carbon source for de novo fatty acid synthesis (Kuhajda, 2000McAndrew, 1986;Swinnen et al., 2006). In rapidly proliferating cells, citrate generated by the tricarboxylic acid (TCA) cycle, either from glucose by glycolysis or glutamine by anaplerosis, is preferentially exported from mitochondria to cytosol and then cleaved by ATP citrate lyase (ACLY) (Icard et al., 2012) to produce cytosolic acetyl coenzyme A (acetyl-CoA), which is the building block for de novo lipid synthesis. As such, ACLY couples energy metabolism with fatty acids synthesis and plays a critical role in supporting cell growth. The function of ACLY in cell growth is supported by the observation that inhibition of ACLY by chemical inhibitors or RNAi dramatically suppresses tumor cell proliferation and induces differentiation in vitro and in vivo (Bauer et al., 2005Hatzivassiliou et al., 2005). In addition, ACLY activity may link metabolic status to histone acetylation by providing acetyl-CoA and, therefore, gene expression (Wellen et al., 2009).

While ACLY is transcriptionally regulated by sterol regulatory element-binding protein 1 (SREBP-1) (Kim et al., 2010), ACLY activity is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Berwick et al., 2002Migita et al., 2008Pierce et al., 1982). Akt can directly phosphorylate and activate ACLY (Bauer et al., 2005Berwick et al., 2002Migita et al., 2008Potapova et al., 2000). Covalent lysine acetylation has recently been found to play a broad and critical role in the regulation of multiple metabolic enzymes (Choudhary et al., 2009Zhao et al., 2010). In this study, we demonstrate that ACLY protein is acetylated on multiple lysine residues in response to high glucose. Acetylation of ACLY blocks its ubiquitinylation and degradation, thus leading to ACLY accumulation and increased fatty acid synthesis. Our observations reveal a crosstalk between protein acetylation and ubiquitylation in the regulation of fatty acid synthesis and cell growth.

Acetylation of ACLY at Lysines 540, 546, and 554

Recent mass spectrometry-based proteomic analyses have potentially identified a large number of acetylated proteins, including ACLY (Figure S1A available online; Choudhary et al., 2009Zhao et al., 2010). We detected the acetylation level of ectopically expressed ACLY followed by western blot using pan-specific anti-acetylated lysine antibody. ACLY was indeed acetylated, and its acetylation was increased by nearly 3-fold after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases, and trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) class I and class II (Figure 1A). Experiments with endogenous ACLY also showed that TSA and NAM treatment enhanced ACLY acetylation (Figure 1B).

Figure 1  ACLY Is Acetylated at Lysines 540, 546, and 554

Ten putative acetylation sites were identified by mass spectrometry analyses (Table S1). We singly mutated each lysine to either a glutamine (Q) or an arginine (R) and found that no single mutation resulted in a significant reduction of ACLY acetylation (data not shown), indicating that ACLY may be acetylated at multiple lysine residues. Three lysine residues, K540, K546, and K554, received high scores in the acetylation proteomic screen and are evolutionarily conserved from C. elegans to mammals (Figure S1A). We generated triple Q and R mutants of K540, K546, and K554 (3KQ and 3KR) and found that both 3KQ and 3KR mutations resulted in a significant (~60%) decrease in ACLY acetylation (Figure 1C), indicating that 3K are the major acetylation sites of ACLY.  Further, we found that the acetylation of endogenous ACLY is clearly increased after treatment of cells with NAM and TSA (Figure 1D). These results demonstrate that ACLY is acetylated at K540, K546, and K554.

Glucose Promotes ACLY Acetylation to Stabilize ACLY

In mammalian cells, glucose is the main carbon source for de novo lipid synthesis. We found that ACLY levels increased with increasing glucose concentration, which also correlated with increased ACLY 3K acetylation (Figure 1E). Furthermore, to confirm whether the glucose level affects ACLY protein stability in vivo, we intraperitoneally injected glucose in BALB/c mice and found that high glucose resulted in a significant increase of ACLY protein levels (Figure 1F).

To determine whether ACLY acetylation affects its protein levels, we treated HeLa and Chang liver cells with NAM and TSA and found an increase in ACLY protein levels (Figure S1G, upper panel). ACLY mRNA levels were not significantly changed by the treatment of NAM and TSA (Figure S1G, lower panel), indicating that this upregulation of ACLY is mostly achieved at the posttranscriptional level. Indeed, ACLY protein was also accumulated in cells treated with the proteasome inhibitor MG132, indicating that ACLY stability could be regulated by the ubiquitin-proteasome pathway (Figure 1G). Blocking deacetylase activity stabilized ACLY (Figure S1H). The stabilization of ACLY induced by high glucose was associated with an increase of ACLY acetylation at K540, K546, and K554. Together, these data support a notion that high glucose induces both ACLY acetylation and protein stabilization and prompted us to ask whether acetylation directly regulates ACLY stability. We then generated ACLYWT, ACLY3KQ, and ACLY3KRstable cells after knocking down the endogenous ACLY. We found that the ACLY3KR or ACLY3KQmutant was more stable than the ACLYWT (Figures 1I and S1I). Collectively, our results suggest that glucose induces acetylation at K540, 546, and 554 to stabilize ACLY.

Acetylation Stabilizes ACLY by Inhibiting Ubiquitylation

To determine the mechanism underlying the acetylation and ACLY protein stability, we first examined ACLY ubiquitylation and found that it was actively ubiquitylated (Figure 2A). Previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). In order to identify the ubiquitylation sites, we tested the ubiquitylation levels of double mutants 540R–546R and 546–554R (Figure S2A). We found that the ubiquitylation of the 540R-546R and 546R-554R mutants is partially decreased, while mutation of K540, K546, and K554 (3KR), which changes all three putative acetylation lysine residues of ACLY to arginine residues, dramatically reduced the ACLY ubiquitylation level (Figures 2B and S2A), indicating that 3K lysines might also be the ubiquitylation target residues. Moreover, inhibition of deacetylases by NAM and TSA decreased ubiquitylation of WT but not 3KQ or 3KR mutant ACLY (Figure 2C). These results implicate an antagonizing role of the acetylation towards the ubiquitylation of ACLY at these three lysine residues.

Figure 2  Acetylation Protects ACLY from Proteasome Degradation by Inhibiting Ubiquitylation

We found that ACLY acetylation was only detected in the nonubiquitylated, but not the ubiquitylated (high-molecular-weight), ACLY species. This result indicates that ACLY acetylation and ubiquitylation are mutually exclusive and is consistent with the model that K540, K546, and K554 are the sites of both ubiquitylation and acetylation. Therefore, acetylation of these lysines would block ubiquitylation.

We also found that glucose upregulates ACLY acetylation at 3K and decreases its ubiquitylation (Figure S2B). High glucose (25 mM) effectively decreased ACLY ubiquitylation, while inhibition of deacetylases clearly diminished its ubiquitylation (Figure 2E). We conclude that acetylation and ubiquitylation occur mutually exclusively at K540, K546, and K554 and that high-glucose-induced acetylation at these three sites blocks ACLY ubiquitylation and degradation.

UBR4 Targets ACLY for Degradation

UBR4 was identified as a putative ACLY-interacting protein by affinity purification coupled with mass spectrometry analysis (data not shown). To address if UBR4 is a potential ACLY E3 ligase, we determined the interaction between ACLY and UBR4 and found that ACLY interacted with the E3 ligase domain of UBR4; this interaction was enhanced by MG132 treatment (Figure 3A). UBR4 knockdown in A549 cells resulted in an increase of endogenous ACLY protein level (Figure 3C). Moreover, UBR4 knockdown significantly stabilized ACLY (Figure 3D) and decreased ACLY ubiquitylation (Figure 3E). Taken together, these results indicate that UBR4 is an ACLY E3 ligase that responds to glucose regulation.

Figure 3  UBR4 Is the E3 Ligase of ACLY

PCAF Acetylates ACLY

PCAF knockdown significantly reduced acetylation of 3K, indicating that PCAF is a potential 3K acetyltransferase in vivo (Figure 4C, upper panel). Furthermore, PCAF knockdown decreased the steady-state level of endogenous ACLY, but not ACLY mRNA (Figure 4C, middle and lower panels). Moreover, we found that PCAF knockdown destabilized ACLY (Figure 4D). In addition, overexpression of PCAF decreases ACLY ubiquitylation (Figure 4E), while PCAF inhibition increases the interaction between UBR4 E3 ligase domain and wild-type ACLY, but not 3KR (Figure 4F). Together, our results indicate that PCAF increases ACLY protein level, possibly via acetylating ACLY at 3K.

Figure 4  PCAF Is the Acetylase of ACLY

SIRT2 Deacetylates ACLY

Figure 5  SIRT2 Decreases ACLY Acetylation and Increases Its Protein Levels In Vivo

Acetylation of ACLY Promotes Cell Proliferation and De Novo Lipid Synthesis

The protein levels of ACLY 3KQ and 3KR were accumulated to a level higher than the wild-type cells upon extended culture in low-glucose medium (Figure S6A, right panel), indicating a growth advantage conferred by ACLY stabilization resulting from the disruption of both acetylation and ubiquitylation at K540, K546, and K554. Cellular acetyl-CoA assay showed that cells expressing 3KQ or 3KR mutant ACLY produce more acetyl-CoA than cells expressing the wild-type ACLY under low glucose (Figures 6B and S6B), further supporting the conclusion that 3KQ or 3KR mutation stabilizes ACLY.

Figure 6  Acetylation of ACLY at 3K Promotes Lipogenesis and Tumor Cell Proliferation

ACLY is a key enzyme in de novo lipid synthesis. Silencing ACLY inhibited the proliferation of multiple cancer cell lines, and this inhibition can be partially rescued by adding extra fatty acids or cholesterol into the culture media (Zaidi et al., 2012). This prompted us to measure extracellular lipid incorporation in A549 cells after knockdown and ectopic expression of ACLY. We found that when cultured in low glucose (2.5 mM), cells expressing wild-type ACLY uptake significantly more phospholipids compared to cells expressing 3KQ or 3KR mutant ACLY (Figures 6C, 6D, and S6D). When cultured in the presence of high glucose (25 mM), however, cells expressing either the wild-type, 3KQ, or 3KR mutant ACLY all have reduced, but similar, uptake of extracellular phospholipids (Figures 6C, 6D, and S6D). The above results are consistent with a model that acetylation of ACLY induced by high glucose increases its stability and stimulates de novo lipid synthesis.

3K Acetylation of ACLY Is Increased in Lung Cancer

ACLY is reported to be upregulated in human lung cancer (Migita et al., 2008). Many small chemicals targeting ACLY have been designed for cancer treatment (Zu et al., 2012). The finding that 3KQ or 3KR mutant increased the ability of ACLY to support A549 lung cancer cell proliferation prompted us to examine 3K acetylation in human lung cancers. We collected a total of 54 pairs of primary human lung cancer samples with adjacent normal lung tissues and performed immunoblotting for ACLY protein levels. This analysis revealed that, when compared to the matched normal lung tissues, 29 pairs showed a significant increase of total ACLY protein using b-actin as a loading control (Figures 7A and S7A). The tumor sample analyses demonstrate that ACLY protein levels are elevated in lung cancers, and 3K acetylation positively correlates with the elevated ACLY protein. These data also indicate that ACLY with 3K acetylation may be potential biomarker for lung cancer diagnosis.

Figure 7
  Acetylation of ACLY at 3K Is Upregulated in Human Lung Carcinoma

Dysregulation of cellular metabolism is a hallmark of cancer (Hanahan and Weinberg, 2011Vander Heiden et al., 2009). Besides elevated glycolysis, increased lipogenesis, especially de novo lipid synthesis, also plays an important role in tumor growth. Because most carbon sources for fatty acid synthesis are from glucose in mammalian cells (Wellen et al., 2009), the channeling of carbon into de novo lipid synthesis as building blocks for tumor cell growth is primarily linked to acetyl-CoA production by ACLY. Moreover, the ACLY-catalyzed reaction consumes ATP. Therefore, as the key cellular energy and carbon source, one may expect a role for glucose in ACLY regulation. In the present study, we have uncovered a mechanism of ACLY regulation by glucose that increases ACLY protein level to meet the enhanced demand of lipogenesis in growing cells, such as tumor cells (Figure 7C). Glucose increases ACLY protein levels by stimulating its acetylation.

Upregulation of ACLY is common in many cancers (Kuhajda, 2000Milgraum et al., 1997Swinnen et al., 2004Yahagi et al., 2005). This is in part due to the transcriptional activation by SREBP-1 resulting from the activation of the PI3K/AKT pathway in cancers (Kim et al., 2010Nadler et al., 2001Wang and Dey, 2006). In this study, we report a mechanism of ACLY regulation at the posttranscriptional level. We propose that acetylation modulated by glucose status plays a crucial role in coordinating the intracellular level of ACLY, hence fatty acid synthesis, and glucose availability. When glucose is sufficient, lipogenesis is enhanced. This can be achieved, at least in part, by the glucose-induced stabilization of ACLY. High glucose increases ACLY acetylation, which inhibits its ubiquitylation and degradation, leading to the accumulation of ACLY and enhanced lipogenesis. In contrast, when glucose is limited, ACLY is not acetylated and thus can be ubiquitylated, leading to ACLY degradation and reduced lipogenesis. Moreover, our data indicate that acetylation and ubiquitylation in ACLY may compete with each other by targeting the same lysine residues at K540, K546, and K554. Consistently, previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). Similar models of different modifications on the same lysine residues have been reported in the regulation of other proteins (Grönroos et al., 2002Li et al., 20022012). We propose that acetylation and ubiquitylation have opposing effects in the regulation of ACLY by competitively modifying the same lysine residues. The acetylation-mimetic 3KQ and the acetylation-deficient 3KR mutants behaved indistinguishably in most biochemical and functional assays, mainly due to the fact that these mutations disrupt lysine ubiquitylation that primarily occurs on these three residues.

ACLY is increased in lung cancer tissues compared to adjacent tissues. Consistently, ACLY acetylation at 3K is also significantly increased in lung cancer tissues. These observations not only confirm ACLY acetylation in vivo, but also suggest that ACLY 3K acetylation may play a role in lung cancer development. Our study reveals a mechanism of ACLY regulation in response to glucose signals.

 

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Nomura DK1Long JZNiessen SHoover HSNg SWCravatt BF.
Cell. 2010 Jan 8; 140(1):49-61
http://dx.doi.org/10.1016.2Fj.cell.2009.11.027

Highlights

  • Monoacylglycerol lipase (MAGL) is elevated in aggressive human cancer cells
  • Loss of MAGL lowers fatty acid levels in cancer cells and impairs pathogenicity
  • MAGL controls a signaling network enriched in protumorigenic lipids
  • A high-fat diet can restore the growth of tumors lacking MAGL in vivo
monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

http://www.cell.com/cms/attachment/1082768/7977146/fx1.jpg

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity—phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.

We show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in non-aggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity — phenotypes that are reversed by an MAGL inhibitor. Interestingly, impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of pro-tumorigenic signals.

The conversion of cells from a normal to cancerous state is accompanied by reprogramming of metabolic pathways (Deberardinis et al., 2008Jones and Thompson, 2009Kroemer and Pouyssegur, 2008), including those that regulate glycolysis (Christofk et al., 2008Gatenby and Gillies, 2004), glutamine-dependent anaplerosis (DeBerardinis et al., 2008DeBerardinis et al., 2007Wise et al., 2008), and the production of lipids (DeBerardinis et al., 2008Menendez and Lupu, 2007). Despite a growing appreciation that dysregulated metabolism is a defining feature of cancer, it remains unclear, in many instances, how such biochemical changes occur and whether they play crucial roles in disease progression and malignancy.

Among dysregulated metabolic pathways, heightened de novo lipid biosynthesis, or the development a “lipogenic” phenotype (Menendez and Lupu, 2007), has been posited to play a major role in cancer. For instance, elevated levels of fatty acid synthase (FAS), the enzyme responsible for fatty acid biosynthesis from acetate and malonyl CoA, are correlated with poor prognosis in breast cancer patients, and inhibition of FAS results in decreased cell proliferation, loss of cell viability, and decreased tumor growth in vivo (Kuhajda et al., 2000Menendez and Lupu, 2007Zhou et al., 2007). FAS may support cancer growth, at least in part, by providing metabolic substrates for energy production (via fatty acid oxidation) (Buzzai et al., 2005Buzzai et al., 2007Liu, 2006). Many other features of lipid biochemistry, however, are also critical for supporting the malignancy of cancer cells, including:

Prominent examples of lipid messengers that contribute to cancer include:

Here, we use functional proteomic methods to discover a lipolytic enzyme, monoacylglycerol lipase (MAGL), that is highly elevated in aggressive cancer cells from multiple tissues of origin. We show that MAGL, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells. The resulting MAGL-FFA pathway feeds into a diverse lipid network enriched in pro-tumorigenic signaling molecules and promotes migration, survival, and in vivo tumor growth. Aggressive cancer cells thus pair lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behavior.

Activity-Based Proteomic Analysis of Hydrolytic Enzymes in Human Cancer Cells

To identify enzyme activities that contribute to cancer pathogenesis, we conducted a functional proteomic analysis of a panel of aggressive and non-aggressive human cancer cell lines from multiple tumors of origin, including melanoma [aggressive (C8161, MUM2B), non-aggressive (MUM2C)], ovarian [aggressive (SKOV3), non-aggressive (OVCAR3)], and breast [aggressive (231MFP), non-aggressive (MCF7)] cancer. Aggressive cancer lines were confirmed to display much greater in vitro migration and in vivo tumor-growth rates compared to their non-aggressive counterparts (Figure S1), as previously shown (Jessani et al., 2004;Jessani et al., 2002Seftor et al., 2002Welch et al., 1991). Proteomes from these cancer lines were screened by activity-based protein profiling (ABPP) using serine hydrolase-directed fluorophosphonate (FP) activity-based probes (Jessani et al., 2002Patricelli et al., 2001). Serine hydrolases are one of the largest and most diverse enzyme classes in the human proteome (representing ~ 1–1.5% of all human proteins) and play important roles in many biochemical processes of potential relevance to cancer, such as proteolysis (McMahon and Kwaan, 2008Puustinen et al., 2009), signal transduction (Puustinen et al., 2009), and lipid metabolism (Menendez and Lupu, 2007Zechner et al., 2005). The goal of this study was to identify hydrolytic enzyme activities that were consistently altered in aggressive versus non-aggressive cancer lines, working under the hypothesis that these conserved enzymatic changes would have a high probability of contributing to the pathogenic state of cancer cells.

Among the more than 50 serine hydrolases detected in this analysis (Tables S13), two enzymes, KIAA1363 and MAGL, were found to be consistently elevated in aggressive cancer cells relative to their non-aggressive counterparts, as judged by spectral counting (Jessani et al., 2005Liu et al., 2004). We confirmed elevations in KIAA1363 and MAGL in aggressive cancer cells by gel-based ABPP, where proteomes are treated with a rhodamine-tagged FP probe and resolved by 1D-SDS-PAGE and in-gel fluorescence scanning (Figure 1A). In both cases, two forms of each enzyme were detected (Figure 1A), due to differential glycoslyation for KIAA1363 (Jessani et al., 2002), and possibly alternative splicing for MAGL (Karlsson et al., 2001). We have previously shown that KIAA1363 plays a role in regulating ether lipid signaling pathways in aggressive cancer cells (Chiang et al., 2006). On the other hand, very little was known about the function of MAGL in cancer.

Figure 1  MAGL is elevated in aggressive cancer cells, where the enzyme regulates monoacylgycerol (MAG) and free fatty acid (FFA) levels

The heightened activity of MAGL in aggressive cancer cells was confirmed using the substrate C20:4 MAG (Figure 1B). Since several enzymes have been shown to display MAG hydrolytic activity (Blankman et al., 2007), we confirmed the contribution that MAGL makes to this process in cancer cells using the potent and selective MAGL inhibitor JZL184 (Long et al., 2009a).

MAGL Regulates Free Fatty Acid Levels in Aggressive Cancer Cells

MAGL is perhaps best recognized for its role in degrading the endogenous cannabinoid 2-arachidonoylglycerol (2-AG, C20:4 MAG), as well as other MAGs, in brain and peripheral tissues (Dinh et al., 2002Long et al., 2009aLong et al., 2009bNomura et al., 2008). Consistent with this established function, blockade of MAGL by JZL184 (1 μM, 4 hr) produced significant elevations in the levels of several MAGs, including 2-AG, in each of the aggressive cancer cell lines (Figure 1C and Figure S2). Interestingly, however, MAGL inhibition also caused significant reductions in the levels of FFAs in aggressive cancer cells (Figure 1D and Figure S2). This surprising finding contrasts with the function of MAGL in normal tissues, where the enzyme does not, in general, control the levels of FFAs (Long et al., 2009aLong et al., 2009b;Nomura et al., 2008).

Metabolic labeling studies using the non-natural C17:0-MAG confirmed that MAGs are converted to LPC and LPE by aggressive cancer cells, and that this metabolic transformation is significantly enhanced by treatment with JZL184 (Figure S1). Finally, JZL184 treatment did not affect the levels of MAGs and FFAs in non-aggressive cancer lines (Figure 1C, D), consistent with the negligible expression of MAGL in these cells (Figure 1A, B).

We next stably knocked down MAGL expression by RNA interference technology using two independent shRNA probes (shMAGL1, shMAGL2), both of which reduced MAGL activity by 70–80% in aggressive cancer lines (Figure 2A, D and Figure S2). Other serine hydrolase activities were unaffected by shMAGL probes (Figure 2A, D and Figures S2), confirming the specificity of these reagents. Both shMAGL probes caused significant elevations in MAGs and corresponding reductions in FFAs in aggressive melanoma (Figure 2B, C), ovarian (Figure 2E, F), and breast cancer cells (Figure S2).

Figure 2  Stable shRNA-mediated knockdown of MAGL lowers FFA levels in aggressive cancer cells.

Together, these data demonstrate that both acute (pharmacological) and stable (shRNA) blockade of MAGL cause elevations in MAGs and reductions in FFAs in aggressive cancer cells. These intriguing findings indicate that MAGL is the principal regulator of FFA levels in aggressive cancer cells. Finally, we confirmed that MAGL activity (Figure 3A, B) and FFA levels (Figure 3C) are also elevated in high-grade primary human ovarian tumors compared to benign or low-grade tumors. Thus, heightened expression of the MAGL-FFA pathway is a prominent feature of both aggressive human cancer cell lines and primary tumors.

Figure 3  High-grade primary human ovarian tumors possess elevated MAGL activity and FFAs compared to benign tumors.

Disruption of MAGL Expression and Activity Impairs Cancer Pathogenicity

shMAGL cancer lines were next examined for alterations in pathogenicity using a set of in vitro and in vivo assays. shMAGL-melanoma (C8161), ovarian (SKOV3), and breast (231MFP) cancer cells exhibited significantly reduced in vitro migration (Figure 4A, F and Figure S2), invasion (Figure 4B, G and Figure S2), and cell survival under serum-starvation conditions (Figure 4C, H and Figure S2). Acute pharmacological blockade of MAGL by JZL184 also decreased cancer cell migration (Figure S2), but not survival, possibly indicating that maximal impairments in cancer aggressiveness require sustained inhibition of MAGL.

Figure 4  shRNA-mediated knockdown and pharmacological inhibition of MAGL impair cancer aggressiveness.

MAGL Overexpression Increases FFAs and the Aggressiveness of Cancer Cells

Stable MAGL-overexpressing (MAGL-OE) and control [expressing an empty vector or a catalytically inactive version of MAGL, where the serine nucleophile was mutated to alanine (S122A)] variants of MUM2C and OVCAR3 cells were generated by retroviral infection and evaluated for their respective MAGL activities by ABPP and C20:4 MAG substrate assays. Both assays confirmed that MAGL-OE cells possess greater than 10-fold elevations in MAGL activity compared to control cells (Figure 5A and Figure S4). MAGL-OE cells also showed significant reductions in MAGs (Figure 5B andFigure S4) and elevated FFAs (Figure 5C and Figure S4). This altered metabolic profile was accompanied by increased migration (Figure 5D and Figure S4), invasion (Figure 5E and Figure S4), and survival (Figure S4) in MAGL-OE cells. None of these effects were observed in cancer cells expressing the S122A MAGL mutant, indicating that they require MAGL activity.  MAGL-OE MUM2C cells also showed enhanced tumor growth in vivo compared to control cells (Figure 5F). Notably, the increased tumor growth rate of MAGL-OE MUM2C cells nearly matched that of aggressive C8161 cells (Figure S4). These data indicate that the ectopic expression of MAGL in non-aggressive cancer cells is sufficient to elevate their FFA levels and promote pathogenicity both in vitro and in vivo.

Figure 5 Ectopic expression of MAGL elevates FFA levels and enhances the in vitro and in vivo pathogenicity of MUM2C melanoma cells.

Metabolic Rescue of Impaired Pathogenicity in MAGL-Disrupted Cancer Cells

MAGL could support the aggressiveness of cancer cells by either reducing the levels of its MAG substrates, elevating the levels of its FFA products, or both. Among MAGs, the principal signaling molecule is the endocannabinoid 2-AG, which activates the CB1 and CB2 receptors (Ahn et al., 2008Mackie and Stella, 2006). The endocannabinoid system has been implicated previously in cancer progression and, depending on the specific study, shown to promote (Sarnataro et al., 2006Zhao et al., 2005) or suppress (Endsley et al., 2007Wang et al., 2008) cancer pathogenesis. Neither a CB1 or CB2 antagonist rescued the migratory defects of shMAGL cancer cells (Figure S5). CB1 and CB2 antagonists also did not affect the levels of MAGs or FFAs in cancer cells (Figure S5).

We then determined whether increased FFA delivery could rectify the tumor growth defect observed for shMAGL cells in vivo. Immune-deficient mice were fed either a normal chow or high-fat diet throughout the duration of a xenograft tumor growth experiment. Notably, the impaired tumor growth rate of shMAGL-C8161 cells was completely rescued in mice fed a high-fat diet. In contrast, shControl-C8161 cells showed equivalent tumor growth rates on a normal versus high-fat diet. The recovery in tumor growth for shMAGL-C8161 cells in the high-fat diet group correlated with significantly increases levels of FFAs in excised tumors (Figure 6D). Collectively, these results indicate that MAGL supports the pathogenic properties of cancer cells by maintaining tonically elevated levels of FFAs.

Figure 6  Recovery of the pathogenic properties of shMAGL cancer cells by treatment with exogenous fatty acids.

MAGL Regulates a Fatty Acid Network Enriched in Pro-Tumorigenic Signals

Studies revealed that neither

  • the MAGL-FFA pathway might serve as a means to regenerate NAD+ (via continual fatty acyl glyceride/FFA recycling) to fuel glycolysis, or
  • increased lipolysis could be to generate FFA substrates for β-oxidation, which may serve as an important energy source for cancer cells (Buzzai et al., 2005), or
  • CPT1 blockade (reduced expression of CPT1 in aggressive cancer cells (data not shown) has been reported previously (Deberardinis et al., 2006))

providing evidence against a role for β-oxidation as a downstream mediator of the pathogenic effects of the MAGL-fatty acid pathway.

Considering that FFAs are fundamental building blocks for the production and remodeling of membrane structures and signaling molecules, perturbations in MAGL might be expected to affect several lipid-dependent biochemical networks important for malignancy. To test this hypothesis, we performed lipidomic analyses of cancer cell models with altered MAGL activity, including comparisons of:

  1. MAGL-OE versus control cancer cells (OVCAR3, MUM2C), and
  2. shMAGL versus shControl cancer cells (SKOV3, C8161).

Complementing these global profiles, we also conducted targeted measurements of specific bioactive lipids (e.g., prostaglandins) that are too low in abundance for detection by standard lipidomic methods. The resulting data sets were then mined to identify a common signature of lipid metabolites regulated by MAGL, which we defined as metabolites that were significantly increased or reduced in MAGL–OE cells and showed the opposite change in shMAGL cells relative to their respective control groups (Figure 7A, B and Table S4).

Figure 7  MAGL regulates a lipid network enriched in pro-tumorigenic signaling molecules.

Most of the lipids in the MAGL-fatty acid network, including several lysophospholipids (LPC, LPA, LPE), ether lipids (MAGE, alkyl LPE), phosphatidic acid (PA), and prostaglandin E2 (PGE2), displayed similar profiles to FFAs, being consistently elevated and reduced in MAGL-OE and shMAGL cells, respectively. Only MAGs were found to show the opposite profile (elevated and reduced in shMAGL and MAGL-OE cells, respectively). Interestingly, virtually this entire lipidomic signature was also observed in aggressive cancer cells when compared to their non-aggressive counterparts (e.g., C8161 versus MUM2C and SKOV3 versus OVCAR3, respectively; Table S4). These findings demonstrate that MAGL regulates a lipid network in aggressive cancer cells that consists of not only FFAs and MAGs, but also a host of secondary lipid metabolites. Increases (rather than decreases) in LPCs and LPEs were observed in JZL184-treated cells (Figure S1 and Table S4). These data indicate that acute and chronic blockade of MAGL generate distinct metabolomic effects in cancer cells, likely reflecting the differential outcomes of short- versus long-term depletion of FFAs.

Within the MAGL-fatty acid network are several pro-tumorigenic lipid messengers, including LPA and PGE2, that have been reported to promote the aggressiveness of cancer cells (Gupta et al., 2007Mills and Moolenaar, 2003). Metabolic labeling studies confirmed that aggressive cancer cells can convert both MAGs and FFAs (Figure S1) to LPA and PGE2 and, for MAGs, this conversion was blocked by JZL184 (Figure S1). Interestingly, treatment with either LPA or PGE2 (100 nM, 4 hr) rescued the impaired migration of shMAGL cancer cells at concentrations that did not affect the migration of shControl cells (Figure 7E).

Heightened lipogenesis is an established early hallmark of dysregulated metabolism and pathogenicity in cancer (Menendez and Lupu, 2007). Cancer lipogenesis appears to be driven principally by FAS, which is elevated in most transformed cells and important for survival and proliferation (De Schrijver et al., 2003;Kuhajda et al., 2000Vazquez-Martin et al., 2008). It is not yet clear how FAS supports cancer growth, but most of the proposed mechanisms invoke pro-tumorigenic functions for the enzyme s fatty acid products and their lipid derivatives (Menendez and Lupu, 2007). This creates a conundrum, since the fatty acid molecules produced by FAS are thought to be rapidly incorporated into neutral- and phospho-lipids, pointing to the need for complementary lipolytic pathways in cancer cells to release stored fatty acids for metabolic and signaling purposes (Prentki and Madiraju, 2008Przybytkowski et al., 2007). Consistent with this hypothesis, we found that acute treatment with the FAS inhibitor C75 (40 μM, 4 h) did not reduce FFA levels in cancer cells (data not shown). Furthermore, aggressive and non-aggressive cancer cells exhibited similar levels of FAS (data not shown), indicating that lipogenesis in the absence of paired lipolysis may be insufficient to confer high levels of malignancy.

Here we show that aggressive cancer cells do indeed acquire the ability to liberate FFAs from neutral lipid stores as a consequence of heightened expression of MAGL. MAGL and its FFA products were found to be elevated in aggressive human cancer cell lines from multiple tissues of origin, as well as in high-grade primary human ovarian tumors. These data suggest that the MAGL-FFA pathway may be a conserved feature of advanced forms of many types of cancer. Further evidence in support of this premise originates from gene expression profiling studies, which have identified increased levels of MAGL in primary human ductal breast tumors compared to less malignant medullary breast tumors (Gjerstorff et al., 2006). The key role that MAGL plays in regulating FFA levels in aggressive cancer cells contrasts with the function of this enzyme in normal tissues, where it mainly controls the levels of MAGs, but not FFAs (Long et al., 2009b). These data thus provide a striking example of the co-opting of an enzyme by cancer cells to serve a distinct metabolic purpose that supports their pathogenic behavior.

Taken together, our results indicate that MAGL serves as key metabolic hub in aggressive cancer cells, where the enzyme regulates a fatty acid network that feeds into a number of pro-tumorigenic signaling pathways.

 

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.8.1  Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling

Komai K1Niwa Y1Sasazawa Y1Simizu S2.
FEBS Lett. 2015 Mar 12; 589(6):738-43
http://dx.doi.org:/10.1016/j.febslet.2015.01.040

Highlights

  • Pirin decreases E-cadherin expression and induces EMT.
  • The induction of EMT by Pirin is achieved through a Bcl3 independent pathway.
  • Pirin may be a novel target for cancer therapy.

Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E-cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle-like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E-cadherin expression similar to wild-type, suggesting that Pirin regulates E-cadherin independently of Bcl3-Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.

Pirin regulates the expression of E-cadherin and EMT markers

In melanoma, Pirin enhances NF-jB activity and increases Slug expression by binding Bcl3 [31], and it may also be involved in adenoid cystic tumor metastasis [23]. Since Slug suppresses E-cadherin transcription and is recognized as a major EMT inducer, we hypothesized that Pirin may regulate EMT through inducing Slug expression. To investigate whether Pirin regulates EMT, we measured E-cadherin expression following Pirin knockdown. As shown in Fig. 1A and B, E-cadherin expression was significantly increased following Pirin knockdown indicating that it may promote EMT. To confirm this, we established Pirin-expressing HeLa cells (Fig. 1C), which inhibited the expression of E-cadherin (Fig. 1D). Additionally, the expression of Occludin, an epithelial marker, was decreased, and several mesenchymal markers, including Fibronectin, N-cadherin, and Vimentin, were increased by Pirin expression (Fig. 1D). These data suggest that Pirin promotes EMT.

Pirin induces EMT-associated cell morphological changes

As mentioned above, cells undergo morphological changes during EMT. Therefore, we next analyzed whether Pirin expression affects cell morphology. Quantitative analysis of morphological changes was based on cell circularity, {4p(area)/(perimeter)2}100, which decreases during EMT-associated morphological changes [34–36]. Indeed, TGF-b or TNF-a exposure induced EMTassociated cell morphological changes in HeLa cells (data not shown). Employing this parameter of circularity, we compared the morphology of our established HeLa/Pirin-GFP cells with control HeLa/GFP cells. Although the control HeLa/GFP cells displayed a cobblestone-like morphology, HeLa/Pirin-GFP cells were elongated in shape (Fig. 2A). Indeed, compared with control cells, the circularity of HeLa/Pirin-GFP cells was significantly decreased (Fig. 2B). To confirm that these observations were dependent on Pirin expression, HeLa/Pirin-GFP cells were treated with an siRNA targeting Pirin. HeLa/Pirin-GFP cells recovered a cobblestone-like morphology (Fig. 2C) and circularity (Fig. 2D) when treated with Pirin siRNA indicating that Pirin expression induces EMT.

Pirin induces cell migration

During EMT cells acquire migratory capabilities. Therefore, we analyzed whether Pirin affects cell migration. HeLa cells were treated with an siRNA targeting Pirin and migration was assessed using a wound healing assay. Although Pirin knockdown had no effect on cell proliferation (data not shown), wound repair was inhibited in Pirin-depleted HeLa cells (Fig. 3A and B) suggesting that Pirin promoted cell migration. Furthermore, camptothecin treatment of HeLa/GFP cells caused decreased cell viability in a dose-dependent manner, whereas HeLa/Pirin-GFP cells were more resistantto drugtreatment (datanot shown).These results suggest that Pirin induces EMT-like phenotypes, such as cell migration and anticancer drug resistance.
Pirin regulates EMT independently of Bcl3-Slug signaling

To investigate whether Pirin controls E-cadherin expression at the transcriptional level, we measured E-cadherin promoter activity with a reporter assay. Indeed, the luciferase reporter analysis indicated that Pirin inhibited E-cadherin promoter activity (Fig. 4A and B). To determine if Bcl3 is involved in Pirin-induced EMT, we tested whether a Pirin mutant defective in Bcl3 binding could inhibit E-cadherin expression. We generated a mutation in the metal-binding cavity of Pirin(E103A) and confirmed that it disrupted Bcl3 binding. In vitro GST pull-down analysis using recombinant Pirin and Bcl3/ARD demonstrated that the Pirin mutant was defective for Bcl3 binding compared to wild-type (Fig. 5A). Interestingly, expression of both wild-type Pirin and the mutant defective in Bcl3 binding reduced E-cadherin gene and protein expression (Fig. 5B and C). Taken together these results indicate that Pirin decreases E-cadherin expression without binding Bcl3, and suggest that Pirin regulates EMT independently of Bcl3-Slug signaling.

Discussion

A characteristic feature of EMT is the disruption of epithelial cell–cell contact, which is achieved by reduced E-cadherin expression. Therefore, revealing the regulatory pathways controlling E-cadherin expression may elucidate the mechanisms of EMT. Several transcription factors regulate E-cadherin transcription. For instance,Snail,Slug,Twist,and Zebact as mastertranscriptional regulators that bind the consensus E-box sequence in the E-cadherin gene promoter and decrease the transcriptional activity [38]. Since Pirin regulates the transcription of Slug [31], we hypothesized that Pirin may also regulate EMT. In this study we demonstrated that Pirin decreases E-cadherin expression, and induces EMT and cancer malignant phenotypes. Since EMT is an initial step of metastasis, Pirin may contribute to cancer progression. We next examined whether the regulation of EMT by Pirin is attributed to Bcl3 binding and the induction of Slug. To this end, we generated a Pirin mutant (E103A) defective for Bcl3 binding (Fig. 5A). Single Fe2+ ion chelating is coordinated by His56, His58, His101, and Glu103 of Pirin, and the N-terminal domain containing these residues is highly conserved between mammals, plants, fungi, and prokaryotic organisms [15,27]. Therefore, it has been predicted that this N-terminal domain containing the metal-binding cavity is important for Pirin function [20,26,31]. Indeed, TPh A inserts into the metal-binding cavity and inhibits binding to Bcl3 suggesting that the interaction occurs with the metal-binding cavity of Pirin [31]. In contrast, Hai Pang suggests that a Pirin–Bcl3– (p50)2 complex forms between acidic regions of the N-terminal Pirin domain at residues 77–82, 97–103 and 124–128 with a basic patch of Bcl3 [27]. In this study, we mutated Glutamic acid 103, a residue common between Hai Pang’s model and Pirin’s metalbinding cavity. Pull-down analysis indicated that an E103A mutant is defectiveinfor Bcl3binding(Fig.5A). Thisis the firstexperimental demonstration showing that Glu103 of Pirin is important Bcl3 binding. However, expression of the E103A mutant suppressed Ecadherin gene expression similarly to wild-type Pirin (Fig. 5B and C). Although the Bcl3–(p50)2 complex participates in oncogene addiction in cervical cells [39,40], expression of Pirin in HeLa cells did not increase Slug expression (data not shown). Therefore, we concludethatPirindecreasesE-cadherinexpressionindependently of Bcl3-Slug signaling. To understand how Pirin suppresses E-cadherin gene expression, we analyzed E-cadherin promoter activity (Fig. 4). Since Pirin decreased the activity of the E-cadherin promoter (995+1), we constructed a series of promoter deletion mutants (795+1, 565+1, 365+1, 175+1) to identify a region important for Pirin-mediated regulation. Expression of Pirin decreased the transcriptional activity of all constructs (Supplementary Fig. S1A), suggesting that Pirin may suppress E-cadherin expression through element(s) in region 175+1. Yan-Nan Liu and colleagues proposed that this region contains four Sp1-binding sites and two E-boxes that regulate E-cadherin expression.

Fig. 1. Pirin regulates E-cadherin gene expression. (A, B) HeLa cells were transfected with siRNA targeting Pirin (siPirin#1 or #2) or control siRNA (siCTRL). Forty-eight hours after transfection, cDNA was used for PCR using primer sets specific against Pirin, E-cadherin and GAPDH (A). Forty-eight hours after transfection, HeLa cells were lysed and the lysates were analyzed by Western blot with the indicated antibodies (B). (C) Lysates from HeLa/Pirin-GFP and HeLa/GFP cells were analyzed by Western blot with the indicated antibodies. (D) cDNA from HeLa/GFP or HeLa/Pirin-GFP cells was used for PCR to determine the effect of Pirin on the expression of EMT marker genes.

Fig. 2. Pirin induces cell morphological changes associated with EMT. (A) Phase contrast and fluorescence microscopic images were taken of HeLa/GFP and HeLa/Pirin-GFP cells. (B) Cell circularity was defined as form factor, {4p(area)/(perimeter)2}100 [%], and calculated using Image J software. A random selection of 100 cells from each condition was measured. (C, D) Phase contrast and fluorescence microscopic images were taken of siRNA-treated HeLa/GFP and HeLa/Pirin-GFP cells. Each cell line was transfected with siPirin#2 or siCTRL. Cells were observed by microscopy 48 h after transfection (C) and circularity was measured (D). Data shown are means ± s.d. ⁄P <0.05, bars 100lm.

Fig. 3. Pirin knockdown suppresses cell migration. (A, B) HeLa cells were transfected with siPirin#2 or siCTRL. An artificial wound was created with a tip 24h after transfection and cells were cultured for an additional 12 h. For quantification, the cells were photographed after 12h of incubation (A) and the area covered by cells was measured using Image J and normalized to control cells (B).

Fig. 4. Pirin regulates E-cadherin promoter activity.(A). HeLacells were transfected with siPirin#2 or siGFP (control) and cultured for 24 h. The E-cadherin promoter construct (995+1) and phRL-TK vectorwere transfected and cellswere cultured for an additional 24 h. Luciferase activities were measured and normalized to Renilla luciferase activity. (B) HeLa cells were transfected with the promoter construct (995+1), phRL-TK vector, and a Pirin expression vector. After 24 h, luciferase activities were measured and normalized to Renilla luciferase activity. Data are the mean ± s.d. ⁄P < 0.05.

Fig. 5. Pirin decreases E-cadherin expression in a Bcl3-independent manner. (A) Purified His6-Pirin and His6-Pirin(E103A) were incubated with Glutathione-Sepharose beads conjugated to GST or GST-Bcl3/ARD. The samples were analyzed by Western blot. (B, C) HeLa cells were transfected with vectors encoding GFP, Pirin-GFP, or Pirin(E103A)GFP. Cells were lysed 48 h after transfection and lysates were analyzed by Western blot (B). RNA collected at 48h was used for RT-PCR with the specified primer sets for each gene (C).

7.7.8.2 1324 PIRIN DOWN-REGULATES THE EAF2/U19 SIGNALING AND RETARDS THE GROWTH INHIBITION INDUCED BY EAF2/U19 IN PROSTATE CANCER CELLS

Zhongjie Qiao, Dan Wang, Zhou Wang
The Journal of Urology Apr 2013; 189(4), Supplement: e541
http://dx.doi.org/10.1016/j.juro.2013.02.2678
EAF2/U19, as the tumor suppressor, has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth in vivo, and its expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification and characterization of the binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells.

7.7.8.3 Pirin Inhibits Cellular Senescence in Melanocytic Cells

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression

Cellular senescence is a physiological process through which normal somatic cells lose their ability to divide and enter an irreversible state of cell cycle arrest, although they remain viable and metabolically active.1,2The specific molecular circuitry underlying the onset of cellular senescence is dependent on the type of stimulus and on the cellular context. A central role is held by the activation of the tumor suppressor proteins p53 and retinoblastoma susceptibility protein (pRB),3–5 which act by interfering with the transcriptional program of the cell and ultimately arresting cell cycle progression.

In the last decade, senescence has been recognized as a major barrier against the development of tumors in mammals.6–8 One of the most prominent in vivo examples is represented by nevi, in which cells proliferate after oncogene activation and then become senescent. Melanoma is a highly aggressive form of neoplasm often observed to derive from nevi, and the transition implies suppression of the mechanisms that sustain the onset and maintenance of senescence.9 In fact, many of the melanoma-associated tumor suppressor genes identified to date are themselves involved in control of senescence, including BRAF (encoding serine/threonine-protein kinase B-raf), CKD4 (cyclin-dependent kinase 4), and CDKN2A (encoding cyclin-dependent kinase inhibitor 2A isoforms p16INK4a and p19ARF).3,10

Nevi frequently harbor oncogenic mutations of the tyrosine kinase BRAF gene, particularly V600E,11 andBRAFV600E is also found in approximately 70% of cutaneous melanomas.12 Expression of BRAFV600E in human melanocytes leads to oncogene-induced senescence,8 which can be considered as a mechanism that protects from malignant progression. In time, some cells may eventually escape senescence, probably through the acquisition of additional genetic abnormalities, thus favoring transformation to melanoma.13

Pirin (PIR) is a highly conserved nuclear protein belonging to the Cupin superfamily14 whose function is, to date, poorly characterized. It has been described as a putative transcriptional regulator on the basis of its physical association with the nuclear I/CCAAT box transcription factor NFI/CTF115 and with the B-cell lymphoma protein, BCL-3, a regulator of NF-κB/Rel activity. A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG).16 Other reports described quercetinase enzymatic activity,17 and regulation of apoptosis18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.

There is evidence of variations in PIR expression levels in different types of malignancies, but a systematic analysis of PIR expression in human tumors has been lacking. We analyzed PIR expression pattern in a collection of normal and neoplastic human tissues and found that it is expressed in scattered melanocytes, virtually absent in more mature regions of nevi, and present at high levels in a subset of melanomas. Functional studies performed in normal and transformed melanocytic cells revealed that PIR ablation results in cellular senescence, and that PIR levels decrease in response to senescence stimuli. Our results suggest that PIR may be a relevant player in the negative control of cellular senescence in PIR-expressing melanomas.

PIR overexpression in melanoma

Figure 3  PIR overexpression in PIR melanoma cells has no effect on proliferation.
PIR Expression Is Down-Regulated by BRAF Activation and Camptothecin Treatment

BRAF mutations are frequent in nevi, and are directly linked to the induction of oncogene-induced senescence. Variations in PIR expression levels were therefore investigated in an experimental model of senescence induced by oncogenic BRAF. Human diploid fibroblasts (TIG3–hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT).28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation (Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

Brian A. Lewis
Biochim et Biophys Acta (BBA) – Gene Regulatory Mechanisms Nov 2013; 1829(11): 1202–1206
http://dx.doi.org/10.1016/j.bbagrm.2013.09.003

Highlights

  • This review article discusses recent advances in the links between O-GlcNAc and transcriptional regulation.
  • Discusses several systems to illustrate O-GlcNAc dynamics: Tet proteins, MLL complexes, circadian clock proteins and RNA pol II.
  • Suggests that promoters are nutrient sensors.

Post-translational modifications play important roles in transcriptional regulation. Among the less understood PTMs is O-GlcNAcylation. Nevertheless, O-GlcNAcylation in the nucleus is found on hundreds of transcription factors and coactivators and is often found in a mutually exclusive ying–yang relationship with phosphorylation. O-GlcNAcylation also links cellular metabolism directly to the proteome, serving as a conduit of metabolic information to the nucleus. This review serves as a brief introduction to O-GlcNAcylation, emphasizing its important thematic roles in transcriptional regulation, and highlights several recent and important additions to the literature that illustrate the connections between O-GlcNAc and transcription.

links between O-GlcNAc and transcriptional regulation.

links between O-GlcNAc and transcriptional regulation.

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr1.sml
links between O-GlcNAc and transcriptional regulation.

systems to illustrate O-GlcNAc dynamics

systems to illustrate O-GlcNAc dynamics

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr2.sml
systems to illustrate O-GlcNAc dynamics

7.7.10 O-GlcNAcylation in cellular functions and human diseases

Yang YR1Suh PG2.
Adv Biol Regul. 2014 Jan; 54:68-73
http://dx.doi.org:/10.1016/j.jbior.2013.09.007

O-GlcNAcylation is dynamic and a ubiquitous post-translational modification. O-GlcNAcylated proteins influence fundamental functions of proteins such as protein-protein interactions, altering protein stability, and changing protein activity. Thus, aberrant regulation of O-GlcNAcylation contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, metabolic disorders, and Alzheimer’s disease. Diverse cellular signaling systems are involved in pathogenesis of these diseases. O-GlcNAcylated proteins occur in many different tissues and cellular compartments and affect specific cell signaling. This review focuses on the O-GlcNAcylation in basic cellular functions and human diseases.

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr2.sml
O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

aberrant regulation of O-GlcNAcylation in disease

aberrant regulation of O-GlcNAcylation in disease

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr3.sml
aberrant regulation of O-GlcNAcylation in disease

 Comment:

Body of review in energetic metabolic pathways in malignant T cells

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.

Dr. Aurel,
Targu Jiu

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Upregulate Tumor Suppressor Pathways

Writer and Curator: Larry H Bernstein, MD, FCAP

 

7.5  Upregulate Tumor Suppressor Pathways

7.5.1 NR4A nuclear receptors are orphans but not lonesome

7.5.2 The interplay of NR4A receptors and the oncogene–tumor suppressor networks in cancer

7.5.3 NLRX1 acts as tumor suppressor by regulating TNF-α induced apoptosis

7.5.4 The Mre11 Complex Suppresses Oncogene-Driven Breast Tumorigenesis and Metastasis

7.5.5 Expression of Stromal Cell-derived Factor 1 and CXCR4 Ligand Receptor System in Pancreatic Cancer

7.5.6 DLC1- a significant GAP in the cancer genome

7.5.7 DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma.

7.5.8 Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma

 

 

7.5.1 NR4A nuclear receptors are orphans but not lonesome

Kurakula K, Koenis DS, van Tiel CM, de Vries CJ.
Biochim Biophys Acta. 2014 Nov; 1843(11):2543-2555
http://dx.doi.org/10.1016/j.bbamcr.2014.06.010

Highlights

  • Nuclear receptors Nur77, Nurr1 and NOR-1 are ‘orphan’ receptors of the NR4A subfamily.
  • The NR4A receptors have no ligands.
  • The known protein–protein interactions of all three NR4A receptors are summarized.
  • Interacting proteins are transcription factors, coregulators or protein kinases.
  • Protein–protein interactions modulate NR4A receptor activity and function.

 

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein–protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77.
Nur77 in the regulation of endocrine signals and steroid hormone synthesis

Nur77 is expressed in endocrine tissues and in organs that are crucial for steroid hormone synthesis such as the adrenal glands, the pituitary gland and the testes. The first functional NurRE was identified in the promoter of the pro-opiomelanocortin (POMC) gene of pituitary derived AtT-20 cells [2]. Nur77 can bind this NurRE either as a homodimer or as a heterodimer with either one of the other two NR4A receptors Nurr1 and NOR-1. Interestingly, it was shown that these heterodimers enhance POMC gene transcription more potently than homodimers of Nur77 do, suggesting that there is interdependency between the NR4A receptors in activating their target genes [3]. The NurRE sequence in the POMC promoter also partially overlaps with a STAT1-3 response element. Philips et al. showed that Nur77 and STAT1-3 bind simultaneously to this so called NurRE-STAT composite site and synergistically enhance transcription of the POMC gene. However, Nur77 and STAT1-3 do not interact directly, which suggests that oneor more facilitatingfactors are involved in NurRE-STAT driven transcription. Mynard et al. showed that this third factor is cAMP response element binding protein (CREB), which binds both STAT1-3 and Nur77 and indirectly enhances transcription of the POMC gene by facilitating the synergistic activation of the NurRE-STAT composite site by STAT1-3 and Nur77 [4]. Nur77also plays animportant role in the steroidogenic acute regulatory protein (StAR)-mediated testosterone production by Leydig cells. StAR is required for the transport of cholesterol through the mitochondrial membrane to initiate steroid hormone synthesis. Nur77 binds to an NBRE in the StAR promoter, which is in close proximity to an AP-1 response element. In response to cAMP stimulation c-Jun and Nur77 synergistically increase StAR gene expression [5], presumably through a direct interaction between c-Jun and the LBD of Nur77 [6]. On the other hand, c-Jun has also been shown to suppress expression of the hydroxylase P450 c17 gene by blocking the DNA-binding activity o fNur77 in response to stimulation of Leydig cells with reactive oxygen species [7].The effect of c-Jun on the transcriptional activity of Nur77 therefore seems to depend on other factors as well. One of these factors could be the atypical nuclear receptor DAX1 (NR0B1), which lacks a DBD and associates with multiple coregulatory proteins. DAX1 binds Nur77 directly and represses its ability to enhance transcription of the previously mentioned P450 c17 gene.

Fig.1.Schematic representation of the domain structure of nuclear receptors. Nuclear receptors are composed of an N-terminal domain (N-term), a central DNA-binding domain (DBD) and a ligand-binding domain (LBD). The amino acid similarity between the individual domains of Nur77 with Nurr1 and NOR-1 is given in percentages below the domains.

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The interactome of NOR-1

NOR-1 is less well studied than Nur77 and Nurr1 and most of the data on interacting proteins of NOR-1 are presented in studies that are mainly focused on its homologues. As a consequence, NOR-1 protein– protein interactions are described with limited detail, for example the HATp300/CBPacetylatesNOR-1similarlyasNur77,however,theeffect on NOR-1 activity has not been described [79]. Likewise, NOR-1 interacts with the co-regulator TIF1β resulting in enhanced NOR-1 activity, but the domain involved in the interaction is unknown [48]. Similar to Nur77, PKC and RSK1/2 were shown to induce NOR-1 mitochondrial translocation [73,79] and DNA-PK binds the DBD of NOR-1. Even though Nurr1 and Nur77 are both essential for optimal DSB repair the function of NOR-1 in this process remains to be studied [68]. Both FHL2 and the peptidyl-prolyl isomerase Pin1 bind the N-terminal domain and DBD of NOR-1, resulting in reduced or enhanced transcriptional activity of NOR-1, respectively [59,64]. Muscat and co-workers performed detailed studies to identify coregulatorsofNOR-1andwerethefirsttorevealtheabsenceofaconventional ligand-binding pocket in the LBD of NOR-1, through molecular modeling and hydrophobicity analysis of the LBD [104]. Based on these analyses, the relative importance of the N-terminal domain of NOR-1 in regulation of the transcriptional activity of NOR-1 became apparent and direct interaction of a number of crucial co-regulators to this domain was shown;SRC-2 (GRIP-1), SRC-1, SRC-3, p300, DRIP250/ TRAP220 and PCAF [104]. The interaction between the N-terminal domain of NOR-1 and TRAP220 is independent of PKA- and PKC phosphorylation sites in TRAP220. Most interestingly, the purine derivative 6-mercaptopurine, which enhances the activity of NR4As without directly binding these nuclear receptors promotes the interaction between NOR-1 and TRAP220 [105]. Both Nur77 and NOR-1 are involved in T-cell receptor mediated apoptosis of developing T cells [106]. During activation of T cells the expressionofNOR-1isinducedandproteinkinaseC(PKC)becomesactive.NOR-1is aPKCsubstratethat isphosphorylatedand subsequently translocatesfromthenucleustothemitochondriawhereitbindsBcl-2. Most interestingly, as already indicated above the interaction between NOR-1/Nur77 and Bcl-2 causes a conformational change in Bcl-2 allowing its BH3 domain to be exposed, resulting in the conversion of Bcl-2 from an anti-apoptotic into a pro-apoptotic protein. For Nur77 it is exactly known which amino acids are involved to provoke the functional switchin Bcl-2, whichis not thecasefor NOR-1 [57,79]. Initially, the homeobox domain containing protein Six3 was identified in a yeast-two-hybrid study as a protein that interacts uniquely withtheDBDandLBDofNOR-1withoutbindingorinhibitingtheactivity of Nur77 or Nurr1. Of interest, NOR-1 and Six3 show overlap in expression in the rat fetal forebrain on embryonic day 18 [107]. In a later study this specificity of Six3 forNOR-1 was not found, rather interaction with all three NR4As was observed [108]. NOR-1 is part of the EWS/NOR-1 fusion protein that is expressed in human extraskeletal myxoid chondrosarcoma tumors. Six3 enhances the activity of NOR-1 (and Nur77 and Nurr1), whereas the activity of EWS/NOR-1 is inhibited and the interaction only requires the DBD of NOR-1. The opposing data in these two studies may be explained by the use of different cell types for the activity assays, as well as the use of Gal4-fusion proteins in the latter study. PARP-1 specifically and effectively interacts with theDBD of NOR-1 independent of the enzymatic activity of PARP-1 [69]. Nurr1 interacts with lower affinity, whereas EWS/NOR-1 and Nur77 do not bind PARP-1, unless the N-terminal domain of Nur77 is deleted. The latter experiment nicely illustrates that the N-terminal domains of Nur77 and EWS/NOR-1 disturb PARP-1 interaction with the DBD. This may be the underlying mechanism of differential function of NOR-1 and the EWS/NOR-1 fusion protein. In line with the binding characteristics, PARP-1 only inhibits the activity of NOR-1 effectively, again independently of the ribose polymerase activity of PARP-1.

Table 5 NOR-1 interacting proteins.

Fig.2. Nur77 and its interacting proteins. Schematic overview of the protein–protein interactions with Nur77 for which the domains of interaction have been elucidated. Details are described in the text and in Tables 1–3, which also contain the full names of the indicated proteins. N-term, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain.

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Fig.3. Nur77 and kinases modulating its activity and localization. A, Overview of the amino-acid sequence of Nur77 with known phosphorylation sites and associated kinases indicated (T= threonine,S= serine). B,Schematic illustration of effects of different kinases on Nur77 transcriptional activity and subcellular localization. See Table3 for definitions of the abbreviations of the kinases shown.

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Discussion and concluding remarks
This review summarizes the currently available knowledge on the protein–protein interactions of the NR4A nuclear receptor family and their downstream effects. When looking at the information gathered in this review three main observations can be made. First, there are a large number of protein–protein interactions that regulate the activity of Nur77 and there is a large variation in the effects of these interactions on the ‘target’ protein, be it Nur77 or the interacting protein itself. These effects include modulation of transcriptional activity, protein stability, post-translational modification and cellular localization: all processes that are tightly regulated by ligand binding in other nuclear receptors. In light of the many interactions it undergoes with other proteins, Nur77 could also be considered to be a molecular ‘chameleon’: a protein that selectively adopts the responsiveness of other proteins by directly interacting with them. Secondly, the protein–protein interactions with Nur77 described in this review have been studied in a wide range of cell types, such as immune cells (T-cells, thymocytes, monocytes and macrophages); somatic cells(neurons,smooth muscle cells,endothelial
cells and hepatocytes) and cancer cells from diverse origins.We reason that a stimulus- and cell type-specific expression pattern of interacting proteins may be decisive in determining both the interactions of NR4 As with other proteins and their activity in general.The well-studied interaction between Nur77 and RXRα, which has unique outcomes depending on both the cell type studied and the stimulus used, is one such interaction that is modulated by stimulus- or cell type- specific auxiliary proteins. Lastly, there is a large amount of overlap in interacting proteins between the three NR4A nuclear receptors. All three domains of the NR4As are involved in interactions with other proteins (Tables 1–5, Fig. 2), and we think that the unstructured N-terminal domains are of special interest as they have the lowest overall amino acid similarity (Fig. 1). Based on this dissimilarity, it could be hypothesized that the N-terminal domain of each NR4A receptor interacts with a unique set of proteins that specifically regulates each of their activities, if it were not for the fact that this review has shown that the interacting partners of the NR4As strongly overlap. However, a closer look at the N-terminal domains of Nur77, Nurr1 and NOR-1 reveals small stretches of relatively high similarity within the amino acid sequences (Fig. 4). The possible importance of these small stretches of high similarity is most readily apparent when looking at phosphorylation sites of the NR4As.

Fig. 4. Amino-acid sequence similarity between the N-terminal domains of the NR4A receptors. The amino-acid sequence of the N-terminal domains of Nur77, Nurr1 and NOR-1 was aligned and the extent of sequence similarity is indicated with colors; e.g. blue indicates the regions where the sequence of the three NR4As is identical. In the Nur77 sequence, the CHEK2 target Thr88, the JNK1 target Ser95, the ERK2 target Thr143, the CK2 target Ser152, and the DNA-PK target Ser164 are indicated with arrows. In the Nurr1 sequence, the ERK2 targets Ser126 and Thr132, and the ERK5 targets Thr168 and Ser177 are indicated with arrows.

 

 

7.5.2 The interplay of NR4A receptors and the oncogene–tumor suppressor networks in cancer

Beard JA, Tenga A, Chen T
Cell Signal. 2015 Feb; 27(2):257-66
http://dx.doi.org/10.1016/j.cellsig.2014.11.009

Highlights

  • The expression and function of NR4As are dysregulated in multiple cancer types.
  • NR4As are positively regulated by oncogenic signaling pathways.
  • NR4As are capable of inhibiting tumor suppressor signaling.
  • The connectedness of NR4As with these pathways mediate their functions in cancer.
  • NR4A agonists and antagonists offer therapeutic strategies for cancer treatment.

Abstract

Nuclear receptor (NR) subfamily 4 group A (NR4A) is a family of three highly homologous orphan nuclear receptors that have multiple physiological and pathological roles, including some in cancer. These NRs are reportedly dysregulated in multiple cancer types, with many studies demonstrating pro-oncogenic roles for NR4A1 (Nur77) and NR4A2 (Nurr1). Additionally, NR4A1 and NR4A3 (Nor-1) are described as tumor suppressors in leukemia. The dysregulation and functions of the NR4A members are due to many factors, including transcriptional regulation, protein-protein interactions, and post-translational modifications. These various levels of intracellular regulation result from the signaling cross-talk of the NR4A members with various signaling pathways, many of which are relevant to cancer and likely explain the family members’ functions in oncogenesis and tumor suppression. In this review, we discuss the multiple functions of the NR4A receptors in cancer and summarize a growing body of scientific literature that describes the interconnectedness of the NR4A receptors with various oncogene and tumor suppressor pathways.

NR4As are positively regulated by oncogenic signaling pathways

NR4A subfamily of nuclear receptors

NR4A subfamily of nuclear receptors

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intracellular regulation result from the signaling cross-talk of the NR4A members

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7.5.3 NLRX1 acts as tumor suppressor by regulating TNF-α induced apoptosis

Singh K, Poteryakhina A, Zheltukhin A, …Chumakov PM, Singh R.
Biochim Biophys Acta. 2015 May; 1853(5):1073-86
http://dx.doi.org/10.1016/j.bbamcr.2015.01.016

Highlights

  • NLRX1 sensitizes cancer cells to TNF induced cell death by regulating Caspase-8.
  • NLRX1 localizes to mitochondria (mt) and regulates TNF induced mt-ROS generation.
  • Mitochondrial association of Caspase-8 with NLRX1 may regulate mt-ETC function.
  • NLRX1 expression in cancer cells suppresses tumorigenicity in nude mice.

Chronic inflammation in tumor microenvironment plays an important role at different stages of tumor development. The specific mechanisms of the association and its role in providing a survival advantage to the tumor cells are not well understood. Mitochondria are emerging as a central platform for the assembly of signaling complexes regulating inflammatory pathways, including the activation of type-I IFN and NF-κB. These complexes in turn may affect metabolic functions of mitochondria and promote tumorigenesis. NLRX1, a mitochondrial NOD-like receptor protein, regulate inflammatory pathways, however its role in regulation of cross talk of cell death and metabolism and its implication in tumorigenesis is not well understood. Here we demonstrate that NLRX1 sensitizes cells to TNF-α induced cell death by activating Caspase-8. In the presence of TNF-α, NLRX1 and active subunits of Caspase-8 are preferentially localized to mitochondria and regulate the mitochondrial ROS generation. NLRX1 regulates mitochondrial Complex I and Complex III activities to maintain ATP levels in the presence of TNF-α. The expression of NLRX1 compromises clonogenicity, anchorage-independent growth, migration of cancer cells in vitro and suppresses tumorigenicity in vivo in nude mice. We conclude that NLRX1 acts as a potential tumor suppressor by regulating the TNF-α induced cell death and metabolism.

 

7.5.4 The Mre11 Complex Suppresses Oncogene-Driven Breast Tumorigenesis and Metastasis

Gupta GPVanness KBarlas AManova-Todorova KOWen YHPetrini JH
Mol Cell. 2013 Nov 7;52(3):353-65
http://dx.doi.org/10.1016%2Fj.molcel.2013.09.001

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic onco-gene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

The DNA damage response (DDR) network comprises DNA repair, DNA damage signaling, apoptosis, and cell-cycle checkpoint functions (Ciccia and Elledge, 2010). Two lines of evidence support the view that the DDR is a barrier to tumorigenesis. Mutations affecting components of the DDR are frequently associated with predisposition to cancer (Ciccia and Elledge, 2010). Also, indices of DDR activation are evident in preneoplastic lesions or in cultured cells harboring activated oncogenes (Bart-kova et al., 2005Gorgoulis et al., 2005). Despite supportive genetic data from in vitro and tumor inoculation studies (Bartkova et al., 2006;Di Micco et al., 2006), causal demonstration that the oncogene-induced DDR suppresses tumorigenesis within a tissue context remains limited (Gorrini et al., 2007Squatrito et al., 2010Takacova et al., 2012). In certain contexts, the role for ataxia telangiectasia mutated (ATM) in suppressing onco-gene-driven tumorigenesis was relatively minor, although these mouse models were limited by the fact that ATM−/− mice are prone to early spontaneous lymphomagenesis (Efeyan et al., 2009).

The mechanism for DDR activation in response to oncogene expression remains incompletely understood, but the prevailing view posits that oncogene activation leads to replication stress in the form of stalled, and subsequently collapsed, DNA replication forks (Halazonetis et al., 2008). Analysis of the ATRSeckel mouse has indicated that ATR may be required for cell viability upon oncogene activation, suggesting that DNA replication stress may indeed underlie these effects of oncogene activation (López-Contreras et al., 2012;Murga et al., 2011Schoppy et al., 2012). However, since ATR promotes viability, rather than elimination of the oncogene-expressing cells, this outcome is not consistent with a barrier function for that component of the DDR. The purpose of this study was to delineate the particular aspects of the DDR network that constitute barriers to oncogenesis using a mouse model of sporadic, oncogene-driven breast cancer.

The Mre11 complex is a sensor of DNA double-strand breaks (Stracker and Petrini, 2011). Hypomorphic mutations in this complex, modeled in the mouse after alleles inherited in ataxiatelangiectasia-like disorder (A-TLD) and Nijmegen breakage syndrome (NBS), have facilitated the elucidation of the Mre11 complex’s role in the ATM-dependent DDR. Here, we utilize these and other mutant mouse strains, individually and in combination, to define the tumor-suppressive functions of the DDR in mammary epithelium.

A Mouse Model of Sporadic, Oncogene-Induced Mammary Neoplasia

Expression of activated NeuT (Bargmann and Weinberg, 1988), the rodent ortholog of the ERBB2/HER2oncogene, in the mammary epithelium of adult mice via the RCAS/MMTVTVA system (Du et al., 2006) results in early DDR activation, and oligoclonal tumors with an average latency of 5 months (Reddy et al., 2010). To delineate the aspects of the DDR primarily relevant for tumor suppression in the face of oncogene activation, we interbred MMTV-TVA mice with a variety of mutant mouse strains with established DDR deficiencies. Age-matched cohorts of female animals (12–18 weeks old) were injected with either RCAS-HA-NeuT or control virus via mammary intraductal injection. The genotypes analyzed wereMre11ATLD1/ATLD1Nbs1ΔBBChk2−/−Nbs1ΔCChk2−/−p53515C/515Cp53−/−, and 53BP1−/−, each of which exhibits defects in DNA-damage-induced cell-cycle checkpoint activation, apoptosis, and/or DNA repair (Figures S1A and S1B available online; Liu et al., 2004Shibata et al., 2010Stracker et al., 20072008Stracker and Petrini, 2011Theunissen et al., 2003Williams et al., 2002). These mouse strains did not exhibit any histopathological deficits in mammary gland development (data not shown), circumventing the potential problem of differences in mammary tissue among the various genetic backgrounds confounding the analyses.

We performed digital quantification of glandular structures relative to total cellular content in the oncogene-expressing mammary glands and normalized this value to the glandular content observed in the matched control mammary glands (Figure 1C). These variations in mammary ductal enlargement, luminal filling, cellular turnover, and glandular density across the different genotypes are summarized in Figure 1D.

NeuT expression in Chk2−/− and Nbs1ΔCChk2−/− mammary epithelium produced hyperplasias that were only modestly dissimilar from WT (Figures 1B–1D; data not shown), suggesting that apoptosis and the intra-S phase checkpoint—diminished in both mutants (Stracker et al., 2008)—do not mediate the early response to oncogene activation. Consistent with that interpretation, p53515C/515C mutants, in which p53-dependent apoptosis is lost (Liu et al., 2004), also exhibited relatively modest hyper-plasia, although some morphological changes were noted (Figures 1B–1D). In contrast, p53−/− mammary glands resembled p53515C/515C morphologically, but exhibited more extensive NeuT-induced hyperplasia (Figures 1B–1D), consistent with additional deficiencies of the null mutant—including, but not limited to, induction of the G1/S checkpoint and senescence pathways.

In contrast to the aforementioned genotypes, oncogene-induced hyperplasia was markedly distinct in Mre11ATLD1/ATLD1 and Nbs1ΔBB mammary glands relative to WT mammary glands (Figures 1B–1D). The Mre11 complex mutant genotypes exhibited florid hyperplasia in response to oncogene expression that frequently filled the lumen of the enlarged mammary ducts. Quantification of hyperplasia across the entire mammary gland revealed that Mre11ATLD1/ATLD1 was associated with the most significant degree of oncogene-induced proliferative change (Figure 1C).

We examined oncogene-dependent activation of the DDR in WT and Mre11ATLD1/ATLD1 mammary hyperplasias. Consistent with prior reports (Reddy et al., 2010), we observed the formation of γH2AX foci and accumulation of 53BP1 nuclear staining in WT hyperplasias after the introduction of NeuT (Figures 2A and 2B). We observed a highly significant, >2-fold reduction in both NeuT-induced γH2AX foci formation and 53BP1 accumulation within Mre11ATLD1/ATLD1 lesions relative to WT (p < 0.0001; Figures 2A and 2B). In contrast to the effects of Mre11 complex hypomorphism, oncogene-dependent DDR activation was unperturbed in p53−/− mammary glands (Figure 2A; data not shown). These data demonstrate that the Mre11 complex is required for DDR activation upon NeuT expression.

The oncogene-driven, Mre11 complex-dependent DDR exhibited dissimilarities from that induced by ionizing radiation (IR). First, oncogene expression in the WT mammary gland resulted in finely punctate 53BP1 staining and did not induce the large foci that develop after irradiation of the mammary gland (Figure S4). In addition, phosphorylation of the ATM target KAP1 at Ser824 was not observed in the oncogene-expressing mammary gland, but was readily detected in IR-treated mammary tissue (Figure 2C). Similarly, we observed significantly less p53 stabilization in mammary epithelial cells after oncogene expression in comparison to irradiated tissue (Figure S4). Hence, the Mre11 complex-mediated response to oncogene activation appears to be qualitatively distinct from the response to clastogen-induced DNA damage.

We examined apoptosis and growth arrest—functional outcomes of DDR activation—in hyperplastic lesions. While NeuT expression was associated with increased proliferation and apoptosis rates relative to control mammary glands, we did not observe a statistically significant difference in TUNEL or Ki67 positivity between WT and Mre11ATLD1/ATLD1 oncogene-induced hyperplasias (Figures 3A and 3B). We observed a 4-fold increase in pHH3-S10 staining in WT versus Mre11ATLD1/ATLD1 hyperplasias (p < 0.001; Figure 3C), which was unexpected given the significantly increased cellularity of Mre11ATLD1/ATLD1 hyperplasias. The pHH3-S10 staining pattern that we observed was punctate, and pHH3-S10-positive nuclei did not exhibit morphological features of mitosis (Figure 3C, inset), suggesting that the pHH3-S10 signal represented pericentromeric staining characteristic of late G2 cells rather than mitotic cells.

Centriole duplication was evident in 84% of pHH3-S10-positive cells, compared to only 16% of pHH3-S10-negative cells (p < 0.0001; Figure 4B), indicating a cell-cycle state that is beyond the G1/S transition. These observations collectively suggest that NeuT expression in mammary epithelium activates a Mre11 complex-dependent G2 arrest or accumulation. Notably, this G2 arrest is distinct from the canonical IR-induced G2/M checkpoint, which is also Mre11 dependent (Theunissen et al., 2003). In that context, pHH3-S10 is not induced, suggesting that the heterochromatin-associated accumulation of this marker is oncogene specific.

The variable and prolonged latency of tumor onset in Mre11ATLD1/ATLD1 animals suggests that additional genetic alterations may be required for NeuT-mediated transformation of mammary epithelial cells. We examined p19Arf expression—a well-established oncogene-induced tumor-suppressive pathway (Sherr, 2001)—in the 3-week-old NeuT-expressing mammary hyperplasias from WT and Mre11ATLD1/ATLD1animals. We observed >10-fold induction of p19Arf after oncogene expression in Mre11ATLD1/ATLD1relative to control-injected mammary glands (Figure 6A). The extent of p19Arf induction in NeuT-expressingWT mammary glands was <50% of that observed in Mre11ATLD1/ATLD1 (p < 0.007, Figure 6A). Notably, there was no difference in HA-NeuT expression levels between the WT and Mre11ATLD1/ATLD1 mice that could account for the elevated levels of p19Arf (Figure S6A). As expected, p53 levels were modestly elevated in Mre11ATLD1/ATLD1 hyperplasias relative to WT (Figure S6B).

Collectively, the findings presented here indicate that the Mre11 complex constitutes an inducible barrier to oncogene-driven neoplasia. In response to oncogene activation, the Mre11 complex mediates a G2 arrest that appears to be qualitatively distinct from that revealed in previous analyses of Mre11 complex-dependent DDR functions (Figure 7EStracker et al., 2004). The arrest is associated with heterochromatin changes, including the appearance of macroH2A and histone H3 (Ser10) phosphorylation. Histone H3 phosphorylation at pericentric heterochromatin begins early in G2 phase and expands as cells enter mitosis (Crosio et al., 2002). That fact, along with the finding that H3 phosphorylation arises in cells that have undergone centriole duplication, indicates that cells in oncogene-expressing hyperplasias accumulate in G2. We cannot exclude the possibility that other NeuT-expressing cells also arrest in G1 without the observed heterochromatic changes. In Mre11ATLD1/ATLD1 mammary epithelium, the NeuT-induced arrest is lost, and macroH2A and histone H3 phosphorylation are not detected in hyperplastic tissue, demonstrating that the G2 accumulation depends on the Mre11 complex.

The Mre11 complex-dependent G2 arrest does not appear permanent, as WT cells are capable at low frequency of progressing to tumors. When the arrest is attenuated, as in Mre11ATLD1/ATLD1, we observe more extensive oncogene-induced mammary hyperplasia, and a significantly greater likelihood of progression to invasive breast cancer. Although previous studies show that the Mre11 complex suppresses genome instability, and thus the risk of spontaneous DNA-damage-associated tumorigenesis (Stracker et al., 2008Theunissen et al., 2003), this study demonstrates that the Mre11 complex also suppresses oncogene-driven neoplasia and tumorigenesis.

An important question concerns the underlying basis of the response to oncogene activation. Given the importance of the Mre11 complex in sensing DNA double-strand breaks and initiating an ATM-dependent DDR, a parsimonious interpretation is that oncogene activation results in DNA damage. Indeed, there are compelling genetic data supporting the induction of DNA replication stress upon oncogene activation (Bartkova et al., 2006Campaner and Amati, 2012Di Micco et al., 2006Dominguez-Sola et al., 2007;López-Contreras and Fernandez-Capetillo, 2010). DNA replication stress is a common precursor of frank DNA damage when forks collapse (Allen et al., 2011), which would readily account for the induction of DNA damage upon oncogene induction.

Potential crosstalk between the oncogene-induced DDR and the Arf tumor suppressor pathways has recently been described (Evangelou et al., 2013Monasor et al., 2013Velimezi et al., 2013). Our data provide direct evidence for a genetic interaction between these pathways during oncogene-driven tumorigenesis. We demonstrate that when Mre11 complex function is impaired, oncogene expression induces Arf expression, and Ink4a-Arf inactivation is commonly observed in the mammary tumors that ensue. The mechanism for how Mre11 hypomorphism promotes oncogene-induced Arf expression remains unclear.  We observe that 40% of the NeuT-induced mammary tumors that developed in Mre11ATLD1/ATLD1 mice had genetic inactivation of the Ink4a-Arf locus, and the remaining tumors exhibited reduced p19Arf expression, suggesting alternative modes of pathway suppression. These findings provide compelling genetic evidence for the cooperative roles of the Mre11 complex and Ink4a-Arf pathways in the suppression of oncogene-driven tumorigenesis and metastasis.

The behavior of the emergent tumors in Mre11ATLD1/ATLD1mice suggests a link between increased chromosomal instability and an elevated rate of metastatic dissemination from the primary tumor. The observation that all of the Ink4a-Arf mutated mammary tumors were lung metastatic also raises the possibility that Arf loss promotes metastatic progression in the context of Mre11 complex impairment.

Our genetic data suggest that functional hypomorphism of this pathway may be a driver of breast tumorigenesis, genomic instability, and metastasis. Given the profound DDR defects associated with Mre11 complex hypomorphism (Stracker and Petrini, 2011), this subset of human breast cancer may exhibit exquisite DNA damage sensitivities that could be therapeutically exploited to improve clinical outcomes.

 

 

7.5.5 Expression of Stromal Cell-derived Factor 1 and CXCR4 Ligand Receptor System in Pancreatic Cancer

Koshiba T, Hosotani R, Miyamoto Y, Ida J, …, Fujii N, Imamura M
Clin Cancer Res Sep; 6(9):3530-5
NR4A subfamily of nuclear receptors
http://clincancerres.aacrjournals.org/content/6/9/3530.long

To examine the expression of the stromal cell-derived factor 1 (SDF-1)/CXCR4 receptor ligand system in pancreatic cancer cells and endothelial cells, we performed immunohistochemical analysis for 52 pancreatic cancer tissue samples with anti-CXCR4 antibody and reverse transcription-PCR analysis for CXCR4 and SDF-1 in five pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, and PANC-1), an endothelial cell line (HUVEC), and eight pancreatic cancer tissues. We then performed cell migration assay on AsPC-1 cells, HUVECs, and CFPAC-1 cells in the presence of SDF-1 or MRC-9 fibroblast cells. Immunoreactive CXCR4 was found mainly in pancreatic cancer cells and endothelial cells of relatively large vessels around a tumorous lesion. The immunopositive ratio in the pancreatic cancer was 71.2%. There was no statistically significant correlation with clinicopathological features. SDF-1 mRNA expressions were detected in all pancreatic cancer tissues but not in pancreatic cancer cell lines and HUVECs; meanwhile, CXCR4 mRNA was detected in all pancreatic cancer tissues, cancer cell lines, and HUVECs. The results indicate that the paracrine mechanism is involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer. In vitro studies demonstrated that SDF-1 significantly increased the migration ability of AsPC-1 and HUVECs, and these effects were inhibited by CXCR4 antagonist T22, and that the coculture system with MRC-9 also increased the migration ability of CFPAC-1 cells, and this effect was significantly inhibited by T22. Our results suggested that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis.

Chemokines belong to the small molecule chemoattractive cytokine family and are grouped into CXC chemokines and CC chemokines, on the basis of the characteristic presence of four conserved cysteine residues (123) . Chemokines mediate the chemical effect on target cells through G-protein-coupled receptors, which are characterized structurally by seven transmembrane spanning domains and are involved in the attraction and activation of mononuclear and polymorphonuclear leukocytes. The effects of CXC chemokines on cancer cells have been investigated in the case of IL3 -8. Several studies have demonstrated the presence of IL-8 and its receptor in tumor tissues, which were involved in vascular endothelial cell proliferation and tumor neovascularization ,(4567) . It was also reported that IL-8 inhibited non-small cell lung cancer proliferation via the autocrine and paracrine pathway (8) . IL-8 produced by malignant melanoma was found to induce cell proliferation via the autocrine pathway in vitro (9) . These studies indicate that IL-8 is involved in the regulation of tumor progression through tumor angiogenesis and/or direct cancer cell growth.

SDF-1 was initially cloned by Tashiro et al. (10) and later identified as a growth factor for B cell progenitors, a chemotactic factor for T cells and monocytes, and in B-cell lymphopoiesis and bone marrow myelopoiesis (111213) . SDF-1 is a member of the CXC subfamily of chemokines, and its chemotactic effect is mediated by the chemokine receptor CXCR4 (12 , 14) . Most of the chemokine receptors interact with pleural ligands, and vice versa, but the SDF-1/CXCR4 receptor ligand system has been shown to involve a one-on-one interaction (15 , 16) . Furthermore, CXCR4 has been shown to function as a coreceptor for T lymphocytotrophic HIV-1 isolates (17) . Recent studies have demonstrated that endothelial cells express CXCR4 and are strongly chemoattracted by SDF-1 (1819,20) . Tachibana et al. (15) reported that in the embryo of CXCR4 or SDF-1 knockout mice larger branches of the superior mesenteric artery were missing and that the resultant abnormal circulatory system led to gastrointestinal hemorrhage and intestinal obstruction. These findings suggest that SDF-1 and CXCR4 are involved in organ vascularization, as well as in the immune and hematopoietic system.

To clarify the role of the SDF-1/CXCR4 receptor ligand system in pancreatic cancer, we have investigated the expression of CXCR4 and SDF-1 with the aid of immunohistochemical analysis and RT-PCR in pancreatic cancer tissue and experimental chemotactic activity of SDF-1 in pancreatic cancer cells and vascular endothelial cells in vitro.

The distribution of CXCR4 protein expression in pancreatic cancer tissue was examined by means of immunohistochemical analysis of pancreatic cancer tissue samples obtained at surgical operation. Fig. 1<$REFLINK> shows representative immunostainings of cancerous and noncancerous regions in pancreatic cancer tissues. Staining of the CXCR4 protein was identified in the cytoplasm and/or cell membrane of cancer cells, but was not detected in the normal acinar cells and ductal cells of noncancerous region in pancreatic cancer tissue. Negative or weak staining for the CXCR4 protein was observed in a majority of the infiltrating inflammatory cells in the specimens. The immunopositive ratio of cancer cells in the pancreatic cancer tissue specimens was 71.2% (37 of 52). Table 1<$REFLINK>summarizes the relationship between CXCR4 expression and clinicopathological features of 52 pancreatic cancers. There was no significant correlation between the expression of CXCR4 protein and the clinicopathological variables examined (i.e., tumor extension, lymph node metastasis, liver metastasis, and Union International Contre Cancer stage). CXCR4 immunoreactivities were observed in endothelial cells of relatively large vessels around the tumorous lesions, but were scarcely found in the endothelial cells of microvessels inside tumorous lesions (Fig. 2, A and B)<$REFLINK> .

We performed RT-PCR using specific primers, as described in“ Materials and Methods,” to confirm CXCR4 and SDF-1 mRNA expression in pancreatic cancer cells, endothelial cells (HUVECs), and pancreatic cancer tissues. CXCR4 mRNA expressions were clearly detected in five pancreatic cancer cell lines, HUVECs, and eight pancreatic cancer tissue samples (Fig. 3a)<$REFLINK> . On the other hand, SDF-1 mRNA expression was not detected in five pancreatic cancer cell lines and HUVECs, but was identified in eight pancreatic cancer tissue samples (Fig. 3b)<$REFLINK> .

Transwell migration assays were performed to examine the effects of SDF-1 on motility of pancreatic cancer cells (AsPC-1) and endothelial cells (HUVEC). At a concentration of 100 ng/ml, SDF-1 induced chemotaxis of AsPC-1 cells, which was approximately double that of the control. One micromolar of T22 (CXCR4 antagonist) and 10 μg/ml of IVR7 (neutralizing CXCR4 antibody) completely blocked the chemotaxis of AsPC-1 induced by 100 ng/ml SDF-1 (Fig. 4a)<$REFLINK> . At a concentration of 100 g/ml SDF-1 induced an approximately quadruple chemotaxis of HUVECs. One micromolar of T22 caused a 33% reduction of the chemotaxis of HUVECs in the presence of containing 100 ng/ml SDF-1 (Fig. 4b)<$REFLINK> .

SDF-1 belongs to the CXC chemokine family and is a ligand for CXCR4. The role of the SDF-1/CXCR4 receptor ligand system has been investigated mainly in the field of immunology, especially in the mechanism of infection of T lymphocytotrophic HIV-1 and for the prevention of HIV-1 infection. Investigators have also paid attention to the role of the SDF-1/CXCR4 receptor ligand system in cancer tissues.

In this study, we first used immunohistochemical methods to examine CXCR4 expression in pancreatic cancer tissues. Immunoreactive CXCR4 was found in the cytoplasm and/or cell membrane of pancreatic cancer cells. Although CXCR4 staining in pancreatic cancer tissue was heterogeneous and showed differences between specimens, it was found mainly in cancer cells: the immunopositive ratio for the pancreatic cancer tissue specimens was 71.2% (37 of 52). There was a tendency for the immunopositive ratio of CXCR4 in tumors with lymph node metastasis or liver metastasis to be higher than in tumors without these features, but no statistically significant correlation with clinicopathological features were found. There is a diversity of views on the role of the SDF-1/CXCR4 receptor ligand system in malignant tissues. In the current study, SDF-1 mRNA expressions were detected in all pancreatic cancer tissues (eight of eight) but were not detected in pancreatic cancer cell lines (zero of five), whereas CXCR4 mRNA was detected in both pancreatic cancer tissues (eight of eight) and cancer cell lines (five of five). The results indicate that the paracrine mechanism may be involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer.

Our results suggest that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis. Because T22 suppressed the migration of both pancreatic cancer cells and endothelial cells in vitro, additional in vivo studies are warranted to examine whether T22 suppresses the tumor spread and tumor angiogenesis to clarify the role of the SDF-1/CXCR4 receptor ligand system in pancreatic cancer.

 

7.5.6 DLC1- a significant GAP in the cancer genome

Aurelia Lahoz and Alan Hall
Genes Dev. 2008 Jul 1; 22(13): 1724–1730
http://dx.doi.org/10.1101.2Fgad.1691408

Rho GTPases are believed to make important contributions to the development and progression of human cancer, but direct evidence in the form of somatic mutations analogous to those affecting Ras has been lacking. A recent study in Genes & Development by Xue and colleagues (1439–1444) now provides in vivo evidence that DLC1, a negative regulator of Rho, is a tumor suppressor gene deleted almost as frequently as p53 in common cancers such as breast, colon, and lung.

Cancer is a complex set of diseases arising from combinations of genetic and epigenetic events, including base mutations, chromosomal rearrangements, DNA methylation, and chromatin modification. Genetic changes were first seen cytologically and revealed gross chromosomal abnormalities, such as translocations, deletions, amplifications (of entire chromosomes or parts of chromosomes), and inversions. Subsequently, DNA sequencing of candidate genes and then whole genomes has uncovered large numbers of more subtle genetic alterations. The recent and continuing successes of sequencing and other nonfunctional based genomic approaches have raised new problems in how to determine which changes have significance for tumor development. This is not a trivial problem and will require combinations of cell-based assays, in vivo animal models, and ultimately clinical intervention.

The identification of the Ras oncogene was the first major triumph of the early application of molecular biology to the cancer problem (Malumbres and Barbacid 2003). Although originally identified as a viral oncogene in a rodent sarcoma-inducing retrovirus, it was the seminal work of the Weinberg and Cooper laboratories in 1981 (Krontiris and Cooper 1981Shih et al. 1981), using DNA transfection assays of human tumor DNA into immortalized mouse fibroblasts, that led to the identification of Ras as a true human oncogene. Several groups went on to show that any one of the three Ras genes (HRASKRAS, and NRAS) could be converted into a human oncogene by a single base mutation leading to a single amino acid substitution in the encoded Ras protein. Ras mutations are found in ∼30% of most, though not all, cancer types and it remains the most frequently mutated dominant oncogene so far identified (Bos 1989). We now know much about the consequences of those amino acid substitutions and the cellular and physiological importance of Ras in controlling proliferation and differentiation. Ras is an example of a regulatory GTPase that cycles between active (GTP-bound) and inactive (GDP-bound) conformations to control biochemical pathways and processes. These molecular switches are activated by guanine nucleotide exchange factors (GEFs), which catalyze exchange of GDP for GTP, and are inactivated by GTPase-activating proteins (GAPs), which promote the otherwise slow, intrinsic GTPase activity of the proteins (Fig. 1). The amino acid substitutions identified in Ras in human cancers are found at codons 12, 61, and to a lesser extent 13, and the common consequence of these changes is to prevent GAP-mediated stimulation of GTP hydrolysis leading to permanent activation of the switch (Trahey and McCormick 1987). Inspection of Figure 1 suggests possible alternative ways in which this molecular switch could be inappropriately activated. For example, activating mutations in one of the nine RasGEF genes or inactivation of one of the eight RasGAP genes could lead to hyperactivation of the switch. To date, no such mutations have been reported in GEF genes in human cancers, but one of the GAPs, neurofibromin, is encoded by the NF1 tumor suppressor gene. Patients with neurofibromatosis type I inherit only one functional NF1 gene and are then predisposed to cancer through complete loss of NF1. In addition, mutational activation of components of downstream signaling pathways (Fig. 1) could bypass the need for Ras and this is clearly the case with somatic mutations in BRAF (which encodes a Ras effector), found most frequently in malignant melanomas (>50%), but also in thyroid, colorectal, and ovarian cancer (Davies et al. 2002Wellbrock et al. 2004).

The Ras GTP.GDP cycle

The Ras GTP.GDP cycle

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732422/bin/1724fig1.jpg

Figure 1. The Ras GTP/GDP cycle. Ras GTPases are molecular switches and the GDP/GTP cycle is controlled by GEFs and GAPs. The output of the switch is through the interaction of Ras.GTP with effector proteins.

Rho GTPases can trigger numerous downstream signaling pathways by interacting with distinct effectors—to date, ∼20 such target proteins have been reported that specifically interact with Rho (Etienne-Manneville and Hall 2002). One of the best-characterized is Rho kinase (ROCK), which regulates myosin II and actin filament contractility, through its ability to phosphorylate and inactivate myosin light chain phosphatase (Fukata et al. 2001). Rho kinase is involved in many aspects of normal cell biology, such as cell cycle, morphogenesis, and migration, and in addition has been shown to participate in the proliferation, invasion, and metastasis of cancer cells (Etienne-Manneville and Hall 2002Sahai and Marshall 2002Narumiya and Yasuda 2006). In the final part of their study, Xue et al. (2008) show that two small molecule Rho kinase inhibitors, Y-27632 and to a lesser extent Fasudil, inhibit in vitro colony formation of p53−/− liver progenitor cells expressing c-Myc and DCL1 shRNA. It should be noted, however, that both Y-27632 and Fasudil inhibit PRK/PKN and citron kinase, two other kinases activated by Rho, so the result is not entirely conclusive (Ishizaki et al. 2000).

Embryonic fibroblasts can be obtained from DLC1−/− mice and these display alterations in the organization of actin filaments and focal adhesion (Durkin et al. 2005). Confusingly, however, these knockout cells have fewer stress fibers and focal adhesions—the opposite of what would have been predicted for the loss of a GAP that regulates Rho. In fact the cytoskeletal and adhesion complex changes seen in DLC−/− fibroblasts appear to be more in keeping with Rac activation. Unfortunately the authors did not examine the levels of either Rho.GTP or Rac.GTP in these cells, which might have provided some insight into this unexpected result. In the absence of tissue-specific mouse knockouts, we must look to work in Drosophila on RhoGAP88C, the fly ortholog of DCL1, to provide some in vivo physiological data. Mutations in RhoGAP88C were first identified as crossveinless-c and result in defects in tissue morphogenesis during development (Denholm et al. 2005). Closer examination suggests that this GAP regulates tubulogenesis and convergent extension, two processes driven by reorganization of the actin cytoskeleton. An additional and provocative observation to emerge from this study is that RhoGAP88C acts through Rho in some tissues, but it acts through Rac and not Rho in others. The in vitro biochemical activity of this GAP has not been determined and so it is possible that it shows a different specificity from its mammalian counterpart. Otherwise, tissue-specific modification of its catalytic activity would need to be invoked, rendering the in vitro assays essentially useless for predicting specificity. Two subsequent studies have concluded that RhoGAP88C is localized basolaterally in epithelial cells and serves to restrict Rho activity to the apical surface and thereby generate morphogenetic tissue remodeling through polarized activation of myosin II (Brodu and Casanova 2006Simoes et al. 2006).

Taken together, a picture emerges of spatially localized DLC1 acting to control Rho activity so as to promote changes in the actin cytoskeleton during cell morphogenesis. The disruption of this pathway might be expected to lead to tissue disorganization during differentiation programs, which could promote inappropriate cell proliferation (Fig. 2).

DLC1 is a tumor suppressor.

DLC1 is a tumor suppressor.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732422/bin/1724fig2.jpg

Figure 2.  DLC1 is a tumor suppressor. Loss of DLC1 leads to deregulated and/or delocalized activation of Rho. This may disrupt tissue morphogenesis leading to inappropriate proliferation. (PM) Plasma membrane.

Directed therapeutic intervention depends on a deep understanding of the relevant signaling pathways through which DLC1 loss is manifest. It is a sobering thought that the signaling pathways downstream from Ras responsible for human cancer are still debated some 25 years after its discovery as a human oncogene and it would be optimistic to believe that identifying Rho pathways will be any easier. Inhibiting the GTPase itself, whether Ras or Rho, is challenging. One of the most promising potential targets for Ras inactivation has been farnesyltransferase (FT), the enzyme required for carboxy-terminal, post-translational modification by a farnesyl lipid (Wright and Philips 2006). FT inhibitors are currently in clinical trials, though the data reported so far are not encouraging. Inhibiting Rho using a similar strategy seems less attractive, since it uses a geranylgeranyltransferase to add a geranylgeranyl group; a much more widespread modification than farnesyl addition. Two other processing enzymes that act on both Ras and Rho, a carboxyl-protease and an isoprenylcysteine carboxyl methyltransferase, are being considered as Ras targets, but in tissue culture at least these seem not to be essential for Rho function (Michaelson et al. 2005). Another possibility that is distinctive to DLC1 might be to attack the epigenetic mechanisms that appear to be commonly used to silence this gene in human cancers. Inhibitors of DNA methyltransferase and histone deacetylase (HDAC) have already been shown to induce the restoration of DLC1 expression in cancer cells, making Zebularine, a new and highly effective DNA demethylating agent, as well as HDAC inhibitors attractive therapeutic approaches (Guan et al. 2006Neureiter et al. 2007Seng et al. 2007Xu et al. 2007). Finally, if it turns out that Rho kinase mediates the key signaling pathway downstream from DLC1 loss, then there is already a huge effort underway to develop small molecule inhibitors of this protein. Rho kinase has been implicated in various forms of cardiovascular disease—such as pulmonary hypertension, myocardial hypertrophy, and atherosclerosis—and in fact one compound, Fasudil, is already being used clinically in Japan for cerebral ischemia (Rikitake and Liao 2005Tawara and Shimokawa 2007). With over a dozen pharmaceutical companies reportedly working on this problem, and if the work from Xue et al. (2008) implicating Rho kinase downstream from DLC1 turns out to be correct, those companies may end up with a blockbuster!

 

7.5.7 DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma.

Xue W, Krasnitz A, Lucito R, Sordella R, … , Zender L, Lowe SW.
Genes Dev. 2008 Jun 1;22(11):1439-44
http://dx.doi.org/10.1101.2Fgad.1672608

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

Tumor suppressor genes act in signaling networks that protect against tumor initiation and progression, and can be inactivated by deletions, point mutations, or promoter hypermethylation. Although tumor suppressors are rarely considered direct drug targets, they can negatively regulate pro-oncogenic signaling proteins that are amenable to small molecule inhibition. For instance, NF1 inhibits the Ras signaling pathway, which is deregulated in many cancers and has been pursued for its therapeutic potential (Downward 2003). Similarly, PTEN inhibits the PI3–kinase pathway, and inhibitors of PI3K pathway components such as PI3K, AKT, and mTORs have entered clinic trials (Luo et al. 2003).

Recurrent chromosomal deletions found in sporadic cancers often contain tumor suppressor genes. For example, PTEN loss on chromosome 10q23 frequently occurs in various cancers and promotes tumorigenesis by deregulating the PI3 kinase pathway (Maser et al. 2007). Similarly, heterozygous deletions on chromosome 8p22 in many hepatocellular carcinomas (HCC) (Jou et al. 2004) and other cancer types, including carcinomas of the breast, prostate, colon, and lung (Matsuyama et al. 2001Durkin et al. 2007). Several genes, including DLC1MTUS1FGL1 and TUSC3, have been identified as candidate tumor suppressors in this region (Yan et al. 2004). Deleted in Liver Cancer 1 (DLC1) is a particularly attractive candidate owing to its genomic deletion, promoter methylation, and underexpressed mRNA in cancer (Yuan et al. 19982003aNg et al. 2000Wong et al. 2003Guan et al. 2006Seng et al. 2007Ying et al. 2007;Zhang et al. 2007Pike et al. 2008; for review, see Durkin et al. 2007).

Despite its potential importance, functional data implicating DLC1 loss in tumorigenesis are lacking. DLC1encodes a RhoGAP protein that catalyzes the conversion of active GTP-bound RhoGTPase (Rho) to the inactive GDP-bound form and thus suppresses Rho activity (Yuan et al. 1998). DLC1 has potent GAP activity for RhoA and limited activity for CDC42 (Wong et al. 2003Healy et al. 2008). When overexpressed, DLC1 inhibits the growth of tumor cells and xenografts (Yuan et al. 2003b2004Zhou et al. 2004Wong et al. 2005Kim et al. 2007), but whether this requires its Rho-GAP activity or other functions remains unresolved (Qian et al. 2007Liao et al. 2007). Most functional studies to date have relied on DLC1 overexpression and, as yet, none have documented that loss of DLC1 promotes transformation in vitro or tumorigenesis in vivo. Indeed, homozygous dlc1 knockout mice die around embryonic day 10.5 (E10.5), and there is no overt phenotype in dlc1 heterozygous mice (Durkin et al. 2005).

Our laboratory recently developed a “mosaic” mouse model whereby liver carcinomas can be rapidly produced with different genetic alterations by manipulation of cultured embryonic liver progenitor cells (hepatoblasts) followed by transplantation into the livers of recipient mice (Zender et al. 20052006). We previously used this model to identify new oncogenes in HCC, which could be characterized in an appropriate biological and genetic context (Zender et al. 2006). Furthermore, using this system, we showed that shRNAs capable of suppressing gene function by RNAi could recapitulate the consequences of tumor suppressor gene loss on liver carcinogenesis (Zender et al. 2005Xue et al. 2007). Here we combine this mosaic model and RNAi to validate DLC1 as a potent tumor suppressor gene and study its action in vivo.

Studies using low-resolution genome scanning methods have identified chromosome 8p deletions as common lesions in liver carcinoma and other tumor types. To confirm and extend these observations, we examined a series of data sets of copy number alterations in HCC obtained using representational oligonucleotide microarray analysis (ROMA), a variation of array-based CGH that enables genome scanning at high resolution (Lucito et al. 2003). In a panel of 86 liver cancers, heterozygous deletions encompassing theDLC1 were observed in 59 tumors (Fig. 1A,B; data not shown). Consistent with previous reports, these deletions were large (>5 Mb), encompassing >20 annotated genes but invariably included the DLC1 locus. Indeed, heterozygous deletions of DLC1 occurred more frequently than those observed for the well-established tumor suppressors such as INK4a/ARFPTEN, and TP53 (Fig. 1C). Furthermore, DLC1deletions were nearly as common as those for TP53 in other major tumor types such as lung, colon, and breast (Fig. 1C). Again, most 8p deletions were large, although in breast cancer DLC1 resided at a local deletion epicenter reminiscent of that surrounding the INK4a/ARF locus on chromosome 9p21 (Fig. 1D,E). Although we did not examine the status of the remaining allele in this tumor cohort, studies suggest that it can be silenced by promoter methylation (Yuan et al. 2003a; for review, see Durkin et al. 2007). Together, these data suggest that DLC1 loss plays an important role in human cancer but, in the absence of functional validation, are not conclusive.

Genetically modified liver progenitors were seeded into the livers of syngeneic recipients to assess their ability to form tumors in situ. In contrast to the modest impact of DLC1 loss in vitro, DLC1 shRNAs significantly accelerated tumor onset in vivo (P value < 0.0001 for shDLC1-1 and P < 0.0005 for shDLC1-2) (Fig. 2D,E). In fact, at 57 d post-transplantation, GFP-positive tumor nodules were observed in the livers of most animals receiving cells harboring DLC1 shRNAs, whereas the control animals showed no macroscopically detectable tumor burden (Fig. 2E). Furthermore, the pathology of tumors derived from DLC1 knockdown resembled aggressive human HCC and displayed a high proliferative index as assessed by Ki67 immunohistochemistry (Fig. 2F). Tumors also expressed the HCC markers α-fetoprotein (AFP) and albumin (Supplemental Fig. S3B). These data demonstrate that loss of DLC1 can efficiently promote the development of HCC.

We also ectopically expressed the murine dlc1 gene in mouse hepatoma cells and tested their ability to form tumors orthotopically. To this end, we cloned a Myc-tagged murine dlc1 cDNA and confirmed its ability to produce a protein of the correct molecular weight (Fig. 3A). A mouse hepatoma cell line harboring a luciferase reporter and expressing oncogenic Ras and undetectable DLC1 (see Fig. 1F, lane 8) was infected with the DLC1-expressing retrovirus or an empty vector. Consistent with the literature (Ng et al. 2000), reintroduction of DLC1 produced a modest effect on proliferation in colony formation assays (Supplemental Fig. S4A,B).

Although RhoA has been identified as a DLC1 effector, overexpression studies suggest that other DLC1 functions can contribute to its anti-proliferative activities (Liao et al. 2007Qian et al. 2007). To determine whether RhoA is required for maintaining tumorigenesis stimulated by DLC1 loss, we tested whether suppression of RhoA in DLC1-suppressed hepatoma lines would impact their expansion as subcutaneous tumors in immunocompromised mice. shRNAs capable of down-regulating RhoA to varying degrees (Fig. 5A) decreased the in vivo growth of two independent murine hepatoma lines with undetectable DLC1 (Fig. 5B, cell lines 1,2; Supplemental Fig. S6A,B). Of note, none of the shRNAs completely suppressed RhoA expression, and their ability to limit tumor expansion was proportional to their knockdown efficiency (Supplemental Fig. S6A). The impact of these shRNAs was less pronounced in hepatoma cell lines with higher DLC1 levels (Fig. 5B, cell lines 3,4; Supplemental Fig. S6C,D). Although complete inhibition of RhoA activity might be generally cytostatic (see Piekny et al. 2005), these data suggest that RhoA is required for maintaining the growth of tumors with attenuated DLC1 activity.

In this study, we combined in vivo RNAi and a mosaic mouse model of HCC to study the impact of DLC1 loss on liver carcinogenesis in mice, which to date has not been possible owing to the embryonic lethality of DLC1 knockout animals. We show that DLC1 loss, when combined with other oncogenic lesions, promotes HCC in vivo and that RhoA activation is both necessary and sufficient for its effects. In our survey of copy number alterations in human tumors, 8p22 deletions encompassing DLC1 occurred in >60% of heptocellular carcinomas as well as a large portion of human lung, breast, and colon carcinomas (see also Durkin et al. 2007). Similarly, RhoA is up-regulated in HCC and many other tumor types (Sahai and Marshall 2002;Fukui et al. 2006). Although other tumor suppressor genes may also reside in the 8p region, our results demonstrate that DLC1 is functionally important and highlight the potential importance of the RhoA signaling network in epithelial cancers.

Molecularly targeted therapies have been devised for inhibiting several oncogenic pathways, including those affected by BCR-ABL, activated Ras and PI3kinase (Downward 2003Luo et al. 2003). Although tumor suppressors are generally not amenable to direct therapeutic targeting, their mutation may confer a cellular dependency on downstream oncogenic proteins that can be inhibited with small molecule drugs. In this regard, the impact of DLC1 loss may parallel that produced by loss of PTEN, which deregulates the PI3K pathway and can sensitize cells to pharmacological inhibitors of downstream effectors such as mTOR (Maser et al. 2007). Our data indicate that RhoA is required for maintaining at least some tumors driven by DLC1 loss, and that cells with disabled DLC1 are particularly sensitive to inhibitors that target at least one RhoA effector. Clearly, more studies will be required to confirm and extend these observations; nevertheless, the high frequency of DLC1 loss in human cancer implies that pharmacologic intervention of the signaling pathways modulated by DLC1 may have broad therapeutic utility.

 

7.5.8 Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma

Feng T, Dzieran J, Gu X, Marhenke S, Vogel A, …, Dooley S, Meindl-Beinker NM.
Clin Sci (Lond). 2015 Jun 1; 128(11):761-74
http://dx.doi.org:/10.1042/CS20140606

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Hepatocellular carcinoma is one of the most common malignancies worldwide, and it has a poor prognosis due to its rapid development and early metastasis. An understanding of tumor metabolism would be helpful for the clinical diagnosis and therapy of hepatocellular carcinoma. Chronic hepatitis B virus infection is the primary risk factor for hepatocellular carcinoma, and the majority of hepatocellular carcinoma cases develop from hepatitis infections and subsequent cirrhosis. Rapid development and early metastasis are the typical characteristics of hepatocellular carcinoma, which always results in a poor prognosis. Therefore, investigating the hepatocarcinogenesis mechanism is very important for decreasing the incidence and mortality of hepatocellular carcinoma. The abnormal metabolism of cancer has been considered an important characteristic of tumors, which could clarify the pathogenesis and provide potential therapeutic targets for clinical treatments. According to the Warburg effect, the deregulated energy metabolism of cancer cells may also modify many related metabolic pathways that influence various biological processes, such as cell proliferation and apoptosis. As a common characteristic of cancer cells, modified metabolism has been the focus of cancer research.

Because of its asymptomatic nature, hepatocellular carcinoma is usually diagnosed at late and advanced stages, for which there are no effective therapies. Thus, biomarkers for early detection and molecular targets for treating hepatocellular carcinoma are urgently needed. Emerging high-throughput metabolomics technologies have been widely applied, aiming at the discovery of candidate biomarkers for cancer staging, prediction of recurrence and prognosis, and treatment selection. Tissue metabolomics is a useful tool for studying the abnormal metabolisms of diseases, and it can provide information about the metabolic modifications and the upstream regulative mechanism in diseases. More importantly, the systemic metabolic characteristics of tissues could provide opportunities for exploring novel diagnostic markers or therapeutic targets for clinical applications. Tissue metabolomics is conducted using a pairwise comparison of different parts of tissue from each patient, which can remove individual differences, such as age, sex, region, etc. The differences between the tumor cells and their surrounding host cells may reflect the interactions of the tumor and the host, which are important clues for studying the invasion and metastasis of tumors. Metabolic profiles, which are affected by many physiological and pathological processes, may provide further insight into the metabolic consequences of this severe liver disease. Small-molecule metabolites have an important role in biological systems and represent attractive candidates to understand hepatocellular carcinoma phenotypes. The power of metabolomics allows an unparalleled opportunity to query the molecular mechanisms of hepatocellular carcinoma.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed/23824744

http://www.ncbi.nlm.nih.gov/pubmed/23150189

http://onlinelibrary.wiley.com/doi/10.1002/hep.26350/abstract

http://www.ncbi.nlm.nih.gov/pubmed/21114800

http://www.ncbi.nlm.nih.gov/pubmed/19305372

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Minimally invasive image-guided therapy for inoperable hepatocellular carcinoma

Curator & Reporter: Dror Nir, PhD

Large organs like the liver are good candidates for focused treatment. The following paper:

Minimally invasive image-guided therapy for inoperable hepatocellular carcinoma: What is the evidence today?

By Beatrijs A. Seinstra1, et. al. published mid-2010, gives a review of the state-of-the-art of the then available methods for local lesions’ ablation. As far as ablation techniques availability, I have found this review very much relevant to today’s technological reality. It is worthwhile noting that in the last couple of years, new imaging-based navigation and guidance applications were introduced into the market holding a promise to improve the accuracy of administrating such treatment. These are subject to clinical validation in large clinical studies.  From the above mentioned publication I have chosen to highlight the parts discussing the importance of imaging-based guidance to the effective application of localized ablation-type therapies.

The clinical need:

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver that accounts for an important health problem worldwide. Primary liver cancer is the sixth most common cancer worldwide with an incidence of 626,000 patients a year, and the third most common cause of cancer-related death [1]. Only 10–15% of HCC patients are suitable candidates for hepatic resection and liver transplantation due to the advanced stage of the disease at time of diagnosis and shortage of donors.

Immerging solution:

In order to provide therapeutic options for patients with inoperable HCC, several minimally invasive image-guided therapies for locoregional treatment have been developed. HCC has a tendency to remain confined to the liver until the disease has advanced, making these treatments particularly attractive.

Minimally invasive image-guided therapies can be divided into the group of the tumor ablative techniques or the group of image-guided catheter-based techniques. Tumor ablative techniques are either based on thermal tumor destruction, as in radiofrequency ablation (RFA), cryoablation, microwave ablation, laser ablation and high-intensity focused ultrasound (HIFU), or chemical tumor destruction, as in percutaneous ethanol injection (PEI). These techniques are mostly used for early stage disease. Image-guided catheter-based techniques rely on intra-arterial delivery of embolic, chemoembolic, or radioembolic agents [22]. These techniques enable treatment of large lesions or whole liver treatment, and are as such used for intermediate stage HCC (Figure 1).

Minimally invasive image-guided ablation techniques and intra-arterial interventions may prolong survival, spare more functioning liver tissue in comparison to surgical resection (which can be very important in cirrhotic patients), allow retreatment if necessary, and may be an effective bridge to transplantation [2327].

During the last 2 decades, minimally invasive image-guided therapies have revolutionized the management of inoperable HCC.

The value of image guidance

Accurate imaging is of great importance during minimally invasive loco-regional therapies to efficiently guide and monitor the treatment. It enables proper placement of instruments, like the probe in case of ablation or the catheter in case of intra-arterial therapy, and accurate monitoring of the progression of the necrotic zone during ablation.

can all be employed. In current clinical practice, placement of the catheter in intra-arterial procedures is usually performed under fluoroscopic guidance, while ablation may be guided by ultrasound, CT or MRI.

  • Ultrasound guidance allows probe insertion from every angle, offers real time visualization and correction for motion artifacts when targeting the tumor, and is low cost. However, the gas created during ablation (or ice in the case of cryoablation) hampers penetration of the ultrasound beams in tissue, causing acoustic shadowing and obscuring image details like the delineation between tumor borders and ablation zone.
  • CT is also frequently used to guide minimally invasive ablation therapy, and is a reliable modality to confirm treatment results. In comparison to US, it provides increased lesion discrimination, a more reliable depiction of ablated/non-ablated interfaces, and a better correlation to pathologic size [28]. However, due to its hypervascularity, small HCCs can only be clearly visualized in the arterial phase for a short period of time. Another disadvantage of CT is the exposure of the patient and physician to ionizing radiation.
  • Combining US imaging for probe placement and CT for ablation monitoring reduces this exposure. At the moment, hybrid systems are being developed, enabling combination of imaging techniques, like ultrasound and CT imaging, thereby improving the registration accuracy during treatment [29]. The interest in MRI-guided ablation is growing, as it produces a high-quality image allowing high-sensitivity tumor detection and accurate identification of the target region with multiplanar imaging.
  • MRI also enables real-time monitoring of the temperature evolution during treatment [3035]. However, MRI is an expensive technique, and MRI-guided ablation is still limited in clinical practice. Currently, the most widely used ablation technique for percutaneous treatment of focal hepatic malignancies is radiofrequency ablation (RFA), which has been shown to be safe and effective for the treatment of early stage HCC [4850]. During RFA, a small electrode is placed within the tumor, and a high-frequency alternating electric current (approximately 400 MHz) is generated, causing ionic agitation within the tissue. ….. Most frequently ultrasound is used for image guidance (Figs. 23), but there are reports of groups who use CT, MRI, or fluoroscopic imaging.
Ultrasound guided RFA. a: HCC lesion in a non-surgical patient pre-treatment (pointed out by arrow). b: Just after start treatment, electrode placed centrally in the tumor. c: Gas formation during ablation causes acoustic shadowing

Ultrasound guided RFA. a: HCC lesion in a non-surgical patient pre-treatment (pointed out by arrow). b: Just after start treatment, electrode placed centrally in the tumor. c: Gas formation during ablation causes acoustic shadowing

Contrast-enhanced CT pre- and post-RFA. Same patient as in Fig. 2. a: Hypervascular lesion (biopsy proven HCC) in right liver lobe (pointed out by arrow) before treatment. b: Ablated lesion directly post ablation, with reactive hyperemia around the RFA lesion

Contrast-enhanced CT pre- and post-RFA. Same patient as in Fig. 2. a: Hypervascular lesion (biopsy proven HCC) in right liver lobe (pointed out by arrow) before treatment. b: Ablated lesion directly post ablation, with reactive hyperemia around the RFA lesion

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Reporter and Curator: Ritu Saxena, PhD

Magnetic Resonance Imaging (MRI) is increasingly used in clinical diagnostics, for a rapidly growing number of indications. The MRI technique is non-invasive and can provide information on the anatomy, function and metabolism of tissues in vivo (Strijkers GJ, et al, Anticancer Agents Med Chem, May 2007;7(3):291-305). Basic contrast in the MRI image scans is as a result of contrast generated by differences in the relaxation times between different regions. Since the intrinsic contrast generated between regions is limited to allow clear and specific diagnosis, MRI contrast agents administered intravenously are increasingly being used to alter image contrast.

Gadoxetic acid, a gadolinium-based compound, is a recently developed hepatobiliary-specific contrast material for MRI that has high sensitivity in the detection of malignant liver tumors. Its salt, gadoxetate disodium, is marketed as Primovist in Europe and Eovist in the United States by Bayer HealthCare Pharmaceuticals. Gadoxetic acid is taken up by hepatocytes and then excreted into the bile ducts (Schuhmann-Giampieri G, et al, Radiology, Apr 1992;183(1):59-64). Therefore, hepatic focal lesions without normal hepatobiliary function are depicted as hypointense areas compared with the well-enhanced hyperintense background liver in the hepatobiliary phase of gadoxetic acid–enhanced MR imaging. In addition, gadoxetic acid can be used in the same way as gadopentetate dimeglumine to evaluate the hemodynamics of hepatic lesions in the dynamic phase after an intravenous bolus injection (Kitao A, et al, Radiology, Sep 2010;256(3):817-26).

Recently, researchers from Kanazawa University Graduate School of Medical Science, (Kanazawa, Japan) analyzed the correlation among biologic features, tumor marker production, and signal intensity at gadoxetic acid-enhanced MR imaging in hepatocellular carcinomas (HCCs). The findings were published in Radiology journal. The research was supported in part by a Grant-in-Aid for Scientific Research (21591549) from the Ministry of Education, Culture, Sports, Science and Technology; and by Health and Labor Sciences Research Grants for “Development of novel molecular markers and imaging modalities for earlier diagnosis of hepatocellular carcinoma.”

Research significance: HCC is the most frequent primary malignant tumor of liver and is the third most common cause of cancer death worldwide. It is the most Hepatocellular.

The accurate detection and characterization of HCC focal lesions is crucial for improving prognosis of patients with HCC.

Research problem: Gadoxetic acid–enhanced MR imaging is highly accurate for diagnosing HCC lesions. As discussed earlier, in this imaging process, hepatic focal lesions without normal hepatobiliary are hypointense as compared with the well-enhanced hyperintense background liver. However, approximately 6%–15% of hypervascular HCCs demonstrate isointensity or hyperintensity (Kitao A, et al, Eur Radiol, Oct 2011;21(10):2056-66).

Hypothesis: The reason for hyperintensity in some HCC lesions was previously shown to be due to overexpression of organic anion transporting polypeptide 8 (OATP8) (Kitao A, et al, Radiology, Sep 2010;256(3):817-26). The authors speculated that there might be a correlation of the tumor marker production and signal intensity (SI) on hepatobiliary phase images, which would reflect distinct genomic and proteomic expression of HCC. Thus, authors stated that “the purpose of this study was to analyze the correlation among the pathologic and biologic features, tumor marker production, with signal intensity (SI) on hepatobiliary phase gadoxetic acid–enhanced MR images of HCC” (Kitao A, et al, Radiology, Dec 2012;265(3):780-9).

Experimental design: From April 2008 to September 2011, 180 surgically resected HCCs in 180 patients (age, 65.0 years ± 10.3 [range, 34–83 years]; 138 men, 42 women) were classified as either hypointense (n = 158) or hyperintense (n = 22) compared with the signal intensity of the background liver on hepatobiliary phase gadoxetic acid–enhanced MR images (Abstract of the study).

Pathologic features were analyzed.

Serum analysis and immunohistochemical staining was performed and following were compared:

  1. Alpha fetoprotein (AFP) – is a main tumor marker of HCCs. AFP is the most abundant plasma protein found in the human fetus and plasma levels decrease rapidly after birth. A level above 500 nanograms/milliliter of AFP in adults can be indicative of hepatocellular carcinoma, germ cell tumors, and metastatic cancers of the liver.
  2. Absence of protein induced by vitamin K or antagonist-II (PIVKA-II) – is a clinically important serum tumor marker. PIVKAII is an incomplete coagulation factor prothrombin II whose production is related to the absence of vitamin K or the presence of the antagonist of vitamin K, which is the cofactor of g carboxylase that converts precursor into prothrombin.

Serum levels of both AFP and PIVKA-II correlate with HCC malignancy and prognosis (Miyaaki H, et al, J Gastroenterol, Dec 2007;42(12):962-8).

Results: The hyperintense HCCs showed significantly higher differentiation grade than the hypointense HCCs (P = .028). There was a significant difference in the proliferation pattern between the hypointense and hyperintense HCCs (P < .001) and the hyperintense HCCs showed a significantly lower rate of portal vein invasion than that of hypointense HCCs (P = .039). The serum levels of tumor markers AFP, AFP-L3, and PIVKA-II were significantly lower in the patients with hyperintense HCCs than in those with

hypointense HCCs (P = .003, .004, and .026). In addition, immunohistochemical analysis revealed that the expression of FP and PIVKA-II was lower in hyperintense than in hypointense HCCs (both P < .001). Also, hyperintense HCCs showed lower recurrence rate than hypointense HCCs (P = .039).

Conclusion: Variation was observed within differently stained lesions of HCC in the hepatobiliary phase gadoxetic acid–enhanced MR images as evident in tumor marker expression, proliferation pattern, differentiation grade, immunohistochemical analysis and recurrence.  The results lead to the hypothesis that hyperintense HCCs in the hepatobiliary phase gadoxetic acid–enhanced MR images might represent a particular type of HCC that is hypervascular and biologically less aggressive as compared to hypovascular HCCs. Interestingly, this research is another great example where tumor heterogeneity has been brought to light (similar to genetic heterogeneity in triple negative breast cancer deciphered by Lehmann BD, et al, 2011). The heterogeneity might be the basis of answers to why a particular therapy fails in a certain tumor type and fortifying evidence for appropriate analysis of the tumor for obtaining the desired tumor response from a particular drug.

Reference:

Kitao A, et al, Radiology, Dec 2012;265(3):780-9

Strijkers GJ, et al, Anticancer Agents Med Chem, May 2007;7(3):291-305

Schuhmann-Giampieri G, et al, Radiology, Apr 1992;183(1):59-64

Kitao A, et al, Radiology, Sep 2010;256(3):817-26

Kitao A, et al, Eur Radiol, Oct 2011;21(10):2056-66

Kitao A, et al, Radiology, Sep 2010;256(3):817-26

Miyaaki H, et al, J Gastroenterol, Dec 2007;42(12):962-8

Lehmann BD, et al, J Clin Invest, 2011;121(7):2750–2767

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