Posts Tagged ‘Wnt signaling pathway’

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 4/23/2019

This has an updated lesson on WNT signaling.  Please click on the following and look at the slides labeled under lesson 10

cell motility 9b lesson_2018_sjw

Remember our lessons on the importance of signal termination.  The CANONICAL WNT signaling (that is the β-catenin dependent signaling)

is terminated by the APC-driven degradation complex.  This leads to the signal messenger  β-catenin being degraded by the proteosome.  Other examples of growth factor signaling that is terminated by a proteosome-directed include the Hedgehog signaling system, which is involved in growth and differentiation as well as WNTs and is implicated in various cancers.

A good article on the Hedgehog signaling pathway is found here:

The Voice of a Pathologist, Cancer Expert: Scientific Interpretation of Images: Cancer Signaling Pathways and Tumor Progression

All images in use for this article are under copyrights with Shutterstock.com

Cancer is expressed through a series of transformations equally involving metabolic enzymes and glucose, fat, and protein metabolism, and gene transcription, as a result of altered gene regulatory and transcription pathways, and also as a result of changes in cell-cell interactions.  These are embodied in the following series of graphics.

Figure 1: Sonic_hedgehog_pathwaySonic_hedgehog_pathway

The Voice of Dr. Larry

The figure shows a modification of nuclear translocation by Sonic hedgehog pathway. The hedgehog proteins have since been implicated in the development of internal organs, midline neurological structures, and the hematopoietic system in humans. The Hh signaling pathway consists of three main components: the receptor patched 1 (PTCH1), the seven transmembrane G-protein coupled receptor smoothened (SMO), and the intracellular glioma-associated oncogene homolog (GLI) family of transcription factors.5The GLI family is composed of three members, including GLI1 (gene activating), GLI2 (gene activating and repressive), and GLI3 (gene repressive).6 In the absence of an activating signal from either Shh, Ihh or Dhh, PTCH1 exerts an inhibitory effect on the signal transducer SMO, preventing any downstream signaling from occurring.7 When Hh ligands bind and activate PTCH1, the inhibition on SMO is released, allowing the translocation of SMO into the cytoplasm and its subsequent activation of the GLI family of transcription factors.


And from the review of  Elaine Y. C. HsiaYirui Gui, and Xiaoyan Zheng   Regulation of Hedgehog Signaling by Ubiquitination  Front Biol (Beijing). 2015 Jun; 10(3): 203–220.

the authors state:

Finally, termination of Hh signaling is also important for controlling the duration of pathway activity. Hh induced ubiquitination and degradation of Ci/Gli is the most well-established mechanism for limiting signal duration, and inhibiting this process can lead to cell patterning disruption and excessive cell proliferation (). In addition to Ci/Gli, a growing body of evidence suggests that ubiquitination also plays critical roles in regulating other Hh signaling components including Ptc, Smo, and Sufu. Thus, ubiquitination serves as a general mechanism in the dynamic regulation of the Hh pathway.

Overview of Hedgehog signaling showing the signal termination by ubiquitnation and subsequent degradation of the Gli transcriptional factors. obtained from Oncotarget 5(10):2881-911 · May 2014. GSK-3B as a Therapeutic Intervention in Cancer


















Note that in absence of Hedgehog ligands Ptch inhibits Smo accumulation and activation but upon binding of Hedgehog ligands (by an autocrine or paracrine fashion) Ptch is now unable to inhibit Smo (evidence exists that Ptch is now targeted for degradation) and Smo can now inhibit Sufu-dependent and GSK-3B dependent induced degradation of Gli factors Gli1 and Gli2.  Also note the Gli1 and Gli2 are transcriptional activators while Gli3 is a transcriptional repressor.

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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Nonhematologic Cancer Stem Cells [11.2.3]

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

Nonhematologic Stem Cells C8orf4 negatively regulates self-renewal of liver cancer stem cells via suppression of NOTCH2 signalling

Pingping Zhu, Yanying Wang, Ying Du, Lei He, Guanling Huang, et al.
Nature Communications May 2015; 6(7122). http://dx.doi.org:/10.1038/ncomms8122

Liver cancer stem cells (CSCs) harbor self-renewal and differentiation properties, accounting for chemotherapy resistance and recurrence. However, the molecular mechanisms to sustain liver CSCs remain largely unknown. In this study, based on analysis of several hepatocellular carcinoma (HCC) transcriptome datasets and our experimental data, we find that C8orf4 is weakly expressed in HCC tumors and liver CSCs. C8orf4 attenuates the self-renewal capacity of liver CSCs and tumor propagation. We show that NOTCH2 is activated in liver CSCs. C8orf4 is located in the cytoplasm of HCC tumor cells and associates with the NOTCH2 intracellular domain, which impedes the nuclear translocation of N2ICD. C8orf4 deletion causes the nuclear translocation of N2ICD that triggers the NOTCH2 signaling, which sustains the stemness of liver CSCs. Finally, NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Altogether, C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Like stem cells, CSCs are characterized by self-renewal and differentiation simultaneously9. Not surprisingly, CSCs share core regulatory genes and developmental pathways with normal tissue stem cells. Accumulating evidence shows that NOTCH, Hedgehog and Wnt signaling pathways are implicated in the regulation of CSC self-renewal4. NOTCH signaling modulates many aspects of metazoan development and tissue stemness1011. NOTCH receptors contain four members (NOTCH1–4) in mammals, which are activated by engagement with various ligands. The aberrant NOTCH signaling was first reported to be involved in the tumorigenesis of human T-cell leukaemia1213. Recently, a number of studies have reported that the NOTCH signaling pathway is implicated in regulating self-renewal of breast stem cells and mammary CSCs1415. However, how the NOTCH signaling regulates the liver CSC self-renewal remains largely unknown.

C8orf4, also called thyroid cancer 1 (TC1), was originally cloned from a papillary thyroid carcinoma and its surrounding normal thyroid tissue16. C8orf4 is ubiquitously expressed across a wide range of vertebrates with the sequence conservation across species. A number of studies have reported that C8orf4 is highly expressed in several tumors and implicated in tumorigenesis171819. In addition, C8orf4 augments Wnt/β-catenin signaling in some cancer cells2021, suggesting it may be involved in the regulation of self-renewal of CSCs. However, the biological function of C8orf4 in the modulation of liver CSC self-renewal is still unknown. Here we show that C8orf4 is weakly expressed in HCC and liver CSCs. NOTCH2 signaling is highly activated in HCC tumors and liver CSCs. C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

C8orf4 is weakly expressed in HCC tissues and liver CSCs

To search for driver genes in the oncogenesis of HCC, we performed genome-wide analyses using several online-available HCC transcriptome datasets by R language and Bioconductor approaches. After analysing gene expression profiles of HCC tumor and peri-tumor tissues, we identified >360 differentially expressed genes from both Park’s cohort (GSE36376; ref. 22) and Wang’s cohort (GSE14520; refs 2324). Of these changed genes, we focused on C8orf4, which was weakly expressed in HCC tumors derived from both Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) (Fig. 1a). Lower expression of C8orf4 was further confirmed in HCC samples by quantitative reverse transcription–PCR (qRT–PCR) and immunoblotting (Fig. 1b,c). In this study, HCC patient samples we used included all subtypes of HCC. In addition, these observations were further validated by immunohistochemical (IHC) staining (Fig. 1d). These data indicate that C8orf4 is weakly expressed in HCC tumor tissues.

C8orf4 is weakly expressed in HCC tumours and liver CSCs

C8orf4 is weakly expressed in HCC tumours and liver CSCs

Figure 1. C8orf4 is weakly expressed in HCC tumours and liver CSCs


(a)C8orf4 is weakly expressed in HCC patients. Using R language and Bioconductor methods, we analyzed C8orf4 expression in HCC tumor and peri-tumor tissues provided by Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) datasets. (b,c) C8orf4 expression levels were verified in HCC patient samples by quantitative RT–PCR (qRT–PCR) (b) and immunoblotting (c). β-actin served as a loading control. 18S: 18S rRNA. (d) HCC samples were assayed by immunohistochemical staining. Scale bar—left: 50 μm; right: 20 μm. (eC8orf4 is weakly expressed in CD13+CD133+ cells sorted from Huh7 cells and primary HCC samples. C8orf4 messenger RNA (mRNA) was measured by qRT–PCR. Six HCC samples got similar results. (fC8orf4 is much more weakly expressed in oncospheres than non-sphere tumor cells. Non-sphere: Huh7 or HCC primary cells that failed to form spheres. (g) HCC sample tissues were co-stained with anti-C8orf4 and anti-CD13 or anti-CD133 antibodies, then counterstained with DAPI for confocal microscopy. White arrows indicate CD13+ or CD133+ cells. Scale bars: 20 μm. For a,b, data are shown as box and whisker plot. Boxes represent interquartile range (IQR); upper and lower edge corresponds to the 75th and 25th percentiles, respectively. Horizontal lines within boxes represent median levels of gene intensity. Whiskers below and above boxes extend to the 5th and 95th percentiles, respectively. For e and f, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. P, peri-tumor; T, tumor.


Notably, C8orf4 was also weakly expressed in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) by analysis of its expression profiles derived from online datasets (GSE14897; ref. 25 and GSE25417; ref. 26) (Supplementary Fig. 1a,b). C8orf4 was also lowly expressed in normal liver stem cells (Supplementary Fig. 1c,d), suggesting that C8orf4 may be involved in the regulation of self-renewal of liver stem cells. Thus, we propose that C8orf4 might play a role in the maintenance of liver CSCs. Since CD13 and CD133 were widely used as liver CSC surface markers, we sorted CD13+CD133+ cells from Huh7 and Hep3B HCC cell lines as well as HCC samples, serving as liver CSCs. We observed that C8orf4 was weakly expressed in liver CSCs enriched from both HCC cell lines and patient samples (Fig. 1e). Six HCC samples were analyzed for these experiments. Similar results were obtained in CD13+CD133+ cells from Hep3B cells. Furthermore, we performed sphere formation experiments using Huh7 cells and HCC primary sample cells, and detected expression levels of C8orf4. We observed that C8orf4 was dramatically reduced in the oncospheres generated by both HCC cell lines and patient samples (Fig. 1f). In addition, we noticed that C8orf4 expression was negatively correlated with liver CSC markers such as CD13 and CD133 in HCC samples (Fig. 1g), suggesting lower expression of C8orf4 in liver CSCs. Moreover, C8orf4 was mainly located in the cytoplasm of tumour cells. Altogether, C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs.

C8orf4 negatively regulates self-renewal of liver CSCs

We then wanted to look at whether C8orf4 plays a critical role in the self-renewal maintenance of liver CSCs. C8orf4 was knocked out in Huh7 cells through a CRISPR/Cas9 system (Fig. 2a). TwoC8orf4-knockout (KO) cell strains were established and C8orf4 was completely deleted in these two strains. C8orf4 deletion dramatically enhanced oncosphere formation (Fig. 2b). We co-stained SOX9, a widely used progenitor marker, and Ki67, a well-known proliferation marker, in C8orf4 KO sphere cells. We found that SOX9 was strongly stained in C8orf4 KO sphere cells (Supplementary Fig. 2a). In contrast, Ki67 staining was not significantly altered in C8orf4 KO sphere cells versus WT sphere cells. We also digested sphere cells and examined the SOX9 and Ki67 expression by flow cytometry. Similar results were achieved (Supplementary Fig. 2b). Importantly, through serial passage of CSC sphere cells, similar observations were obtained in the fourth generation oncosphere assay (Supplementary Fig. 2c,d). These data suggest that C8orf4 is involved in the regulation of liver CSC self-renewal.

(not shown)

Figure 2: C8orf4 knockout enhances self-renewal of liver CSCs.


  • C8orf4-deficient Huh7 cells were established using a CRISPR/Cas9 system. T7 endonuclease I cleavage confirmed the efficiency of sgRNA (left panel, white arrowheads), and C8orf4-knockout efficiency was confirmed by western blot (right panel). Two knockout cell lines were used.  C8KO#1:C8orf4KO#1;  C8KO#2C8orf4KO#2. (bC8orf4-deficient cells enhanced sphere formation activity. Calculated ratios are shown in the right panel. (cC8orf4-deficient or WT Huh7 cells (1 × 106) were injected into BALB/c nude mice. Tumor sizes were observed every 5 days. (dC8orf4 deficiency enhances tumor-initiating capacity. Diluted cell numbers of Huh7 cells were implanted into BALB/c nude mice for tumor initiation. Percentages of tumor-formation mice were calculated (left panel), and frequency of tumor-initiating cells was calculated using extreme limiting dilution analysis (right panel). Error bars represent the 95% confidence intervals of the estimation. (e) Expression levels of CD13 andCD133 were analyzed in C8orf4-knockout Huh7 cells. (f) C8orf4 was silenced in HCC primary cells and C8orf4 depletion enhanced sphere formation activity. Calculated ratios are shown at the right panel. Three HCC specimens obtained similar results. (g) C8orf4-overexpressing Huh7 cells were established (left panel). C8orf4-overexpressing Huh7 cells and control Huh7 cells were cultured for sphere formation. (h,i) Xenograft tumor growth (h) and frequency of tumor-initiating cells (i) for C8orf4-overexpressing Huh7 cells were analyzed as c,d. (j) C8orf4 overexpression reduces expression of CD133 and CD13 in Huh7 cells. (k) C8orf4 was transfected in HCC primary cells and cultured for sphere formation. Three HCC patient samples obtained similar results. Scale bars: b,f,g,k, 500 μm. Student’s t-test was used for statistical analysis,    *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data represent at least three independent experiments. oeC8orf4, overexpression of C8orf4; oeVec, overexpression vector.

In addition, C8orf4-deficient Huh7 cells overtly increased xenograft tumour growth (Fig. 2c). We then performed sphere formation and digested oncospheres formed by C8orf4-deficient or WT cells into single-cell suspension, then subcutaneously implanted 1 × 104, 1 × 103, 1 × 102 and 10 cells into BALB/c nude mice. Tumour formation was examined for tumour-initiating capacity at the third month. C8orf4 deficiency remarkably enhanced tumour-initiating capacity and liver CSC ratios (Fig. 2d). In addition, C8orf4 deletion significantly enhanced expression levels of the liver CSC markers such as CD13 and CD133 (Fig. 2e). We also silenced C8orf4 in HCC primary cells using a lentivirus infection system and established C8orf4-silenced cells. Two pairs of short hairpin RNA (shRNA) sequences obtained similar knockdown efficiency. C8orf4 knockdown remarkably promoted sphere formation and xenograft tumour growth (Fig. 2f and Supplementary Fig. 2e). These data indicate that C8orf4 deletion potentiates the self-renewal of liver CSCs.
We next overexpressed C8orf4 in Huh7 cells and HCC primary cells using lentivirus infection. We observed that C8orf4 overexpression in Huh7 cells remarkably reduced sphere formation and xenograft tumour growth (Fig. 2g,h). In addition, C8orf4 overexpression remarkably reduced tumour-initiating capacity and expression of liver CSC markers (Fig. 2i,j). Similar results were observed by C8orf4 overexpression in HCC primary cells (Fig. 2k). We tested three HCC samples with similar results. Overall, C8orf4 negatively regulates the maintenance of liver CSC self-renewal and tumour propagation.

C8orf4 suppresses NOTCH2 signaling in liver CSCs

To further determine the underlying mechanism of C8orf4 in the regulation of liver CSCs, we analyzed three major self-renewal signaling pathways, including Wnt/β-catenin, Hedgehog and NOTCH pathways, in C8orf4-deleted Huh7 cells and HCC primary cells. We found that only NOTCH target genes were remarkably upregulated in C8orf4-deficient cells (Fig. 3a), whereasC8orf4 deficiency did not significantly affect the Wnt/β-catenin or the Hedgehog pathway. Given that the NOTCH family receptors have four members, we wanted to determine which NOTCH member was involved in the C8orf4-mediated suppression of liver CSC stemness. We noticed that only NOTCH2 was highly expressed in both Huh7 cells and HCC samples (Fig. 3b). In addition, this result was also confirmed by analysis of NOTCH expression levels derived from Wang’s cohort (GSE14520) and Petel’s cohort (E-TABM-36; ref. 27) (Fig. 3c). Moreover, we analysed expression profiles of C8orf4 and NOTCH target genes using Park’s cohort (GSE36376) and Wurmbach’s cohort (GSE6764; ref. 28). These cohort datasets provided several Notch signaling and its target genes. HEY1NRARP and HES6 genes were highly expressed in HCC tumour tissues (GSE6764; ref. 28), which were further confirmed in HCC samples by real-time PCR (Supplementary Fig. 3a,b). Furthermore, HEY1NRARP and HES6 genes have been reported to be relatively specific NOTCH target genes. We then examined these three genes as the NOTCH2 target genes throughout this study. We found that the C8orf4 expression level was negatively correlated with the expression levels of HEY1 and HES6, suggesting that C8orf4 inhibited NOTCH signaling in HCC patients (Fig. 3d). Finally these results were further confirmed in HCC samples by qRT-PCR (Fig. 3e). To further explore the activation status of NOTCH2 signaling in liver CSCs, we examined the expression levels of NOTCH downstream target genes in oncospheres and CD13+CD133+ cells derived from both Huh7 cells and HCC cells. We observed that NOTCH target genes were highly expressed in liver CSCs (Fig. 3f,g). These observations were verified by immunoblotting (Fig. 3h). In addition, the expression levels of NRARPHES6 and HEY1 were positively related to the expression levels of EpCAM and CD133 derived from Zhang’s cohort (GSE25097; ref. 29) and Wang’s cohort (GSE14520; Supplementary Fig. 3c,d). These data suggest that the NOTCH2 signaling plays a critical role in the maintenance of self-renewal of liver CSCs.

(not shown)

Figure 3: C8orf4 suppresses NOTCH2 signaling in liver CSCs.


(aC8orf4 deficiency or depletion activates NOTCH signaling. The indicated major stemness signalling pathways were analysed in C8orf4-knockout Huh7 cells (left panel) and C8orf4-silenced primary cells of HCC samples (right panel). (b) Four receptor members of NOTCH family were examined in both Huh7 cells (left panel) and 29 pairs of HCC samples (right panel). (cNOTCH receptors were analyzed from Wang’s cohort (left panel) and Petel’s cohort (right panel) datasets. (dHEY1 and HES6 were highly expressed in C8orf4low samples by analysis of Park’s cohort (upper panel) and Wurmbach’s cohort (lower panel). (e) Expression levels of HEY1 and HES6 along with C8orf4 were analysed in HCC samples by qRT–PCR. (f,g) Expression levels of NRARPHEY1 and HES6 in spheres generated by Huh7 cells and HCC primary cells (f) and in CD13+CD133+ cells sorted from Huh7 cells and HCC primary cells (g). Non-sphere: Huh7 cells or HCC cells that failed to form spheres. (h) HEY1, HES6 and NRARP expression in sphere and non-sphere cells was detected by immunoblotting. β-actin was used as a loading control. For c,d, data are shown as box and whisker plot. Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For a,b,f,g, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

C8orf4 interacts with NOTCH2 that is critical for liver CSCs

On ligand–receptor binding, the NOTCH receptor experiences a proteolytic cleavage by metalloprotease and γ-secretase, releasing a NOTCH extracellular domain (NECD) and a NOTCH intracellular domain (NICD), respectively30. Then the active NICD undergoes nuclear translocation and activates the expression of NOTCH downstream target genes31.Then we constructed the NOTCH2 extracellular domain (N2ECD) and intracellular domain (N2ICD) and examined the interaction with C8orf4 via a yeast two-hybrid approach. Interestingly, we found that C8orf4 interacted with N2ICD, but not N2ECD (Fig. 4a). The interaction was validated by co-immunoprecipitation (Fig. 4b). Through domain mapping, the ankyrin repeat domain of NOTCH2 was essential and sufficient for its association with C8orf4 (Fig. 4c). Taken together, C8orf4 interacts with the N2ICD domain of NOTCH2.

Figure 4: C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs.

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs


(a) C8orf4 interacts with N2ICD. Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD) fused C8orf4 and Gal4-activating domain (AD) fused N2ICD. p53 and large T antigen were used as a positive control. (b) Recombinant Flag-N2ICD and GFP–C8orf4 were incubated for co-immunoprecipitation. (c) The ankyrin repeat AR domain is essential and sufficient for the interaction of C8orf4 with N2ICD. Various N2ICD truncation constructs were co-transfected with GFP–C8orf4 for domain mapping. NLS: nuclear location signal. (d) NOTCH2 was knocked down in Huh7 cells and detected by qRT–PCR and immunoblotting. (e) NOTCH2-silenced Huh7 cells were cultured for sphere formation assays. Two pairs of shRNAs against NOTCH2 obtained similar results. (f,g) Xenograft tumor growth (f) and frequency of tumor-initiating cells (g) for NOTCH2-silenced Huh7 cells were analyzed. (h) NOTCH2 was silenced in HCC primary cells and NOTCH2 depletion declined sphere formation activity. Three HCC specimens obtained similar results. (i) Sphere formation capacity was examined in differently treated HCC primary cells. (j) HCC primary cells were treated with indicated lentivirus and implanted into BALB/c nude mice for xenograft tumor growth assays. Scale bars: e,h,i, 500 μm, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation; NS, not significant.

To further verify the role of NOTCH2 in the maintenance of liver CSC self-renewal, we knocked down NOTCH2 in Huh7 cells and established stably depleted cell lines by two pairs of NOTCH2 shRNAs (Fig. 4d). NOTCH2 knockdown dramatically reduced sphere formation (Fig. 4e), as well as attenuated xenograft tumor growth and tumor-initiating capacity (Fig. 4f,g). Similar observations were achieved in NOTCH2-depleted HCC primary cells (Fig. 4h). In addition, we found that simultaneous knockdown of NOTCH2 and overexpression of C8orf4 failed to reduce sphere formation capacity compared with individual knockdown of NOTCH2 (Fig. 4i), suggesting that NOTCH2 and C8orf4 affected sphere formation through the same pathway. Meanwhile, C8orf4 knockdown failed to rescue the sphere formation ability of NOTCH2-depleted HCC primary cells (Fig. 4i). Similar observations were obtained in Huh7 cells (Supplementary Fig. 4). Finally, NOTCH2 depletion in C8orf4-silenced Huh7 cells or HCC primary cells also abrogated the C8orf4 depletion-mediated enhancement of xenograft tumor growth (Fig. 4j), suggesting that C8orf4 acted as upstream of NOTCH2 signaling. These data suggest that C8orf4 suppresses the liver CSC stemness through inhibiting the NOTCH2 signaling pathway.

C8orf4 blocks nuclear translocation of N2ICD

As shown in Fig. 1g, C8orf4 was mainly localized in the cytoplasm in tumor cells of HCC samples. To confirm these observations, we stained C8orf4 in several HCC cell lines and noticed that C8orf4 also resided in the cytoplasm of Huh7 cells and Hep3B cells (Fig. 5a and Supplementary Fig. 5a). These results were further validated by cellular fractionation (Fig. 5b). Importantly, C8orf4 KO led to nuclear translocation of N2ICD (Fig. 5c). In addition, we also examined the intracellular location of N2ICD in Huh7 spheres. We found that C8orf4 deletion caused complete nuclear translocation of N2ICD in oncosphere cells (Fig. 5d,e), while N2ICD was mainly located in the cytoplasm of WT oncosphere cells. However, we found that C8orf4 KO did not affect subcellular localization of β-catenin (Supplementary Fig. 5b,c). Through luciferase assays, C8orf4 transfection did not significantly influence promoter transcription activity of Wnt target genes such as TCF1, LEF and SOX4 (Supplementary Fig. 5d). These data indicate that C8orf4 resides in the cytoplasm of HCC cells and inhibits nuclear translocation of N2ICD.

C8orf4 deletion causes the nuclear translocation of N2ICD

C8orf4 deletion causes the nuclear translocation of N2ICD

Figure 5: C8orf4 deletion causes the nuclear translocation of N2ICD.


(a) C8orf4 resides in the cytoplasm of Huh7 cells. Huh7 cells were permeabilized and stained with anti-C8orf4 antibody, then counterstained with PI for confocal microscopy. (b) Cellular fractionation was performed and detected by immunoblotting. (c,d) C8orf4 knockout causes the nuclear translocation of N2ICD. C8orf4-deficient Huh7 cells (c) and sphere cells (d) were permeabilized and stained with anti-C8orf4 and anti-N2ICD antibodies, then counterstained with DAPI followed by confocal microscopy. (e) Cellular fractionation was performed in C8orf4-deficient sphere and WT sphere cells followed by immunoblotting. (f) C8orf4-deficient Huh7 cells were implanted into BALB/c nude mice. Xenograft tumors were analyzed by immunohistochemical staining. Red arrowheads denote nuclear translocation of N2ICD. (g) C8orf4-overexpressing Huh7 cells were permeabilized for immunofluorescence staining. (h) Cellular fractionation was performed in C8orf4-overexpressing Huh7 cells for immunoblotting. (i,j) C8orf4 was overexpressed in N2ICD-overexpressing Huh7 cells followed by immunofluorescence staining (i) and immunoblotting (j). (k) NOTCH target genes were measured in cells treated as in i by real-time PCR. Scale bars: a,c,d,g,i, 10 μm; f, 40 μm. Student’s t-test was used for statistical analysis, **P<0.01;***P<0.001, data are shown as mean±s.d.. Data represent at least three independent experiments.

To further determine whether C8orf4 inhibits the NOTCH2 signaling in the propagation of xenograft tumors, we examined the distribution of N2ICD and NOTCH2 target gene activation inC8orf4-deficient xenograft tumor tissues. We found that C8orf4-deficient tumors displayed much more nuclear translocation of N2ICD compared with WT tumors (Fig. 5f). Expectedly, C8orf4-deficient tumors showed elevated expression levels of NOTCH2 target genes such as HEY1, HES6 and NRARP (Supplementary Fig. 5e). Furthermore, C8orf4 overexpression blocked the nuclear translocation of N2ICD (Fig. 5g,h). Consequently, C8orf4-overexpressing tumors showed much less N2ICD nuclear translocation and reduced expression levels of NOTCH2 target genes compared with control tumors (Supplementary Fig. 5f,g). Of note, C8orf4 overexpression in N2ICD-overexpressing Huh7 cells still blocked nuclear translocation of N2ICD (Fig. 5i,j). Consequently, C8orf4 overexpression abolished the activation of Notch2 signaling (Fig. 5k). These results suggest that C8orf4 deletion causes the nuclear translocation of N2ICD leading to activation of NOTCH2 signaling.

NOTCH2 signalling is required for the stemness of liver CSCs

To further verify the role of NRARP and HEY1 in the maintenance of liver CSC self-renewal, we knocked down these two genes in Huh7 cells and established stably depleted cell lines by two pairs of shRNAs. As expected, NRARP knockdown dramatically reduced sphere formation (Fig. 6a,b). NRARP knockdown also attenuated tumor-initiating capacity and liver CSC ratios (Fig. 6c). Similar results were achieved in NRARP-silenced HCC primary cells (Fig. 6d,e). Similarly, HEY1 silencing remarkably reduced sphere formation derived from Huh7 and HCC primary cells (Fig. 6f–i), as well as declined xenograft tumor growth and tumor-initiating capacity (Supplementary Fig. 6a,b). In sum, NOTCH2 signaling is required for the maintenance of liver CSC self-renewal.

(not shown)

Figure 6: Depletion of NRARP and HEY1 impairs stemness of liver CSCs.


(a,b) NRARP-silenced Huh7 cells were established (a) and showed reduced sphere formation capacity (b). Two pairs of shRNAs against NRARP obtained similar results. (c) NRARP-silenced Huh7 cells decline tumour-initiating capacity (left panel) and reduce liver CSC frequency (right panel). Error bars represent the 95% confidence intervals of the estimation. (d,e) NRARP was knocked down in HCC primary cells (d) and sphere formation was detected (e). Three HCC samples were tested with similar results. (f,g) HEY1-silenced Huh7 cells were established (f) and sphere formation was assayed (g). Two pairs of shRNAs against HEY1 obtained similar results. (h,i) HEY1 was knocked down in HCC primary cells (h) and HEY1 depletion impaired sphere formation capacity (i). Three HCC samples were tested with similar results. Scale bars: b,e,g,i, 500 μm. For a,b,di, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01;  ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

NOTCH2 signaling is correlated with HCC severity

As shown above, the NOTCH2 signaling was highly activated in liver CSCs and involved in the regulation of liver CSC stemness. We further examined the relationship of NOTCH2 signaling with the progression of HCC. First, we analyzed NOTCH2 activation levels in HCC tumor tissues and peri-tumor tissues derived from Park’s cohort (GSE36376). We observed that HEY1HES6 and NRARP were highly expressed in the tumor tissues of HCC patients (Fig. 7a). Consistently, high expression levels of HEY1HES6 and NRARP in HCC tumors were validated by Zhang’s cohort (GSE25097) (Fig. 7b). Importantly, high expression of these three genes was confirmed in HCC samples through quantitative RT–PCR (Fig. 7c), as well as immunoblotting (Fig. 7d). To confirm a causative link between low C8orf4 expression level and nuclear N2ICD, we examined 93 HCC samples (31 peri-tumor, 37 early stage of HCC patients and 25 advanced stage of HCC patients) with immunohistochemistry staining. We observed that nuclear staining of N2ICD appeared in ~10% tumor cells in the majority of early HCC patients we tested (Fig. 7e,f). In advanced HCC patients, nuclear staining of N2ICD in tumor cells increased to ~30% in almost all the advanced HCC patients we examined. Consequently, HEY1 staining existed in ~10% tumor cells with scattered distribution and increased to 30% tumor cells in the advanced HCC patients (Fig. 7e). Consistently, low expression of C8orf4 was well correlated with activation of NOTCH2 signaling (Fig. 7e,f).

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

Figure 7: NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients.


(a,b) NOTCH target genes were highly expressed in HCC tumour tissues derived from Park’s cohort (a) and Zhang’s cohort (b). (c) High expression levels of NOTCH target genes in HCC tumor tissues were verified by qRT–PCR. (d) HEY1 expression in HCC tumor tissues was detected by western blot. (e) IHC staining for N2ICD, C8orf4 and HEY1. These images represent 93 HCC samples. Scale bars, 50 μm. (f) IHC images were calculated using Image-Pro Plus 6. (g) Expression levels of NOTCH target genes were elevated in HCC tumors and advanced HCC patients derived from Wang’s cohort. (hHEY1 expression level was positively correlated with prognosis prediction of HCC patients analyzed by Petel’s cohort and Wang’s cohort. HCC samples were divided into two groups according to HEY1 expression levels followed by Kaplan–Meier survival analysis. For ac, data are shown as box and whisker plot, Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For f,g, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001; data are shown as mean ± standard deviation. Experiments were repeated at least three times. aHCC, advanced HCC; CL, cirrhosis liver; eHCC, early HCC; IL, inflammatory liver; NL, normal liver; NS, not significant.

Serial passages of colonies or sphere formation in vitro, as well as transplantation of tumor cells, are frequently used to assess the long-term self-renewal capacities of CSCs32. We used HCC primary cells for serial passage growth in vitro and tested the expression levels of C8orf4HEY1 and SOX9. We found that C8orf4 expression was gradually reduced over serial passages in oncosphere cells (Supplementary Fig. 7a). Consequently, the expression of NOTCH2 targets such as HEY1 and SOX9 was gradually increased in oncosphere cells during serial passages (Supplementary Fig. 7b). In addition, N2ICD nuclear translocation appeared in oncosphere cells with high expression of HEY1 plus low expression of C8orf4 (termed as C8orf4/N2ICDnuc/HEY1+cells) (Supplementary Fig. 7c). These data suggest that the C8orf4/N2ICDnuc/HEY1+ fraction cells represent a subset of liver CSCs.

Through analyzing Wang’s cohort (GSE54238), we noticed that the NOTCH2 activation levels were positively correlated with the development and progression of HCC (Fig. 7g). By contrast, the NOTCH2 pathway was not activated in inflammation liver, cirrhosis liver and normal liver (Fig. 7f). Consistently, similar observations were achieved by analysis of Zhang’s cohort (GSE25097) (Supplementary Fig. 7d). In addition, the NOTCH2 activation levels were consistent with clinicopathological stages of HCC patients derived from Wang’s cohort (GSE14520) (Supplementary Fig. 7e). Finally, HCC patients with higher expression of HEY1 displayed worse prognosis derived from Petel’s cohort (E-TABM-36) and Wang’s cohort (GSE14520) (Fig. 7h). These two cohorts (E-TABM-36 and GSE14520) have survival information of HCC patients. Taken together, the NOTCH2 activation levels in tumor tissues are consistent with clinical severity and prognosis of HCC patients.


CSC have been identified in many solid tumors, including breast, lung, brain, liver, colon, prostate and bladder cancers4633. CSCs have similar characteristics associated with normal tissue stem cells, including self-renewal, differentiation and the ability to form new tumors. CSCs may be responsible for cancer relapse and metastasis due to their invasive and drug-resistant capacities34. Thus, targeting CSCs may become a promising therapeutic strategy to deadly malignancies3536. However, it remains largely unknown about hepatic CSC biology. In this study, we used CD13 and CD133 to enrich CD13+CD133+
subpopulation cells as liver CSCs. Based on analysis of several online-available HCC transcriptome datasets, we found that C8orf4 is weakly expressed in HCC tumors as well as in CD13+CD133+ liver CSCs. NOTCH2 signaling is required for the maintenance of liver CSC self-renewal. C8orf4 resides in the cytoplasm of tumor cells and interacts with N2ICD, blocking the nuclear translocation of N2ICD. Lower expression of C8orf4 causes nuclear translocation of N2ICD that activates NOTCH2 signaling in liver CSCs. NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Therefore, C8orf4 negatively regulates self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Elucidating signaling pathways that maintains self-renewal of liver CSCs is pivotal for the understanding of hepatic CSC biology and the development of novel therapies against HCC. Several signaling pathways, such as Wnt/β-catenin, transforming growth factor-beta, AKT and STAT3 pathways, have been defined to be implicated in the regulation of liver CSCs37. Not surprisingly, some liver CSC subsets and normal tissue stem cells may share core regulatory genes and common signaling pathways. The NOTCH signaling pathway plays an important role in development via cell-fate determination, proliferation and cell survival3839. The NOTCH family receptors contain four members in mammals (NOTCH1–4), which are activated by binding to their corresponding ligands. A large body of evidence provides that NOTCH signaling is implicated in carcinogenesis40. However, the role of NOTCH signaling in liver cancer is controversial. A previous study reported that NOTCH1 signaling suppresses tumor growth of HCC41. Recently, several reports showed that NOTCH signaling enhances liver tumor initiation424344. Importantly, a recent study showed that various NOTCH receptors have differential functions in the development of liver cancer45. Here we demonstrate that NOTCH2 signaling is activated in HCC tumor tissues and liver CSCs, which is required for the maintenance of liver CSC self-renewal.

C8orf4, also known as TC1, was originally cloned from a papillary thyroid cancer16, 46. The copy number variations of C8orf4 are associated with acute myeloid leukemia and other hematological malignancies19, 47. C8orf4 has been reported to be implicated in various cancers. C8orf4 was highly expressed in thyroid cancer, gastric cancer and breast cancer16, 20, 46. C8orf4 has been reported to enhance Wnt/β-catenin signaling in cancer cells that is associated with poor prognosis20, 21. However, C8orf4 is downregulated in colon cancer48. In this study, we show that C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs. Our observations were confirmed by two HCC cohort datasets. Importantly, C8orf4 negatively regulates the NOTCH2 signaling to suppress the self-renewal of liver CSCs. Therefore, C8orf4 may exert distinct functions in the regulation of various malignancies.

NOTCH receptors consist of noncovalently bound extracellular and transmembrane domains. Once binding with membrane-bound Delta or Jagged ligands, the NOTCH receptors undergoes a proteolytic step by metalloprotease and γ-secretase, generating NECD and NICD fragments11, 31. The NICD, a soluble fragment, is released in the cytoplasm on proteolysis. Then the NICD translocates to the nucleus and binds to the transcription initiation complex, leading to activation of NOTCH-associated target genes49. However, it is largely unclear how the NICD is regulated during NOTCH signaling activation. Here we show that N2ICD binds to C8orf4 in the cytoplasm of liver non-CSC tumor cells, which impedes the nuclear translocation of N2ICD. By contrast, in liver CSCs, lower expression of C8orf4 causes the nuclear translocation of N2ICD, leading to activation of NOTCH signaling.

CSCs or tumour-initiating cells, behave like tissue stem cells in that they are capable of self-renewal and of giving rise to hierarchical organization of heterogeneous cancer cells4. Thus, CSCs harbour the stem cell properties of self-renewal and differentiation. Actually, the CSC model cannot account for tumorigenesis in all tumours. CSCs could undergo genetic evolution, and the non-CSCs might switch to CSC-like cells4. These results highlight the dynamic nature of CSCs, suggesting that the clonal evolution and CSC models can act in concert for tumorigenesis. Furthermore, low C8orf4 expression in tumor cells results in overall Notch2 activation, which then may have more of a progenitor signature and be more aggressive. These cells would likely have a growth advantage in non-adherent conditions and express many of the stemness markers. The dynamic nature of CSCs or persistent NOTCH2 activation may contribute to the high number of C8orf4/N2ICDnuc/HEY1+ cells in advanced HCC tumors and correlation in the patient cohort.

A recent study showed that NOTCH2 and its ligand Jag1 are highly expressed in human HCC tumors, suggesting activation of NOTCH2 signaling in HCC45. In addition, inhibiting NOTCH2 or Jag1 dramatically reduces tumor burden and growth. However, suppression of NOTCH3 has no effect on tumor growth. Dill et al.43 reported that Notch2 is an oncogene in HCC. Notch2-driven HCC are poorly differentiated with a high expression level of the progenitor marker Sox9, indicating a critical role of Notch2 signaling in liver CSCs. Here we found that NOTCH2 and its target genes such as NRARP, HEY1 and HES6 are highly expressed in HCC samples. In addition, depletion of NRARP and HEY1 impairs the stemness maintenance of liver CSCs and tumor propagation. Moreover, the expression levels of NRARP, HEY1 and HES6 in tumors are positively correlated with clinical severity and prognosis of HCC patients. Finally, the NOTCH2 activation status is positively related to the clinicopathological stages of HCC patients. Altogether, C8orf4 and NOTCH2 signaling can be detected for the diagnosis and prognosis prediction of HCC patients, as well as used as targets for eradicating liver CSCs for future therapy. Quantifying the Landscape for Development and Cancer from a Core Cancer Stem Cell Circuit

The authors developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. The landscape exhibits four basins of attraction, representing cancer stem cell, stem cell, cancer and normal cell states. They also uncovered certain key genes and regulations responsible for determining the switching between different states. [Cancer Res]

Chunhe Li and Jin Wang
Cancer Res May 13, 2015; 75(10).

Cancer presents a serious threat to human health. The understanding of the cell fate determination during development and tumor genesis remains challenging in current cancer biology. It was suggested that cancer stem cell (CSC) may arise from normal stem cells, or be transformed from normal differentiated cells. This gives hints on the connection between cancer and development. However, the molecular mechanisms of these cell type transitions and the CSC formation remain elusive. We quantified landscape, dominant paths and switching rates between cell types from a core gene regulatory network for cancer and development. Stem cell, CSC, cancer, and normal cell types emerge as basins of attraction on associated landscape. The dominant paths quantify the transition processes among CSC, stem cell, normal cell and cancer cell attractors. Transition actions of the dominant paths are shown to be closely related to switching rates between cell types, but not always to the barriers in between, due to the presence of the curl flux. During the process of P53 gene activation, landscape topography changes gradually from a CSC attractor to a normal cell attractor. This confirms the roles of P53 of preventing the formation of CSC, through suppressing self-renewal and inducing differentiation. By global sensitivity analysis according to landscape topography and action, we identified key regulations determining cell type switchings and suggested testable predictions. From landscape view, the emergence of the CSCs and the associated switching to other cell types are the results of underlying interactions among cancer and developmental marker genes. This indicates that the cancer and development are intimately connected. This landscape and flux theoretical framework provides a quantitative way to understand the underlying mechanisms of CSC formation and interplay between cancer and development. Major Findings: We developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. Landscape exhibits four basins of attraction, representing CSC, stem cell, cancer and normal cell states. We quantified the kinetic rate and paths between different attractor states. We uncovered certain key genes and regulations responsible for determining the switching between different states. IMP3 Promotes Stem-Like Properties in Triple-Negative Breast Cancer by Regulating SLUG

Scientists observed that insulin-like growth factor-2 mRNA binding protein 3 (IMP3) expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and demonstrated that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells. [Oncogene]

S Samanta, H Sun, H L Goel, B Pursell, C Chang, A Khan, et al.
 , (18 May 2015) |

IMP3 (insulin-like growth factor-2 mRNA binding protein 3) is an oncofetal protein whose expression is prognostic for poor outcome in several cancers. Although IMP3 is expressed preferentially in triple-negative breast cancer (TNBC), its function is poorly understood. We observed that IMP3 expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and we demonstrate that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells (CSCs). The mechanism by which IMP3 contributes to this phenotype involves its ability to induce the stem cell factor SOX2. IMP3 does not interact with SOX2 mRNA significantly or regulate SOX2 expression directly. We discovered that IMP3 binds avidly to SNAI2 (SLUG) mRNA and regulates its expression by binding to the 5′ UTR. This finding is significant because SLUG has been implicated in breast CSCs and TNBC. Moreover, we show that SOX2 is a transcriptional target of SLUG. These data establish a novel mechanism of breast tumor initiation involving IMP3 and they provide a rationale for its association with aggressive disease and poor outcome. Type II Transglutaminase Stimulates Epidermal Cancer Stem Cell Epithelial-Mesenchymal Transition

Researchers investigated the role of type II transglutaminase (TG2) in regulating epithelial mesenchymal transition (EMT) in epidermal cancer stem cells. They showed that TG2 knockdown or treatment with TG2 inhibitor, resulted in a reduced EMT marker expression, and reduced cell migration and invasion. [Oncotarget]

ML Fisher, G Adhikary, W Xu, C Kerr, JW Keillor, RL Ecker
Oncotarget May 08, 2015;

Type II transglutaminase (TG2) is a multifunctional protein that has recently been implicated as having a role in ECS cell survival. In the present study we investigate the role of TG2 in regulating epithelial mesenchymal transition (EMT) in ECS cells. Our studies show that TG2 knockdown or treatment with TG2 inhibitor, results in a reduced EMT marker expression, and reduced cell migration and invasion. TG2 has several activities, but the most prominent are its transamidase and GTP binding activity. Analysis of a series of TG2 mutants reveals that TG2 GTP binding activity, but not the transamidase activity, is required for expression of EMT markers (Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α), and increased ECS cell invasion and migration. This coupled with reduced expression of E-cadherin. Additional studies indicate that NFϰB signaling, which has been implicated as mediating TG2 impact on EMT in breast cancer cells, is not involved in TG2 regulation of EMT in skin cancer. These studies suggest that TG2 is required for maintenance of ECS cell EMT, invasion and migration, and suggests that inhibiting TG2 GTP binding/G-protein related activity may reduce skin cancer tumor survival.

Epidermal squamous cell carcinoma (SCC) is among the most common cancers and the frequency is increasing at a rapid rate [1,2]. SCC is treated by surgical excision, but the rate of recurrence approaches 10% and the recurrent tumors are aggressive and difficult to treat [2]. We propose that human epidermal cancer stem (ECS) cells survive at the site of tumor excision, that these cells give rise to tumor regrowth, and that therapies targeted to kill ECS cells constitute a viable anti-cancer strategy. An important goal in this context is identifying and inhibiting activity of key proteins that are essential for ECS cell survival. Working towards this goal, we have developed systems for propagation of human ECS cells [3]. These cells display properties of cancer stem cells including self-renew and high level expression of stem cell marker proteins [3].

In the present study we demonstrate that ECS cells express proteins characteristic of cells undergoing EMT (epithelial-mesenchymal transition). EMT is a morphogenetic process whereby epithelial cells lose epithelial properties and assume mesenchymal characteristics [4]. The epithelial cells lose cell-cell contact and polarity, and assume a mesenchymal migratory phenotype. There are three types of EMT. This first is an embryonic process, during gastrulation, when the epithelial sheet gives rise to the mesoderm [5]. The second is a growth factor and cytokine-stimulated EMT that occurs at sites of tissue injury to facilitate wound repair [6]. The third is associated with epithelial cancer cell acquisition of a mesenchymal migratory/invasive phenotype. This process mimics normal EMT, but is not as well controlled and coordinated [478]. A number of transcription factors (ZEB1, ZEB2, snail, slug, and twist) that are expressed during EMT suppress expression of epithelial makers, including E-cadherin, desmoplakin and claudins [4]. Snail proteins also activate expression of vimentin, fibronectin and metalloproteinases [4]. Snail factors are not present in normal epithelial cells, but are present in the tumor cells and are prognostic factors for poor survival [4].

An important goal is identifying factors that provide overarching control of EMT in cancer stem cells. In this context, several recent papers implicate type II transglutaminase (TG2) as a regulator of EMT [912]. TG2, the best studied transglutaminase, was isolated in 1957 from guinea pig liver extract as an enzyme involved in the covalent crosslinks proteins via formation of isopeptide bonds [13]. However, subsequent studies reveal that TG2 also serves as a scaffolding protein, regulates cell adhesion, and modulates signal transduction as a GTP binding protein that participates in G protein signaling [14]. TG2 is markedly overexpressed in cancer cells, is involved in cancer development [1518], and has been implicated in maintaining and enhancing EMT in breast and ovarian cancer [10121920]. The G protein function may have an important role in these processes [102123].

In the present manuscript we study the role of TG2 in regulating EMT in human ECS cells. Our studies show that TG2 is highly enriched in ECS cells. We further show that these cells express EMT markers and that TG2 is required to maintain EMT protein expression. TG2 knockdown, or treatment with TG2 inhibitor, reduces EMT marker expression and ECS cell survival, invasion and migration. TG2 GTP binding activity is absolutely required for maintenance of EMT protein expression and EMT-related responses. However, in contrast to breast cancer [910], we show that TG2 regulation of EMT is not mediated via NFκB signaling.

TG2 is required for expression of EMT markers

EMT is a property of tumor stem cells that confers an ability to migrate and invade surrounding tissue [2426]. We first examined whether ECS cells express EMT markers. Non-stem cancer cells and ECS cells, derived from the SCC-13 cancer cell line, were analyzed for expression of EMT markers. Fig. 1A shows that a host of EMT transcriptional regulators, including Twist, Snail and Slug, are increased in ECS cells (spheroid) as compared to non-stem cancer cells (monolayer). This is associated with increased levels of vimentin, fibronectin and N-cadherin, which are mesenchymal proteins, and reduced expression of E-cadherin, an epithelial marker. HIF-1α, an additional marker frequently associated with EMT, is also elevated. We next examined whether TG2 is required to maintain EMT marker expression. SCC-13 cell-derived ECS cells were grown in the presence of control- or TG2-siRNA, to reduce TG2, and the impact on EMT marker level was measured. Fig. 1B shows that loss of TG2 is associated with reduced expression of Twist, Snail, vimentin and HIF-1α. To further assess the role of TG2, we utilized SCC13-Control-shRNA and SCC13-TG2-shRNA2 cell lines. These lines were produced by infection of SCC-13 cells with lentiviruses encoding control- or TG2-specific shRNA. Fig. 1C shows that SCC13-TG2-shRNA2 cells express markedly reduced levels of TG2 and that this is associated with reduced expression of EMT associated transcription factors and target proteins, and increased expression of E-cadherin. To confirm this, we grew SCC13-Control-shRNA and SCC13-TG2-shRNA2 cells as monolayer cultures for immunostain detection of EMT markers. As shown in Fig. 2A, TG2 levels are reduced in TG2-shRNA expressing cells, and this is associated with the anticipated changes in epithelial and mesenchymal marker expression.

Tumor cells that express EMT markers display enhanced migration and invasion ability [2426]. We therefore examined the impact of TG2 reduction on these responses. To measure invasion, control-shRNA and TG2-shRNA cells were monitored for ability to move through matrigel. Fig. 2B shows that loss of TG2 reduces movement through matrigel by 50%. We further show that this is associated with a reduction in cell migration using a monolayer culture wound closure assay. The control cells close the wound completely within 14 h, while TG2 knockdown reduces closure rate (Fig. 2C).

TG2 inhibitor reduces EMT marker expression and EMT functional responses

NC9 is a recently developed TG2-specific inhibitor [2728]. We therefore asked whether pharmacologic inhibition of TG2 suppresses EMT. SCC-13 cells were treated with 0 or 20 μM NC9. Fig. 3A shows that NC9 treatment reduces EMT transcription factor (Twist, Snail, Slug) and EMT marker (vimentin, fibronectin, N-cadherin, HIF-1α) levels. Consistent with these changes, the level of the epithelial marker, E-cadherin, is elevated. Fig. 3B and 3C show that pharmacologic inhibition of TG2 activity also reduces EMT biological response. Invasion (Fig. 3B) and cell migration (Fig. 3C) are also reduced.

Identification of TG2 functional domain required for EMT

We next performed studies to identify the functional domains and activities required for TG2 regulation of EMT. TG2 is a multifunctional enzyme that serves as a scaffolding protein, as a transamidase, as a kinase, and as a GTP binding protein [21]. The two best studied functions are the transamidase and GTP binding/G-protein related activities [21]. Transamidase activity is observed in the presence of elevated intracellular calcium, while GTP binding-related signaling is favored by low calcium conditions (reviewed in [21]). To identify the TG2 activity required for EMT, we measured the ability of wild-type and mutant TG2 to restore EMT in SCC13-TG2-shRNA2 cells, which have reduced TG2 expression (Fig. 4A). SCC13-TG2-shRNA2 cells display reduced expression of EMT markers including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α, and increased expression of the epithelial maker, E-cadherin, as compared to SCC13-Control-shRNA cells. Expression of wild-type TG2, or the TG2-C277S or TG2-W241A mutants, restores marker expression in SCC13-TG2-shRNA2 cells (Fig. 4A). TG2-C277S and TG2-W241A lack transamidase activity [10,2931]. In contrast, TG2-R580A, which lacks G-protein activity [2931], and TG2-Y516F, which retains only partial G-protein activity [30], do not efficiently restore marker expression. These findings suggest that the TG2 GTP binding function is required for EMT.

We next assayed the ability of the TG2 mutants to restore EMT functional responses-invasion and migration. Fig. 4B4C shows that wild-type TG2, TG2-C277S and TG2-W241A restore the ability of SCC13-TG2-shRNA2 cells to invade matrigel, but TG2-R580A and Y516F are less active. Fig. 4D shows a similar finding for cell migration, in that the TG2-R580A and Y517F mutant are only partially able to restore SCC13-TG2-shRNA2 cell migration. These findings suggest that TG2 GTP binding/G-protein related activity is required for EMT-related migration and invasion by skin cancer cells.

Role of TG2 in regulating EMT in A431 cells

The number of available epidermis-derived squamous cell carcinoma cell lines is limited, and so we compared our findings with A431 cells. A431 cells are squamous cell carcinoma cells established from human vulvar skin. A431 cells were grown as monolayer (non-stem cancer cells) and spheroids (ECS cells) and after 10 d the cells were harvested and assayed for expression of TG2 and EMT makers. Fig. 5A shows that TG2 levels are elevated in ECS cells and that this is associated with increased levels of mesenchymal markers, including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α. In contrast, E-cadherin levels are reduced. We next examined the impact of TG2 knockdown on EMT marker expression. Fig. 5B shows that mesenchymal markers are globally reduced and E-cadherin level is increased. As a biological endpoint of EMT, we examine the impact of TG2 knockdown on spheroid formation and found that TG2 loss leads to reduced spheroid formation (Fig. 5C). We next examined the impact of NC9 treatment on EMT and found a reduction in EMT markers expression associated with an increase in epithelial (E-cadherin) marker level (Fig. 5D). This loss of EMT marker expression is associated with reduced matrigel invasion (Fig. 5E), reduced spheroid formation (Fig. 5F) and reduced cell migration (Fig. 5G).

Role of NFκB

Previous studies in breast [183236], ovarian cancer [123738], and epidermoid carcinoma [11] indicate that NFκB signaling mediates TG2 impact on EMT. We therefore assessed the role of NFκB in skin cancer cells. As shown in Fig. 6A, the increase in TG2 level observed in ECS cells (spheroids) is associated with reduced NFκB level. In addition, NFκB level is increased in TG2 knockdown cells (Fig. 6B). Thus, increased NFκB is not associated with increased TG2. We next assessed the impact of NFκB knockdown on TG2 control of EMT marker expression. Fig. 6C shows that TG2 is required for increased expression of EMT markers (HIF-1α, snail, twist, N-cadherin, vimentin and fibronectin) and reduced expression of the E-cadherin epithelial marker; however, knockdown of NFκB expression does not interfere with TG2 regulation of these endpoints. We next examined the effect of TG2 knockdown on NFκB and IκBα localization. The fluorescence images in Fig. 6D suggest that TG2 knockdown with TG2-siRNA does not alter the intracellular localization of NFκB or IκBα. This is confirmed by subcellular fractionation assay (Fig. 6E) which compares NFκB level in SCC13-TG2-Control and SCC13-TG2-shRNA2 (TG2 knockdown) cells. We also monitored NFκB subcellular distribution following treatment with NC9, the TG2 inhibitor. Fig. 6F shows that cytoplasmic/nuclear distribution of NFκB is not altered by NC9. Finally, we monitored the impact of TG2 expression on NFκB binding to a canonical NFκB-response element. Increased NFκB binding to the response element is a direct measure of NFκB activity [10]. Fig. 6G shows that overall binding is reduced in nuclear (N) extract prepared from ECS cells (spheroids) as compared to non-stem cancer cells (monolayer), and that NFkB binding, as indicated by gel supershift assay, is also slightly reduced in ECS cell extracts. These findings indicate that NFkB binding is slightly reduced in ECS cells, which are TG2-enriched (Fig. 1A).

We next monitored the role of NFκB on biological endpoints of EMT. Fig. 7A and 7B show that TG2 knockdown reduces migration through matrigel, but NFκB knockdown has no impact. Likewise, TG2 knockdown reduces wound closure, but NFκB knockdown does not. These findings suggest that NFκB does not mediate the pro-EMT actions of TG2 in epidermal squamous cell carcinoma.

The metastatic cascade, from primary tumor to metastasis, is a complex process involving multiple pathways and signaling cascades [3941]. Cells that complete the metastatic cascade migrate away from the primary tumor through the blood to a distant site and there form a secondary tumor. Identifying the mechanisms that allow cells to survive this journey and form secondary tumors is an important goal. The processes involved in epithelial-mesenchymal transition (EMT) are important cancer therapy targets, as EMT is associated with enhanced cancer cell migration and stem cell self-renewal. EMT regulators, including Snail, Twist, Slug, are increased in expression in EMT and control expression of genes associated with the EMT phenotype [42].

TG2 is required for EMT

We have characterized a population of ECS cells derived from epidermal squamous cell carcinoma [3]. The present studies show that these cells, which display enhanced migration and invasion, possess elevated levels of TG2. Moreover, these cells are enriched in expression of transcription factors associated with EMT (Snail, Slug, and Twist, HIF-1α) as well as mesenchymal structural proteins including vimentin, fibronectin and N-cadherin. Consistent with a shift to mesenchymal phenotype, E-cadherin, an epithelial marker, is reduced in level. Additional studies show that TG2 knockdown results in a marked reduction in EMT marker expression and that this is associated with reduced ability of the cells to migrate to close a scratch wound and reduced movement in matrigel invasion assays. We also examined the impact of treatment with a TG2 inhibitor. NC9 is an irreversible active site inhibitor of TG2, that locks the enzyme in an open conformation [284345]. NC9 treatment of ECS cells results in decreased levels of Snail, Slug and Twist. These transcription factors suppress E-cadherin expression [46] and their decline in level is associated with increased levels of E-cadherin. NC9 inhibition of TG2 also reduces expression of vimentin, fibronectin and N-cadherin, and these changes are associated with reduced cell migration and reduced invasion through matrigel.

(Figures are not shown)

We also examined the role of TG2 in A431 squamous cell carcinoma cells derived from the vulva epithelium. TG2 is elevated in A431-derived ECS cells, as are EMT markers, and knockdown of TG2, with TG2-siRNA, reduces EMT marker expression and spheroid formation. Studies with NC9 indicate that NC9 inhibits A431 spheroid formation, EMT, migration and invasion. These studies indicate that TG2 is also required for EMT and migration and invasion in A431 cells. Based on these findings we conclude that TG2 is essential for EMT, migration and invasion, and is likely to contribute to metastasis in squamous cell carcinoma.

TG2 GTP binding activity is required for EMT

TG2 is a multifunctional enzyme that can act as a transamidase, GTP binding protein, protein disulfide isomerase, protein kinase, protein scaffold, and DNA hydrolase [21294447]. The two most studied functions are the transamidase and GTP binding functions [294447]. To identify the TG2 activity responsible for induction of EMT, we studied the ability of TG2 mutants to restore EMT in SCC13-TG2-shRNA2 cells, which express low levels of TG2 and do not express elevated levels of EMT markers or display EMT-related biological responses. These studies show that wild-type TG2 restores EMT marker expression and the ability of the cells to migrate on plastic and invade matrigel. TG2 mutants that retain GTP binding activity (TG2-C277S and TG2-W241A) also restore EMT. In contrast, TG2-R580A, which lacks GTP binding function, does not restore EMT. This evidence suggests that the GTP binding function is essential for TG2 induction of the EMT phenotype in ECS cells. Recent reports suggest that the TG2 is important for maintenance of stem cell survival in breast [91017] and ovarian [123848] cancer cells. Moreover, our findings are in agreement of those of Mehta and colleagues who reported that the TG2 GTP binding function, but not the crosslinking function, is required for TG2 induction of EMT in breast cancer cells [10].

TG2, NFκB signaling and EMT

To gain further insight into the mechanism of TG2 mediated EMT, we examined the role of NFκB. NFκB has been implicated as mediating EMT in breast, ovarian, and pancreatic cancer; however, NFκB may have a unique role in epidermal squamous cell carcinoma. In keratinocytes, NFκB has been implicated in keratinocyte dysplasia and hyperproliferation [49]. However, inhibition of NFκB function has also been shown to predispose murine epidermis to cancer [50]. Here we show that TG2 levels are elevated and NFκB levels are reduced in ECS cells as compared to non-stem cancer cells, and that TG2 knockdown is associated with increased NFκB level. In addition, TG2 knockdown, or inhibition of TG2 by treatment with NC9, does not altered the nuclear/cytoplasmic distribution of NFκB. We further show that elevated levels of TG2 in spheroid culture results in a slight reduction in NFκB binding to the NFκB response element, as measured by gel mobility supershift assay. These molecular assays strongly suggest that NFκB does not mediate the action of TG2 in epidermal cancer stem cells. Moreover, knockdown of NFκB-p65 in TG2 positive cells does not result in a reduction in Snail, Slug and Twist, or mesenchymal marker proteins expression, and concurrent knockdown of TG2 and NFκB does not reduce EMT marker protein levels beyond that of TG2 knockdown alone. These findings suggest that NFκB is not an intermediary in TG2-stimulated EMT in ECS cells. This is in contrast to the required role of NFκB in mediating TG2 induction of cell survival and EMT in breast cancer cells [183233] and ovarian cancer [123738] and epidermoid carcinoma [11]. CD24+ Ovarian Cancer Cells are Enriched for Cancer Initiating Cells and Dependent on JAK2 Signaling for Growth and Metastasis

Investigators showed that CD24+ and CD133+ cells have increased tumorsphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24– cells, had significantly greater tumor initiation and tumor growth capacity. [Mol Cancer Ther]

D Burgos-OjedaR Wu, K McLean, Yu-Chih Chen, M Talpaz, et al.
Molec Cancer Ther May 12, 2015; 14(5)

Ovarian cancer is known to be composed of distinct populations of cancer cells, some of which demonstrate increased capacity for cancer initiation and/or metastasis. The study of human cancer cell populations is difficult due to long requirements for tumor growth, inter-patient variability and the need for tumor growth in immune-deficient mice. We therefore characterized the cancer initiation capacity of distinct cancer cell populations in a transgenic murine model of ovarian cancer. In this model, conditional deletion of Apc, Pten, and Trp53 in the ovarian surface epithelium (OSE) results in the generation of high grade metastatic ovarian carcinomas. Cell lines derived from these murine tumors express numerous putative stem cell markers including CD24, CD44, CD90, CD117, CD133 and ALDH. We show that CD24+ and CD133+ cells have increased tumor sphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24- cells, had significantly greater tumor initiation and tumor growth capacity. No preferential tumor initiating or growth capacity was observed for CD44+, CD90+, CD117+, or ALDH+ versus their negative counterparts. We have found that CD24+ cells, compared to CD24- cells, have increased phosphorylation of STAT3 and increased expression of STAT3 target Nanog and c-myc. JAK2 inhibition of STAT3 phosphorylation preferentially induced cytotoxicity in CD24+ cells. In vivo JAK2 inhibitor therapy dramatically reduced tumor metastases, and prolonged overall survival. These findings indicate that CD24+ cells play a role in tumor migration and metastasis and support JAK2 as a therapeutic target in ovarian cancer. EpCAM-Antibody-Labeled Noncytotoxic Polymer Vesicles for Cancer Stem Cells-Targeted Delivery of Anticancer Drug and siRNA

Researchers designed and synthesized a novel anti-epithelial cell adhesion molecule (EpCAM)-monoclonal-antibody-labeled cancer stem cells (CSCs)-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA. [Biomacromolecules]

Jing Chen , Qiuming Liu , Jiangang Xiao , and Jianzhong Du
Biomacromolecules May 19, 2015. (just published)

Cancer stem cells (CSCs) have the capability to initiate tumor, to sustain tumor growth, to maintain the heterogeneity of tumor, and are closely linked to the failure of chemotherapy due to their self-renewal and multilineage differentiation capability with an innate resistance to cytotoxic agents. Herein, we designed and synthesized a novel anti-EpCAM (epithelial cell adhesion molecule)-monoclonal-antibody-labeled CSCs-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA (to overcome CSCs drug resistance by silencing the expression of oncogenes). This vesicle shows high delivery efficacy of both anticancer drug doxorubicin hydrochloride (DOX∙HCl) and siRNA to the CSCs because it is labeled by the monoclonal antibodies to the CSCs-surface-specific marker. Compared to non-CSCs-targeting vesicles, the DOX∙HCl or siRNA loaded CSCs-targeting vesicles exhibited much better CSCs killing and tumor growth inhibition capabilities with lower toxicity to normal cells (IC50,DOX decreased by 80%), demonstrating promising potential applications in nanomedicine. Survival of Skin Cancer Stem Cells Requires the Ezh2 Polycomb Group Protein

Investigators showed that Ezh2 is required for epidermal cancer stem (ECS) cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. They also showed that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduced Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. [Carcinogenesis]

G Adhikary, D Grun, S Balasubramanian, C Kerr, J Huang and RL Eckert
Carcinogenesis (2015)

Polycomb group (PcG) proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation, and that treatment with Ezh2 inhibitors may be a strategy for reducing epidermal cancer stem cell survival and suppressing tumor formation. Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer

R Thakur, R Trivedi, N Rastogi, M Singh & DP Mishra
Scientific Reports May 14, 2015; 5(10194)

Cancer stem cells (CSCs) are responsible for aggressive tumor growth, metastasis and therapy resistance. In this study, we evaluated the effects of Shikonin (Shk) on breast cancer and found its anti-CSC potential. Shk treatment decreased the expression of various epithelial to mesenchymal transition (EMT) and CSC associated markers. Kinase profiling array and western blot analysis indicated that Shk inhibits STAT3, FAK and Src activation. Inhibition of these signaling proteins using standard inhibitors revealed that STAT3 inhibition affected CSCs properties more significantly than FAK or Src inhibition. We observed a significant decrease in cell migration upon FAK and Src inhibition and decrease in invasion upon inhibition of STAT3, FAK and Src. Combined inhibition of STAT3 with Src or FAK reduced the mammosphere formation, migration and invasion more significantly than the individual inhibitions. These observations indicated that the anti-breast cancer properties of Shk are due to its potential to inhibit multiple signaling proteins. Shk also reduced the activation and expression of STAT3, FAK and Src in vivo and reduced tumorigenicity, growth and metastasis of 4T1 cells. Collectively, this study underscores the translational relevance of using a single inhibitor (Shk) for compromising multiple tumor-associated signaling pathways to check cancer metastasis and stem cell load.

Breast cancer is the most common endocrine cancer and the second leading cause of cancer-related deaths in women. In spite of the diverse therapeutic regimens available for breast cancer treatment, development of chemo-resistance and disease relapse is constantly on the rise. The most common cause of disease relapse and chemo-resistance is attributed to the presence of stem cell like cells (or CSCs) in tumor tissues12. CSCs represent a small population within the tumor mass, capable of inducing independent tumors in vivo and are hard to eradicate2. Multiple signaling pathways including Receptor Tyrosine Kinase (RTKs), Wnt/β-catenin, TGF-β, STAT3, Integrin/FAK, Notch and Hedgehog signaling pathway helps in maintaining the stem cell programs in normal as well as in cancer cells3456. These pathways also support the epithelial-mesenchymal transition (EMT) and expression of various drug transporters in cancer cells. Cells undergoing EMT are known to acquire stem cell and chemo-resistant traits7. Thus, the induction of EMT programs, drug resistance and stem cell like properties are interlinked7. Commonly used anti-cancer drugs eradicate most of the tumor cells, but CSCs due to their robust survival mechanisms remain viable and lead to disease relapse8. Studies carried out on patient derived tumor samples and in vivo mouse models have demonstrated that the CSCs metastasize very efficiently than non-CSCs91011. Therefore, drugs capable of compromising CSCs proliferation and self-renewal are urgently required as the inhibition of CSC will induce the inhibition of tumor growth, chemo-resistance, metastasis and metastatic colonization in breast cancer.

Shikonin, a natural dietary component is a potent anti-cancer compound1213. Previous studies have shown that Shk inhibits the cancer cell growth, migration, invasion and tumorigenic potential12. Shk has good bioavailability, less toxicity and favorable pharmacokinetic and pharmacodynamic profiles in vivo12. In a recent report, it was shown that the prolonged exposure of Shk to cancer cells does not cause chemo-resistance13.Other studies have shown that it inhibits the expression of various key inflammatory cytokines and associated signaling pathways1214. It decreases the expression of TNFα, IL12, IL6, IL1β, IL2, IFNγ, inhibits ERK1/2 and JNK signaling and reduces the expression of NFκB and STAT3 transcription factors1415. It inhibits proteasome and also modulates the cancer cell metabolism by inhibiting tumor specific pyurvate kinase-M214,1516. Skh causes cell cycle arrest and induces necroptosis in various cancer types14. Shk also inhibits the expression of MMP9, integrin β1 and decreases invasive potential of cancer cells1417. Collectively, Shk modulates various signaling pathways and elicits anti-cancer responses in a variety of cancer types.

In breast cancer, Shk has been reported to induce the cell death and inhibit cell migration, but the mechanisms responsible for its effect are not well studied1819. Signaling pathways modulated by Shk in cancerous and non-cancerous models have previously been shown important for breast cancer growth, metastasis and tumorigenicity20. Therefore in the current study, we investigated the effect of Shk on various hallmark associated properties of breast cancer cells, including migration, invasion, clonogenicity, cancer stem cell load and in vivo tumor growth and metastasis.

Shk inhibits cancer hallmarks in breast cancer cell lines and primary cells

We first examined the effect of Shk on various cancer hallmark capabilities (proliferation, invasion, migration, colony and mammosphere forming potential) in breast cancer cells. MTT assay was used to find out effect of Shk on viability of breast cancer cells. Semi-confluent cultures were exposed to various concentrations of Shk for 24 h. Shk showed specific anti-breast cancer activity with IC50 values ranging from 1.38 μM to 8.3 μM in MDA-MB 231, MDA-MB 468, BT-20, MCF7, T47D, SK-BR-3 and 4T1 cells (Fig. 1A). Whereas the IC50 values in non-cancerous HEK-293 and human PBMCs were significantly higher indicating that it is relatively safe for normal cells (Fig. S1A). Shk was found to induce necroptotic cell death consistent with previous reports (Fig. S1B). Treatment of breast cancer cells for 24 h with 1.25 μM, 2.5 μM and 5.0 μM of Shk significantly reduced their colony forming potential (Fig. 1B). To check the effect of Shk on the heterogeneous cancer cell population, we tested it on patient derived primary breast cancer cells. Shk reduced the viability and colony forming potential of primary breast cancer cells in dose dependent manner (Fig. 1C,D). Further we checked its effects on migration and invasion of breast cancer cells. Shk (2.5 μM) significantly inhibited the migration of MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cells (Fig. 1E). It also inhibited the cell invasion in dose dependent manner (Fig. 1F and S1CS1DS1E,S1F). We further examined its effect on mammosphere formation. MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cell mammosphere cultures were grown in presence or absence of 1.25 μM, 2.5 μM and 5.0 μM Shk for 24 h. After 8 days of culture, a dose dependent decrease in the mammosphere forming potential of these cells was observed (Figs. 1G,H). Collectively, these results indicated that Shk effectively inhibits the various hallmarks associated with aggressive breast cancer.

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Figure 1: Shk inhibits multiple cancer hallmarks

Shk reduces cancer stem cell load in breast cancer

As Shk exhibited strong anti-mammosphere forming potential; therefore it was further examined for its anti-cancer stem cell (CSC) properties. Cancer stem cell loads in breast cancer cells were assessed using Aldefluor assay which measures ALDH1 expression. MDA-MB 231 cells with the highest number of ALDH1+ cells were selected for further studies (Fig. S2A). We also checked the correlation between ALDH1 expression and mammosphere formation. Sorted ALDH1+ cells were subjected to mammosphere cultures. ALDH1+ cells formed highest number of mammospheres compared to ALDH1-/low and parent cell population, indicating that ALDH1+ cells are enriched in CSCs (Fig. S2B). Shk reduced the Aldefluor positive cells in MDA-MB 231 cells after 24 h of treatment (Fig. 2A,B). Next, we examined the effect of Shk on the expression of stem cell (Sox2, Oct3/4, Nanog, AldhA1 and c-Myc) and EMT (Snail, Slug, ZEB1, Twist, β-Catenin) markers, associated with the sustenance of breast CSCs. Shk (2.5 μM) treatment for 24 h reduced the expression of these markers (Fig. 2C and S2D). Shk also reduced protein expression of these markers in dose dependent manner (Fig. 2D,E and S2C).

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Figure 2: Shk decreases stem cell load in breast cancer cells and enriched CD44+,CD24−/low breast cancer stem cells.

To further confirm anti-CSC properties of Shk, we checked the effect of shikonin on the load of CD44+ CD24− breast CSCs in MCF7 cells grown on matrigel. Shikonin reduced CD44+ CD24− cell load in dose dependent manner after 24 h of treatment (Fig S2E). We also tested its effects on the enriched CSC population. CD44+ CD24− cells were enriched from MCF7 cells using MagCellect CD24− CD44+ Breast CSC Isolation Kit (Fig. S2F). Enriched CSCs formed highest number of mammosphere in comparison to parent MCF7 cell population or negatively selected CD24+ cells (Fig. S2G). Enriched CSCs were treated with indicated doses of Shk (0.625 μM, 1.25 μM and 2.5 μM) for 24 h and were either analyzed for ALDH1 positivity or subjected to colony or mammosphere formation. 2.5 μM dose of Shk reduced ALDH1+ cells by 50% and inhibited colony and mammosphere formation (Fig. S2H2F2G and 2H). Shk also reduced the mRNA expression of CSC markers in CD44+ CD24− cells and patient derived primary cancer cells (Fig. 2I,J). These results collectively indicated that Shk inhibits CSC load and associated programs in breast cancer.

Shk is a potent inhibitor of STAT3 and poorly inhibits FAK and Src

To identify the molecular mechanism responsible for anti-cancer properties of Shk, we used a human phospho-kinase antibody array to study a subset of phosphorylation events in MDA-MB 231 cells after 6h of treatment with 2.5 μM Shk. Amongst the 46 phospho-antibodies spotted on the array, the relative extent of phosphorylation of three proteins decreased to about ≳ 2 fold (STAT3, 3.3 fold; FAK, 2.5 fold and Src, 1.8 fold) upon Shk treatment (Fig. 3A,B). These proteins (STAT3, FAK and Src) are known to regulate CSC proliferation and self renewal212223. Therefore, we focused on these proteins and the result of kinase-array was confirmed by western blotting. Shk effectively inhibits STAT3 at early time point (1 h) while activation of FAK and Src decreased on or after 3 h (Fig. 3C) confirming Shk as a potent inhibitor of STAT3. Shk also reduced the protein expression of STAT3, FAK and Src at 24 h (Fig. 3C).

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Figure 3. Shk inhibits STAT3, FAK and Src signaling pathways.

We also observed that Shk does not inhibit JAK2 at initial time-points (Fig. 3C). This raised a possibility that Shk either regulates STAT3 independent of JAK2 or it binds directly to STAT3. To check the first probability, we activated STAT3 by treating the cells with IL6 (100 ng ml−1) for 1 h followed by treatment with Shk (2.5 μM) for 1 h. Both immunofluorescence and western-blotting results showed that Shk inhibited activated STAT3 without inhibiting JAK2 (Fig. S3AS3B) confirming that Shk inhibits JAK2 mediated activation of STAT3 possibly by binding directly to STAT3. For further confirmation, we performed an in silico molecular docking analysis to examine binding of Shk with the STAT3 SH2 domain. In a major conformational cluster, Shk occupied Lys-707, Lys-709 and Phe-710 binding sites in the STAT3 SH2 domain similar to the STAT3 standard inhibitor S3I-201 (Fig. S3C and S3D). The binding energy of Shk to STAT3 was −4.20 kcal mol−1. Collectively, these results showed that Shk potently inhibits STAT3 activation and also attenuates FAK and Src activation.

STAT3, Src and FAK are differentially expressed and activated in breast CSCs (BCSCs)

STAT3 and FAK are known to play an important role in proliferation and self-renewal of CSCs in various cancer types including breast cancer212224. Src also support CSC phenotype in some cancer types, but there are limited reports of its involvement in breast cancer25. Therefore, we checked the expression and activation of STAT3, FAK and Src in CSCs and non-CSCs. Here we used two methods to enrich the CSCs and non-CSCs. In the first method, the MDA-MB 231 cells were subjected to mammosphere formation for 96 h. After 96 h, mammosphere and non-mammosphere forming cells were clearly visible (Fig. 4A). These mammosphere and non-mammosphere forming cells were separated by using a 70 micron cell strainer. Mammospheres were subjected to two subculture cycles to enrich CSCs. With each passage, the viable single cells (non-mammosphere forming cells) and mammospheres were collected in RIPA lysis buffer and western blotting was done (Fig. 4B). We found that the activation and expression of the STAT3, FAK and Src is higher in enriched mammosphere cultures (Fig. 4C). In the second method, CD44+ CD24− cells were isolated from MCF7 cultures using MagCellect Breast CSC Isolation Kit. STAT3, FAK and Src activation and their mRNA and protein expression were assessed in enriched CSCs and were compared to parent MCF7 cell population. STAT3, FAK and Src all were differentially activated in CSCs (Fig. 4E). High mRNA as well as protein expressions of all the three genes was also observed in CSCs (Fig. 4D,E). Collectively, these results indicate that STAT3, FAK and Src are over expressed and activated in BCSCs.

Figure 4: STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

  • Representative picture indicating mammosphere and single suspended cells. (B) Schematic outline of mammosphere enrichment. (C) Protein expression and activation of STAT3, FAK and Src was determined in single suspended cells (non-mammosphere forming cells) and mammospheres by western blot. The full size blots corresponding to the cropped blot images are given in  S10. (D) Gene expression of STAT3, FAK and Src was determined in MCF7 parent population and CD44+ CD24−/low MCF7 cells using PCR. The full agarose gel images corresponding to the cropped images are given in Fig. S10. (E) Protein expression and activation of STAT3, FAK and Src was in CD44+ 24− cells and parent population.
STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.


STAT3 is important for mammosphere formation and CSC programs in breast cancer

As our results indicated that the expression and activation of STAT3, FAK and Src is high in BCSCs and Shk is capable of inhibiting these signaling proteins; therefore to find out functional relevance of each protein and associated effects on their pharmacological inhibition by Shk, we used specific inhibitors against these three. Effect of these inhibitors was first tested on the mammosphere forming potential of MDA-MB 231, MDA-MB 468 and MCF7 cells. A drastic reduction in the mammosphere formation was observed upon STAT3 inhibition. FAK and Src inhibition also reduced the primary and secondary mammosphere formation but STAT3 inhibition showed most potent effect (Fig. 5A and S4). Further, we also checked the effect of these inhibitors on the expression of various CSC and EMT related markers in MDA-MB 231 cells. STAT3 inhibition decreased the expression of most of the CSC and EMT markers (Fig. 5B). These two findings indicated that STAT3 inhibition is more effective in reducing mammosphere forming potential and weakens major CSC programs and the anti-CSC potential of Shk is possibly due to its strong STAT3 inhibitory effect.
(not shown)

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

Figure 5: STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer.


(A) Bar graph represents number of mammospheres formed from 2500 cells in presence and absence of indicated treatments. MDA-MB 231, MDA-MB 468 and MCF7 24 h mammosphere cultures were treated with Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). After 24 h, treatments were removed and cells were allowed to grow in fresh mammosphere culture media for 8 days. (B) Expression of various stem cell and EMT related transcription factors and markers were detected using western blotting in MDA-MB 231 cells with or without indicated treatments. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) MDA-MB 231, MDA-MB 468 and MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. Bar graph represents average of three independent experiments. (D) MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. After 24 h, cells were treated with DMSO (untreated) or Shk (treated) as indicated in the bar graph. Data are shown as the mean ±SD. (*) p < 0.05 and (**) p < 0.01.

To further check the involvement of these pathways in CSCs, we cultured MDA-MB 231, MDA-MB 468 and MCF7 cells in the presence of either IL6 (100ng ml−1), EGF (25 ng ml−1) or Fibronectin (1 μg ml−1) coated surface for two population doublings. Cells were then subjected to mammosphere formation. In IL6 pre-treated cultures, there was a sharp rise in mammosphere formation, indicating that the STAT3 activation shifts CSC and non-CSC dynamics towards CSCs (Fig. 5C). IL6 is previously known to induce the conversion of non-CSC to CSC via STAT3 activation26. In MCF7 cells, mammosphere forming potential after IL6 pre-treatment increased nearly by three fold. Therefore, we further checked the effectiveness of Shk on mammosphere forming potential in pre-treated MCF7 cells. It was found that Shk inhibits mammosphere formation most effectively in IL6 pre-treated cultures (Fig. 5D). However, in EGF and Fibronectin pre-treated cultures, Shk was relatively less effective. This was possibly due to its weak FAK and Src inhibitory potential. Collectively, these results illustrated that STAT3 activation is significantly correlated with the mammosphere forming potential of breast cancer cells and its inhibition by a standard inhibitor or Shk potently reduce the mammosphere formation.

Shk inhibit CSCs load by disrupting the STAT3-Oct3/4 axis

In breast cancer, STAT3 mediated expression of Oct3/4 is a major regulator of CSC self-renewal2627. As we observed that both Shk and STAT3 inhibitors decreased the Oct3/4 expression (Figs. 2C and 5B), we further checked the effect of STAT3 activation on ALDH1+ CSCs and Oct3/4 expression. On IL6 pre-treatment, number of ALDH1+ cells increased in all three (MDA-MB 231, MDA-MB 468 and MCF7) cancer cells (Fig. 6A). MCF7 cells showed highest increase. Therefore, to check the effect of STAT3 inhibition on CSC load, we incubated IL6 pre-treated MCF7 cells with Shk and STAT3 inhibitor for 24 h and analyzed for ALDH1 positivity. It was observed that both Shk and STAT3 inhibitor reduced the IL6 induced ALDH1 positivity from 10% to < 2% (Fig. 6B). These results suggested that Shk induced inhibition of STAT3 and decrease in BCSC load is interlinked. We further checked the effect of STAT3 activation status on Oct3/4 expression in MDA-MB 231, MDA-MB 468 and MCF7 cells. We observed that expression of Oct3/4 increases with the increase in STAT3 activation (Fig. 6C–E).

(not shown)

Figure 6: STAT3 activation status and its effect on cancer stem cell load

STAT3 transcriptional activity is important in maintaining CSC programs2829. Therefore, we also examined the effect of Shk on STAT3 promoter activity. STAT3 reporter assay was performed in presence of IL6 and Shk; it was found that Shk reduced the promoter activity of STAT3 in a dose dependent manner (Fig. S5). Collectively, these results showed that Shk mediated STAT3 inhibition are responsible for decrease in CSC load and Oct3/4 associated stem cell programs.

Shk inhibits mammosphere formation, migration and invasion through inhibition of STAT3, FAK and Src in breast cancer cells

As the earlier results (Fig. 1) showed that Shk inhibits cell migration and invasion in breast cancer cells, we further examined the effect of STAT3, FAK and Src inhibitors on cell migration and invasion in MDA-MB 231 cells. It was found that STAT3 inhibitor poorly inhibits cell migration while both Src and FAK inhibitors were effective in reducing cell migration (Fig. 7A). All the three inhibitors decreased the cell invasion and MMP9 expression significantly (Fig. 7B and S6). It was also observed that effect of all these inhibitors, except STAT3 inhibitor on mammosphere formation and FAK inhibitor on cell migration, were not comparable to that of Shk. Shk inhibited all these properties more effectively than individual inhibition of STAT3, FAK and Src. This made us to assume that the ability of Shk to inhibit multiple signaling molecules simultaneously is the reason behind its potent anti-cancer effect. To check this notion, we combined STAT3, FAK and Src inhibitors with each other and examined the effect of combinations on invasion, migration and mammosphere forming potential in MDA-MB 231 cells. We observed further decrease in cell migration and invasion on combining STAT3 and FAK, STAT3 and Src, or FAK and Src (Figs. 7A,B). Combination of FAK and Src was not very effective in inhibiting mammosphere formation in MDA-MB 231 cells and CD44+ CD24− MCF7 CSCs. However, their combination with STAT3 decreased the mammosphere forming potential equivalent to that of Shk (Fig. 7C,D). We also compared the mammosphere forming potential of Shk with Salinomycin (another anti-CSC agent) and found that at 2.5 μM dose of Shk was almost two times more potent than Salinomycin (Fig. S7). Collectively, these results indicated that Shk inhibits multiple signaling proteins (STAT3, FAK and Src) to compromise various aggressive breast cancer hallmarks.

Figure 7: Combination of FAK, Src and STAT3 inhibitors is more potent than individual inhibition against various cancer hallmarks.




  • Cell migration and (B) cell invasion potential of MDA-MB 231 cells was assessed in the presence of Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). Various combinations of these inhibitors were also used STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM + AZM 475271; 10 μM) and FAK+Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cell migration and cell invasion was assessed through scratch cell migration assay and transwell invasion after 24 h of treatments. (C,D) Mammosphere forming potential of MDA-MB 231 cells and CD44+ CD24−/low enriched MCF7 cells was assessed in presence of similar combination of STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM+ AZM 475271; 10 μM) and FAK + Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cells were subjected to mammosphere cultures for 24 h and treated with the indicated inhibitors for next 24 h, followed by media change and growth of mammospheres were monitored for next 8 days. Data are shown as the mean ±SD. (**) p < 0.01.

Shk inhibits breast cancer growth, metastasis and decreases tumorigenicity

To explore whether Shk may have therapeutic potential for breast cancer treatment in vivo, we tested Shk against 4T1-induced breast cancer syngenic mouse model. 4T1 cells (mouse breast cancer cells) are capable of growing fast and metastasize efficiently in vivo30. Prior to the in vivo experiments, we checked the effect of Shk on ALDH1 positivity and on activation of STAT3, FAK and Src in 4T1 cells in vitro. Shk effectively decreased the ALDH1+ cells and inhibited STAT3, FAK and Src in 4T1 cells in vitro (Fig. S8A and S8B). For in vivo tumor generation, 1 × 106 cells were injected subcutaneously in the fourth nipple mammary fat pad of BALB/c mice. When the average size of tumors reached around 50 mm3, mice were divided into three groups, vehicle and two Shk treated groups each received either 2.5 mg Kg−1 or 5.0 mg Kg−1 Shk. Shk was administered via the intraperitoneal injection on every alternate day. It significantly suppressed the tumor growth in 4T1 induced syngenic mouse model (Fig. 8A). The average reduction in 4T1 tumor growth was 49.78% and 89.73% in 2.5 mg Kg−1 and 5.0 mg Kg−1 groups respectively compared with the vehicle treated group (Fig. 8A). No considerable change in body weight of the treated group animals was observed (Fig. S9A). We further examined the effect of Shk on the tumor initiating potential of breast cancer cells. 4T1 induced tumors were excised from the control and treatment groups on the second day after 4th dose of Shk was administered. Tumors were dissociated; cells were allowed to adhere and then re-injected into new animals for secondary tumor formation. Growth of secondary tumors was monitored till day 15 post-reinjection. Shk treated groups showed a marked decrease in secondary tumor formation (Fig. 8D). We also observed a drastic reduction in the number of metastatic nodules in the lungs of treatment group animals (Fig. 8F). The reduction in the metastatic load was not proportional to the decrease in tumor sizes; however within the treatment group, some animals with small tumors were carrying higher number of metastatic nodules. As FAK is an important mediator of cancer metastasis and metastatic colonization, we further examined the effects of Shk on metastatic colonization. For this, 1 × 105 4T1 cells were injected to BALB/c mice through tail vein. Animals were divided into three groups, as indicated above. Shk and vehicle were administered through intraperitoneal injections at alternate days starting from the 2nd day post tail vein injections till 33rd day. The average reduction in total number of metastatic nodules was 88.6% – 90.5% in Shk treated mice compared to vehicle control (Fig. 8F). An inset picture (Fig. 8A lower panel) represents lung morphology of vehicle control and treated groups. We further examined the activation and expression status of STAT3, FAK and Src between vehicle control and treated group tumors. There were low expression and activation of STAT3, FAK and Src in treated tumors as compared to the vehicle control (Fig. 8B,C). Similar trend was observed in ALDH1 expressions (Fig. 8B). Further, the mice tumor sections were subjected to immunohistochemistry, immunofluorescence and hematoxylin and eosin (H&E) staining to study histology and expression of key proteins being examined in this study. Fig. 8G shows representative images of H&E staining, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), STAT3 and Oct3/4 immunostaining. PCNA expression was low while TUNEL positive cells were high in tumor tissues of Shk treated groups. STAT3 and Oct3/4 expression was low in Shk treated groups. These results collectively demonstrated that Shk modulates the expression and activation of STAT3, FAK and Src in vivo and is effective in suppressing tumorigenic potential and metastasis in syngenic mouse model.

Figure 8: Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo.

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo


  • Shk inhibited 4T1 tumor growth. Bar graph represents the average tumor volumes in vehicle control and Shk treated tumor bearing mice (n = 6). (*) p < 0.05 and (**) p < 0.01. Inset picture of upper panel represents tumor sizes and lower pane represents lung morphology in vehicle control and Shk treatment groups. (B) Western blot examination of indicated proteins for their expression and activation in vehicle control and treated tumor groups. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) Gene expression of stem cell and EMT markers in tumor tissues excised from the vehicle control and Shk treated groups (n = 3). (D) Number of secondary tumors formed after injecting indicated cell dilutions from Vehicle treated and Shk treated 4T1 tumors. (E) Number of lung nodules formed in mice injected with 4T1 mouse mammary tumor cells in the mammary fat pad and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks (n = 6). (F) Number of lung nodules in mice injected with 4T1 mouse mammary tumor cells through tail vein and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks. (n = 8) (G) Representative panel of the histological H&E staining, immunofluorescence staining for the STAT3, Oct3/4, cell proliferation marker PCNA and DNA damage indicator-TUNEL staining of tumor sections from vehicle and treatment groups.

Recent studies have shown that aggressiveness, therapy resistance and disease relapse in breast cancer is attributed to a small population of CSCs involved in continuous self-renewal and differentiation through signaling pathways similar to that of the normal stem cells31. Therapeutic targeting of CSCs therefore, has profound clinical implications for cancer treatment31. Recent studies indicated that therapies / agents targeting both differentiated cancer cells and CSCs may possibly have significant therapeutic advantages32. Therefore, it is imperative to look for novel therapeutic agents with lesser side effects urgently for effective targeting of CSCs. In search of novel, nontoxic anti-CSC agents, attention has been focused on natural agents in recent times33,34. In this study, we have used a natural napthoquinone compound, Shk with established antitumorigenic, favorable pharmacokinetic and toxicity profiles and report for the first time its potent anti-CSC properties. Shk significantly inhibits breast cancer cell proliferation in vitroex vivoand in vivo. It decreases the cell migration and invasion of breast cancer cells in vivo, as well as inhibits tumorigenicity, metastasis and metastatic colonization in a syngenic mouse model of breast cancer in vivo. These finding suggest a strong potential of Shk in breast cancer therapy.

We assessed the effect of Shk on the CSC load in breast cancer cells through various functional assays (tumorsphere in vitro and syngenic mouse model of breast cancer in vivo) and quantification of specific stem cell markers. In breast cancer, CD44+ CD24− cells and ALDH1+ cells are considered to be BCSCs2125. Shk significantly decreased the mammosphere formation (Fig. 1HS1G and 2H), ALDH1+ cell and CD44+ CD24− cell loads in vitro (Fig. 2BS2E and S2H). It also reduced the expression of CSC markers (Oct3/4, Sox2, Nanog, c-Myc and Aldh1) in vivo andin vitro (Fig. 2C,DS2C and S2D). These genes are known to regulate stem cell programs and in cancer, they are established promoters and regulators of CSC phenotype353637383940. Decrease in the expression of these genes on Shk treatment indicates its potential to suppress CSC programs. Tumor initiating potential (tumorigenicity) is the bona fide measure of CSCs. Reduction in the tumorigenic potential of cells isolated form Shk treated tumors indicates in vivoanti-CSC effects of Shk.

We further demonstrated that Shk is a potent inhibitor of STAT3 and it also inhibits FAK and Src (Fig. 3A–C). Its STAT3 inhibitory property was found to be responsible for its anti-CSC effects (Figs. 6B and 7B). STAT3 and FAK inhibitors are previously known to compromise CSC growth41,42. Here, we found that pharmacological inhibition of STAT3 was more effective in compromising CSC load than FAK and Src inhibitions (Fig. 5A). STAT3 activation through IL6 increases mammosphere formation more significantly than Src and FAK activation through EGF and Fibronectin (Fig. 5C). This indicates that IL6-STAT3 axis is a key regulator of BCSC dynamics. Ovatodiolide Sensitizes Aggressive Breast Cancer Cells to Doxorubicin Anticancer Activity, Eliminates Their Cancer Stem Cell-Like Phenotype, and Reduces Doxorubicin-Associated Toxicity

Investigators evaluated the usability of ovatodiolide (Ova) in sensitizing triple negative breast cancer (TNBC) cells to doxorubicin (Doxo), cytotoxicity, so as to reduce Doxo effective dose and consequently its adverse effects. Ova-sensitized TNBC cells also lost their cancer stem cell-like phenotype evidenced by significant dissolution and necrosis of formed mammospheres, as well as their terminal differentiation. [Cancer Lett] Glabridin Inhibits Cancer Stem Cell-Like Properties of Human Breast Cancer Cells: An Epigenetic Regulation of miR-148a/SMAd2 Signaling

The authors report that glabridin (GLA) attenuated the cancer stem cell (CSC)-like properties through microRNA-148a (miR-148a)/transforming growth factor beta-SMAD2 signal pathway in vitro and in vivo. In MDA-MB-231 and Hs-578T breast cancer cell lines, GLA enhanced the expression of miR-148a through DNA demethylation. [Mol Carcinog] Ginsenoside Rh2 Inhibits Cancer Stem-Like Cells in Skin Squamous Cell Carcinoma

The effects of ginsenoside Rh2 (GRh2) on Lgr5-positive cancer stem cells (CSCs) were determined by flow cytometry and by tumor sphere formation. Scientists found that GRh2 dose-dependently reduced skin squamous cell carcinoma viability, possibly through reduced the number of Lgr5-positive CSCs. [Cell Physiol Biochem]

Liu S. Chen M. Li P. Wu Y. Chang C. Qiu Y. Cao L. Liu Z. Jia C.
Cell Physiol Biochem 2015;36:499-508

Background/Aims: Treatments targeting cancer stem cells (CSCs) are most effective cancer therapy, whereas determination of CSCs is challenging. We have recently reported that Lgr5-positive cells are cancer stem cells (CSCs) in human skin squamous cell carcinoma (SCC). Ginsenoside Rh2 (GRh2) has been shown to significantly inhibit growth of some types of cancers, whereas its effects on the SCC have not been examined. Methods: Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by fow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for Atg7 and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Results: We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability. Conclusion: Taken together, our data suggest that GRh2 inhibited SCC growth, possibly through reduced the number of Lgr5-positive CSCs. This may be conducted through an interaction Carcinoma account for more than 80% of all types of cancer worldwide, and squamous cell carcinoma (SCC) is the most frequent carcinoma. Skin SCC causes a lot of mortality yearly, which requires a better understanding of the molecular carcinogesis of skin SCC for developing efficient therapy [1,2]. Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng, and has proven therapeutic effects on inflammation [3] and a number of cancers [4,5,6,7,8,9,10,11,12,13,14], whereas its effects on the skin SCC have not been examined.

Cancer stem cells (CSCs) are cancer cells with great similarity to normal stem cells, e.g., the ability to give rise to various cell types in a particular cancer [15,16]. CSCs are highly tumorigenic, compared to other non-CSCs. CSCs appear to persist in tumors as a distinct population and CSCs are believed to be responsible for cancer relapse and metastasis after primary tumor resection [15,16,17,18]. Recently, the appreciation of the critical roles of CSCs in cancer therapy have been continuously increasing, although the identification of CSCs in a particular cancer is still challenging.

To date, different cell surface proteins have been used to isolate CSCs from a variety of cancers by flow cytometry. Among these markers for identification of CSCs, the most popular ones are prominin-1 (CD133), side population (SP) and increased activity of aldehyde dehydrogenase (ALDH). CD133 is originally detected in hematopoietic stem cells, endothelial progenitor cells and neuronal and glial stem cells. Later on, CD133 has been shown to be expressed in the CSCs from some tumors [19,20,21,22,23], but with exceptions [24]. SP is a sub-population of cells that efflux chemotherapy drugs, which accounts for the resistance of cancer to chemotherapy. Hoechst (HO) has been experimentally used for isolation of SP cells, while the enrichment of CSCs by SP appears to be limited [25]. Increased activity of ALDH, a detoxifying enzyme responsible for the oxidation of intracellular aldehydes [26,27], has also been used to identify CSCs, using aldefluor assay [28,29]. However, ALDH has also been detected in other cell types, which creates doubts on the purity of CSCs using ALDH method [30,31]. Moreover, all these methods appear to be lack of cancer specificity.

The Wnt target gene Lgr5 has been recently identified as a stem cell marker of the intestinal epithelium, and of the hair follicle [32,33]. Recently, we reported that Lgr5 may be a potential CSC marker for skin SCC [34]. We detected extremely high Lgr5 levels in the resected skin SCC specimen from the patients. In vitro, Lgr5-positive SCC cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Elimination of Lgr5-positive SCC cells completely inhibited cancer cell growth in vitro.

Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by flow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for autophagy-related protein 7 (Atg7) and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Atg7 was identified based on homology to Pichia pastoris GSA7 and Saccharomyces cerevisiae APG7. In the yeast, the protein appears to be required for fusion of peroxisomal and vacuolar membranes. The protein shows homology to the ATP-binding and catalytic sites of the E1 ubiquitin activating enzymes. Atg7 is a mediator of autophagosomal biogenesis, and is a putative regulator of autophagic function [35,36,37,38]. We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability.

Transduction of SCC cells with GFP under Lgr5 promoter

We have recently shown that Lgr5 is CSC marker for skin SCC [34]. In order to examine the role of GRh2 on SCC cells, as well as a possible effect on CSCs, we transduced human skin SCC cells A431 [34] with a lentivirus carrying GFP reporter under Lgr5 promoter (Fig. 1A). The Lgr5-positive cells were green fluorescent in culture (Fig. 1B), and could be analyzed or isolated by flow cytometry, based on GFP (Fig. 1C).

(not shown)

Fig. 1. Transduction of SCC cells with GFP under Lgr5 promoter. (A) The structure of lentivirus carrying GFP reporter under Lgr5 promoter. (B) The pLgr5-GFP-transduced A431 cells in culture. Lgr5-positive cells were green fluorescent. Nuclear staining was done by DAPI. (C) Representative flow chart for analyzing pLgr5-GFP-transduced A431 cells by flow cytometry based on GFP. Gated cells were Lgr5-positive cells. Scar bar is 20µm.

GRh2 dose-dependently inhibits SCC cell growth

Then, we examined the effect of GRh2 on the viability of SCC cells. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. We found that from 0.01mg/ml to 1mg/ml, GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (Fig. 2A), or a MTT assay (Fig. 2B). Next, we questioned whether GRh2 may have a specific effect on CSCs in SCC cells. Thus, we analyzed GFP+ cells, which represent Lgr5-positive CSCs in pLgr5-GFP-transduced A431 cells after GRh2 treatment. We found that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (Fig. 2C), and by quantification (Fig. 2D). We also examined the capability of the GRh2-treated cells in the formation of tumor sphere. We found that GRh2 dose-dependently deceased the formation of tumor sphere-like structure, by quantification (Fig. 2E), and by representative images (Fig. 2F). Together, these data suggest that GRh2 dose-dependently inhibited SCC cell growth, possibly through inhibition of CSCs.

Fig. 2. GRh2 dose-dependently inhibits SCC cell growth. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. (A-B) GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (A), or a MTT assay (B). (C-D) GFP+ cells after GRh2 treatment were analyzed by flow cytometry, showing that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (C), and by quantification (D). The capability of the GRh2-treated cells to form tumor sphere-like structures was examined, shown by quantification (E), and by representative images (F). *p


GRh2 treatment decreases β-catenin and increases autophagy in SCC cells

We analyzed the molecular mechanisms underlying the cancer inhibitory effects of GRh2 on SCC cells. We thus examined the growth-regulatory proteins in SCC. From a variety of proteins, we found that GRh2 treatment dose-dependently decreases β-catenin, and dose-dependently upregulated autophagy-related proteins Beclin, Atg7 and increased the ratio of LC3 II to LC3 I, by quantification (Fig. 3A), and by representative Western blots (Fig.3B). Since β-catenin signaling is a strong cell-growth stimulator and autophagy can usually lead to stop of cell-growth and cell death, we feel that the alteration in these pathways may be responsible for the GRh2-mediated suppression of SCC growth.

(not shown)

Figure 3. GRh2 treatment decreases β-catenin and increases autophagy in SCC cells.


Inhibition of autophagy abolishes the effects of GRh2 on β-catenin

In order to find out the relationship between β-catenin and autophagy in this model, we inhibited autophagy using a shRNA for Atg7, and examined its effect on the changes of β-catenin by GRh2. First, the inhibition of Atg7 in A431 cells by shAtg7 was confirmed by RT-qPCR (Fig. 4A), and by Western blot (Fig. 4B). Inhibition of Atg7 resulted in abolishment of the effects of GRh2 on other autophagy-associated proteins (Fig. 4B), and resulted in abolishment of the inhibitory effect of GRh2 on β-catenin (Fig. 4B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 4C). Together, inhibition of autophagy abolishes the effects of GRh2 on β-catenin. Thus, the regulation of GRh2 on β-catenin needs autophagy-associated proteins.

Fig. 4. Inhibition of autophagy abolishes the effects of GRh2 on β-catenin.

A431 cells were transfected with shRNA for Atg7, or scrambled sequence (scr) as a control. (A) RT-qPCR for Atg7. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p


Overexpression of β-catenin abolishes the effects of GRh2 on autophagy

Next, we inhibited the effects of GRh2 on β-catenin by overexpression of β-catenin in A431 cells. First, the overexpression of β-catenin in A431 cells was confirmed by RT-qPCR (Fig. 5A), and by Western blot (Fig. 5B). Overexpression of β-catenin resulted in abolishment of the effects of GRh2 on autophagy-associated proteins (Fig. 5B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 5C). Together, inhibition of β-catenin signaling abolishes the effects of GRh2 on autophagy. Thus, the regulation of GRh2 on autophagy needs β-catenin signaling. This model is thus summarized in a schematic (Fig. 6), suggesting that GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.


Fig. 5. Overexpression of β-catenin abolishes the effects of GRh2 on autophagy. A431 cells were transfected with β-catenin, or scrambled sequence (scr) as a control. (A) RT-qPCR for β-catenin. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p


Fig. 6. Schematic of the model. GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.

Understanding the cancer molecular biology of skin SCC and identification of an effective treatment are both critical for improving the current therapy [1]. Lgr5 has been recently identified as a novel stem cell marker of the intestinal epithelium and the hair follicle, in which Lgr5 is expressed in actively cycling cells [32,33]. Moreover, we recently showed that Lgr5-positive are CSCs in skin SCC [34]. Thus, specific targeting Lgr5-positive cells may be a promising therapy for skin SCC.

In the current study, we analyzed the effects of GRh2 on the viability of SCC. Importantly, we not only found that GRh2 dose-dependently decreases SCC cell viability, but also dose-dependently decreased the number of Lgr5-positive CSCs in SCC cells. These data suggest that the CSCs in SCC may be more susceptible for the GRh2 treatment, and the decreases in CSCs may result in the decreased viability in total SCC cells. This point was supported by following mechanism studies. Activated β-catenin signaling by WNT/GSK3β prevents degradation of β-catenin and induces its nuclear translocation [39]. Nuclear β-catenin thus activates c-myc, cyclinD1 and c-jun to promote cell proliferation, and activates Bcl-2 to inhibit apoptosis [39]. High β-catenin levels thus are a signature of CSCs. Therefore, it is not surprising that CSCs are more affected than other cells when GRh2 targets β-catenin signaling.

In addition, GRh2 appears to target autophagy. Although altered metabolism may be beneficial to the cancer cells, it can create an increased demand for nutrients to support cell growth and proliferation, which creates metabolic stress and subsequently induces autophagy, a catabolic process leading to degradation of cellular components through the lysosomal system [40]. Cancer cells use autophagy as a survival strategy to provide essential biomolecules that are required for cell viability under metabolic stress [40]. However, autophagy not only results in a staring in cell growth, but also may result in cell death [40]. Increases in autophagy may substantially decrease cancer cell growth. Thus, GRh2 has its inhibitory effect on skin SCC cells through a combined effect on cell proliferation (by decreasing β-catenin) and autophagy [40].

Interestingly, our data suggest an interaction between β-catenin and autophagy. This finding is consistent with previous reports showing that autophagy negatively modulates Wnt/β-catenin signaling by promoting Dvl instability [41,42], and with other studies showing that β-catenin regulates autophagy [38,43,44].

Of note, we have checked other SCC lines and essentially got same results. Together with our previous reports showing that Lgr5-positive cells are CSCs in skin SCC [34], these findings thus highlight a future engagement of Lgr5-directed GRh2 therapy, which could be performed in a sufficiently frequent manner, to substantially improve the current treatment for skin SCC.

Normal vs Cancer Thyroid Stem Cells: The Road to Transformation
The authors discuss new insights into thyroid stem cells as a potential source of cancer formation in light of the available information on the oncogenic role of genetic modifications that occur during thyroid cancer development. Understanding the fine mechanisms that regulate tumor transformation may provide new ground for clinical intervention in terms of prevention, diagnosis and therapy. [Oncogene] Abstract
Cancer Stem Cells: A Potential Target for Cancer Therapy
The identification of cancer stem cells (CSCs) and a better understanding of the complex characteristics of CSCs will provide invaluable diagnostic, therapeutic and prognostic targets for clinical application. The authors introduce the dysregulated properties of CSCs in cancers and discuss the possible challenges in targeting CSCs for cancer treatment. [Cell Mol Life Sci] Abstract
Targeting Cancer Stem Cells Using Immunologic Approaches
Wicha, M; Chang, A; Yingxin, X; Xiaolian, Z; Ning, N; Liu, Shuang, Q, L; Pan, Q
Stem Cells 2015-04-15 4.15 | Apr 22
Targeting Notch, Hedgehog, and Wnt Pathways in Cancer Stem Cells: Clinical Update
Ivy, P; Takebe, N
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Hypoxia-Inducible Factors in Cancer Stem Cells and Inflammation
Liu, Y; Peng, G
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NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions
Tang, D; Chao, HP; Wang, J; Yang, Tao; Jeter, C
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Two Genes Control Breast Cancer Stem Cell Proliferation and Tumor Properties

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Targeting the Wnt Pathway

Writer and Curator: Larry H Bernstein, MD, FCAP 

Trageting the Wnt Pathway [7.11]

Word Cloud created by Noam Steiner Tomer 8/10/2020

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

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.




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.

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


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


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],

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


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

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.



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.


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.


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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

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


  • 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.

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.


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.


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.


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.


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.


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.


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


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

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

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.


  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

    also this is a reference which should be linked to great article by Emma Hill

    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
(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)
(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)
(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)
(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
(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.
(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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Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Author and Curator: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN

This article is a presentation of recently published work on the basis for control of mesodermal and cardiovascular differentiation of embryonic stem cells, which has taken on increasing importance in the treatment of cardiovascular disease, with particular application to heart failure due to any cause, but with particular relevance to significant loss of myocardium, as may occur with acute myocardial infarction with more than 60% occlusion of the left anterior descending artery, near the osteum, with or without adjacent artery involvement, resulting in major loss of cardiac contractile force and insufficient ejection fraction. The article is of high interest and makes the following points:

  1. Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal
  2. Ablation of Jmjd3 in mouse embryonic stem cells compromised mesoderm and subsequent endothelial and cardiac differentiation 
  3. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin
  4. β-catenin s critical for Wnt signal–induced mesoderm differentiation. 

Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

K Ohtani, C Zhao, G Dobreva, Y Manavski, B Kluge, T Braun, MA Rieger, AM Zeiher and S Dimmeler

I The Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany (K.O., C.Z., Y.M., B.K., S.D.); Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (G.D., T.B.); Department of Hematology/Oncology, Internal Medicine
II LOEWE Center for Cell and Gene Therapy, University of Frankfurt, Frankfurt, Germany (M.A.R.); and Department of Cardiology, Internal Medicine
III University of Frankfurt, Frankfurt, Germany (A.M.Z.).
This manuscript was sent to Benoit Bruneau, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Stefanie Dimmeler,  Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany. E-mail dimmeler@em.uni-frankfurt.de
 Circ Res. 2013;113:856-862;  http://dx.doi.org/10.1161/CIRCRESAHA.113.302035   http://circres.ahajournals.org/content/113/7/856  


Rationale: The developmental role of the H3K27 demethylases Jmjd3, especially its epigenetic regulation at target genes in response to upstream developmental signaling, is unclear.

Objective: To determine the role of Jmjd3 during mesoderm and cardiovascular lineage commitment.

Methods and Results: Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal but compromised mesoderm and subsequent endothelial and cardiac differentiation. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin, which is critical for Wnt signal–induced mesoderm differentiation.

Conclusions: These data demonstrate that Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment. (Circ Res. 2013;113:856-862.)

  • Key Words: Brachyury protein ■ embryonic stem cells ■ epigenomics ■ Jmjd3 protein, mouse ■ mesodermn  –  Wnt signaling pathway


Post-translational modifications of histone proteins represent essential epigenetic control mechanisms that can either allow or repress gene expression.1 Trimethylation of H3K27 is mediated by Polycomb group proteins and represses gene expression.2 The JmjC domain–containing proteins, UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) and Jmjd3 (jumonji domain–containing protein 3, Kdm6b), not only act as demethylases to remove the repressive H3K27me3 marks, but also exhibit additional demethylase-independent functions.3–6 Jmjd3 is induced and participates in Hox gene expression during development,7 neuronal differentiation,8,9 and inflammation,5,10–12 and recent data suggest that Jmjd3 inhibits reprogramming by inducing cellular senescence.13 Because previous studies suggest that H3K27me3 regulates endothelial gene expression in adult proangiogenic cells,14 we addressed the function of Jmjd3 in cardiovascular lineage differentiation of embryonic stem cells (ESCs).


A detailed description of the experimental procedure is provided in the Online Data Supplement.  The online-only Data Supplement is available with this article at   http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 113.302035/-/DC1.

Cell Culture

Plasmid Construction and Stable Transfection

The full-length Jmjd3, the mutants, and Brachyury were cloned into pEF1 vector (Invitrogen). The linearized plasmids were transfected in Jmjd3−/− ESCs using the Amaxa nucleofection system (Lonza).

Chromatin Immunoprecipitation

Nonstandard Abbreviations and Acronyms

EB                       embryoid body

ESC                    embryonic stem cell

WI                        wild type


Jmjd3 knockout ESCs were generated by 2 rounds of gene targeting (Online Figure IA and IB). We obtained 7 Jmjd3−/− ESC clones, which lacked Jmjd3 mRNA and protein expression. All of the clones showed slightly increased global H3K27me3, but the expression of pluripotency genes, the morphology, the growth kinetic, and survival was indistinguishable from wild-type (WT) ESCs (Figure 1A–1C; Online Figure IC–IF). No significant changes of repressive H3K27me3 marks at the promoters of pluripotency genes were detected in Jmjd3−/− compared with WT ESCs (Online Figure IH). When

  • spontaneous differentiation was induced by leukemia inhibitory factor withdrawal,
  • Jmjd3 expression increased in WT ESCs (Figure 1D).
  • EBs derived from Jmjd3−/− ESCs were slightly smaller in size compared with WT EBs (Figure 1E).

mRNA expression profiling of Jmjd3−/− and WT ESC clones at day 4 after induction of differentiation showed

  • a distinct expression pattern of lineage-specific genes (Online Figure IIA).

Gene ontology functional analyses revealed a significant repression of genes that are involved in mesoderm development (Figure 1F; Online Figure IIB). Moreover,

  • repressed gene sets in Jmjd3−/− EBs were shown to be related to cardiac and vascular development,
  • consistent with impaired mesoderm differentiation (Figure 1F; Online Figure IIB).

Validation of the microarray results showed a similar reduction of pluripotency gene expression after leukemia inhibitory factor withdrawal in Jmjd3−/− compared with WT ESCs (Figure 1G). However, depletion of Jmjd3 substantially compromised the induction of mesodermal genes (Figure 1G). Especially, the pan-mesoderm marker, Brachyury, and the early mesoendoderm marker, Mixl1, were profoundly increased at day 4 of differentiation in WT ESCs, but not in Jmjd3−/− ESCs (Figure 1G). Moreover, the mesoendodermal marker, Eomes, and endodermal markers, such as Sox17 and FoxA2, were significantly suppressed, which is consistent with a very recent study showing that Jmjd3 is required for endoderm differentiation.19 Ectodermal markers were not significantly changed in Jmjd3−/− ESCs when using the spontaneous differentiation protocol (Figure 1G).

Because Jmjd3−/− ESCs showed a prominent inhibition of mesodermal markers after leukemia inhibitory factor withdrawal, we next questioned whether this phenotype can also be observed when directing differentiation of mesoderm using 2 different protocols. Consistent with our findings,

  • Jmjd3−/− ESCs showed a reduced expression of mesodermal marker genes when using the protocol for mesoderm differentiation described by Gadue et al20 (data not shown). Moreover,
  • mesoderm differentiation was significantly suppressed when Jmjd3−/− ESCs were cultured on OP9 stromal cells, which support mesodermal differentiation21 (Figure 2A).

Whereas WT ESCs showed the typical time-dependent increase in Brachyury+ cells, Jmjd3−/− ESCs generated significantly less Brachyury+ mesodermal cells (Figure 2B). Moreover, fluorescence activated cell sorting analysis revealed that fetal liver kinase (Flk)1+ vascular endothelial-cadherin−mesodermal cells were generated in WT ESCs but were reduced when Jmjd3−/− ESCs were used (Figure 2C). Interestingly, the formation of vascular endothelial-cadherin+ Flk+ cells was also significantly reduced by 96±1% and 88±3% in the 2 Jmjd3−/− ESC clones compared with WT ESCs (P<0.01), prompting us to explore the role of Jmjd3 in vascular differentiation further.
Endothelial differentiation was induced by a cytokine cocktail18 and was associated with a significant upregulation of Jmjd3 expression (Online Figure IIIA). Jmjd3−/− ESCs showed a marked reduction of endothelial differentiation as evidenced by

  • significantly reduced mRNA levels of the endothelial marker vascular endothelial-cadherin and endothelial-specific receptor tyrosine kinase Tie2 (Figure 3A).
  • The formation of endothelial marker expressing vascular structures after induction of endothelial differentiation was abolished in Jmjd3−/− ESCs (Figure 3B; Online Figure IIIB).
  • The impaired endothelial differentiation of Jmjd3−/− cells was partially rescued by the overexpression of Brachyury (Online Figure IIIC and IIID), suggesting that the inhibition of mesoderm formation, at least in part, contributes to the impaired endothelial commitment.

Because genes involved in heart development and morphogenesis were significantly downregulated in Jmjd3−/− ESCs on differentiation (Figure 1F; Online Figure II), we additionally determined the capacity of Jmjd3−/− ESCs to generate cardio-myocytes by inducing cardiac differentiation.17

  • Expression of cardiac progenitor cell markers, Mesp1 and Pdgfra, was inhibited in Jmjd3−/− ESCs compared with WT ESCs (Figure 3C).

Moreover, after plating on gelatin-coated dishes,

  • the Jmjd3−/− ESCs showed an impaired formation of EBs and
  • only 20% of EBs were contracting (Figure 3D).

Consistently, expression of the cardiac transcription factor Mef2c, the marker of working myocardium Nppa, and cardiac structural proteins TnT2 and α-myosin heavy chain were downregulated in Jmjd3−/− ESCs (Figure 3E and 3F; Online Figure IIIE).

  • Next, we addressed whether the impaired mesoderm differentiation observed in Jmjd3−/− ESCs might be mediated by an increase of repressive H3K27me3 marks at the promoters of developmental regulators. Of the various promoters studied, only Brachyury and Mixl1 showed a significant augmentation of H3K27me3 marks in Jmjd3−/− ESCs on differentiation (Figure 4A; Online Figure IVA). Consistently, the recruitment of RNA polymerase II to the transcription start sites of the promoters of Brachyury and Mixl1 was also significantly reduced (Online Figure IVC). In addition, Jmjd3 deficiency repressed polymerase II recruitment to the Flk1 and Mesp1 promoter but the inactivation of these promoters was not associated with changes in H3K27me3 marks (Figure IVA and IVC). These data were confirmed using protocols
  • that induce mesoderm differentiation by addition of Wnt3a (data not shown).20 Under these conditions,
  • Jmjd3−/− ESCs showed a 1.81±0.23-fold (P<0.05) enrichment of H3K27me3 marks at the Brachyury promoter compared with WT ESCs.

To determine whether the demethylase activity of Jmjd3 controls Brachyury expression by reducing repressive H3K27me3 marks during differentiation, we overexpressed full-length Jmjd3, the carboxyl-terminal part, including the JmjC-domain (cJmjd3: amino acids, 1141–1641), and a carboxyl-terminal mutant construct, which includes a point mutation (cJmjd3H1388A) to inactivate demethylase activity. Overexpression of full-length Jmjd3 and the carboxyl-terminal part of Jmjd3 in Jmjd3−/− ESCs partially rescued the expression of Brachyury on differentiation (Figure 4B and 4C). Howver, the inactive carboxyl-terminal part of Jmjd3 failed to rescue the impaired Brachyury expression in Jmjd3−/− ESCs (Figure 4C), suggesting that

  • the demethylase activity of Jmjd3 is required for the activation of the Brachyury promoter.

Because canonical Wnt signaling regulates the expression of Brachyury during development22,23 and Wnt/B-catenin–dependent genes were suppressed in Jmjd3−/− EBs compared with WT EBs (Online Figure V), we further explored whether Jmjd3 might interact with B-catenin signaling. Indeed,

  • B-catenin recruitment to the Brachyury promoter was significantly suppressed in Jmjd3−/− ESCs (Figure 4D) and
  • was rescued by Jmjd3 overexpression (Figure 4E).

Similar results were obtained when using the protocol for direct mesoderm differentiation described by Gadue et al20 (data not shown). To determine whether Jmjd3 might interact with B-catenin, we performed coimmunoprecipitation studies and showed that

  • Jmjd3 interacts with B-catenin in human embryonic kidney 293 cell and differentiated EBs (Figure 4F; Online Figure VI).

To assess a direct effect of Jmjd3 on B-catenin responsive promoter activity, we used a luciferase reporter assay. Coexpression of lymphoid enhancer binding factor 1 and the constitutive active form of B-catenin harboring a nuclear localization signal resulted in the activation of lymphoid enhancer binding factor 1 luciferase reporter activity in WT ESCs, but

  • this transcriptional activation was markedly impaired in Jmjd3−/− ESCs (Figure 4G).


The data of the present study demonstrate that

  • deletion of Jmjd3 in ESCs does not affect self-renewal but
  • significantly impairs the formation of mesoderm on induction of differentiation.

The findings that Jmjd3 is not required for ESC maintenance are consistent with the dispensability of the Polycomb complex and the related demethylase UTX for self-renewal.1

  • The requirement of Jmjd3 for mesoderm differentiation was shown in spontaneous differentiation, as well as
  • when more specifically inducing mesoderm differentiation by the OP9 coculture system or under serum-free conditions followed by Wnt3a stimulation.
  • Jmjd3 deficiency profoundly suppressed the expression of Brachury, which is essential for mesoderm differentiation.

In the absence of Jmjd3,

  • repressive H3K27me3 marks at the Brachyury promoter are significantly increased, and
  • the recruitment of B-catenin, which is a prerequisite for Wnt-induced mesoderm differentiation, is impaired.
  • In addition, Jmjd3 is interacting with B-catenin and is contributing to B-catenin– dependent promoter activation.

This is consistent with the recent findings that cofactors can form a complex with B-catenin/ lymphoid enhancer binding factor 1

  • at Tcf/lymphoid enhancer binding factor 1 binding sites
  • at B-catenin–dependent promoters and
  • synergize with canonical Wnt signaling.24

Interestingly, a demethylase-independent regulation of B-catenin–dependent gene expression was recently described for UTX.25 However, our data provide evidence that

  • Brachyury expression in Jmjd3−/− ESCs is only rescued by catalytically active Jmjd3, which has maintained the demethylase activity.

On the basis of these findings, we propose a model in which Jmjd3 is recruited to the Brachury promoter to remove repressive H3K27me3 marks and on Wnt stimulation additionally promotes B-catenin–dependent promoter activation (Figure 4H). Such a model is similar to the recently described function of Jmjd3 in endoderm differentiation, whereby Jmjd3 associates with Tbx3 and is recruited to the poised promoter of Eomes, to mediate chromatin remodeling allowing subsequent induction of endoderm differentiation induced by activin A.19 The present study additionally demonstrates that

  • Jmjd3 contributes to endothelial and cardiac differentiation.
  • endothelial differentiation was profoundly impaired,

a finding that is consistent with previous findings in adult progenitor cells, showing a high H3K27me3 at endothelial genes.14 The relatively modest inhibition of cardiomyocyte differentiation in Jmjd3−/−  ESCs may be, in part, explained by a compensatory effect of UTX which was shown to regulate cardiac development.26 Together, our study provides first evidence for the regulation of B-catenin–dependent Wnt target genes by Jmjd3 during differentiation of ESCs.  However, the in vivo relevance of the findings is still unclear. The Jmjd3−/− mice that we have generated out of the ESCs, used in the present study, showed embryonic lethality before E6.5, suggesting a crucial role of Jmjd3 in early embryonic development.


Novelty and Significance

What Is Known?

•            Cell fate decisions require well-controlled changes in gene expression that are tightly controlled by epigenetic modulators.

•            The post-transcriptional modifications of histone proteins epigeneti-cally regulate gene expression.

•            Trimethylation of lysine 27 at histone K3 (H3K27me3) silences gene expression and can be reversed by the demethylase Jmjd3.

What New Information Does This Article Contribute?

•            The histone demethylase Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment of mouse embryonic stem cells.

•            This effect is partially mediated by a silencing of the mesodermal regulator Brachyury.

•            Ablation of Jmjd3 further reduces β-catenin recruitment to the Brachyury promoter, which interferes with Wnt signaling that is required for proper mesoderm differentiation.

The differentiation of stem cells to specific lineages requires a well-defined modulation of gene expression programs, which is often controlled by epigenetic mechanisms. Although several epi-genetically active enzymes and complexes have been described, the function of the histone demethylase Jmjd3 for cardiovascu¬lar lineage commitment was unknown. Using mouse embryonic stem cells as a model, we now show that the demethylase Jmjd3 is required for mesoderm differentiation and for the differentia¬tion of embryonic stem cells to the vascular and cardiac lineage. We further identified the mechanism and showed that ablation of Jmjd3 resulted in a silencing of the Brachyury promoter that is associated with an increase in H3K27me3 marks. In addition, Jmjd3 was shown to facilitate the recruitment of β-catenin to the Brachyury promoter, which contributes to the Wnt-dependent ac-tivation of mesoderm differentiation. Together these data describe a novel epigenetic mechanism that controls cell fate decision.

Supplemental Methods

Generation of Jmjd3 knockout ES cell lines

Mouse genomic DNA encompassing the murine Jmjd3 gene region were isolated by PCR amplification and used to generate short (1.6kb) and long (6.2kb) arms of homology. The targeting vectors were constructed by inserting a loxP site together with an FRT flanked neomycin selection cassette within the intron 5 and a single distal loxP within the intron 3. This targeting strategy results in the deletion of 600bp coding sequences encoding for the ATG methionine codon and produces a frame shift of JmjC domain existing exon 19-21 required for Jmjd3 demethylase activity. The targeting vector was electroporated in 129Sv ES cells. G418 resistant ES cell clones were screened for homologous recombination by PCR analysis and targeting was verified by Southern blot analysis. Homozygous Jmjd3lox/lox ES cells were generated by electroporation of heterozygous Jmjd3lox/+ ES cells with the same targeting vector as above except that the neomycin resistance gene was replaced by puromycin gene using the Nucleofector (Lonza). Double-allele-recombined ES cells were selected for puromycin (1.3µg/mL, Invitrogen). Correct targeting of homozygous Jmjd3lox/lox ES clones were determined by PCR. To obtain Jmjd3-/- ES cells, Jmjd3lox/lox ES cells were electroporated with Cre-recombinase plasmid vector and loss of targeting cassettes was evaluated by loss of resistance of G418 and puromycin. Correct targeting of homozygous Jmjd3-/- ES cells was determined by PCR.

Reporter gene assays

3xLEF1 reporter plasmid, LEF1 expression construct and NLS-13-catenin were kind gifts from Rudolf Grosschedl. Mouse ES cells were seeded (5×104) on gelatin coated 24-well. After 24 hours of plating, 3xLEF1 reporter plasmid, LEF1, and NLS-13-catenin plasmids were transiently transfected with FugeneHD (Promega). 13-galactosidase plasmid was co-transfected for normalization of transfection efficiency. Each group was transfected in triplicates. 48 hours after transfection, cells were harvested. Cell lysis and luciferase assay were performed following the protocol of Luciferase Reporter Assay System (Promega). 13-galactosidase assays were performed using CPRG (Sigma) as substrate and the absorbance at 600nm was measured. Luciferase activity was normalized to 13-galactosidase activity.

Manuscript References

  1. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32, 491-502 (2008).
  2. Sargent, C.Y., Berguig, G.Y. & McDevitt, T.C. Cardiomyogenic differentiation of embryoid bodies is promoted by rotary orbital suspension culture. Tissue engineering. Part A 15, 331­342 (2009).
  3. Gadue, P., Huber, T.L., Paddison, P.J. & Keller, G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A 103, 16806-16811 (2006).
  4. Ohtani, K. et al. Epigenetic regulation of endothelial lineage committed genes in pro-angiogenic hematopoietic and endothelial progenitor cells. Circ Res 109, 1219-1229 (2011).
  5. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A.P. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13, 3185-3190 (1999).

Selected Figures   (Figure may not be shown)

Figure 1. Aberrant differentiation of Jmjd3−/− embryonic stem cells (ESCs). A, Quantitative polymerase chain reaction analysis of Jmjd3 in wild-type (WT) and Jmjd3−/− ESCs. B, Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3−/− ESCs. Histone H3 is used as a loading control. Quantification is shown in the right (n=3–5). C, Top, Morphology of WT and Jmjd3−/− ESCs on feeder cells. Bottom, Alkaline phosphatase staining of undifferentiated WT and Jmjd3−/− ESCs. D, Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. α-Tubulin is used as a loading control. E, Bright field image of embryoid bodies at day 5. Scale bar, 200 μm. F, Gene ontology analysis for >2-fold repressed genes in Jmjd3−/− ESCs compared with WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and P values for the significance of enrichment are shown on the x-axis. G, Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3−/− ESCs after spontaneous differentiation by leukemia inhibitory factor withdrawal (n=4). Flk indicates fetal liver kinase.

Figure 2. Jmjd3−/− embryonic stem cells (ESCs) show an impaired ability to differentiate into mesoderm. A, Schematic illustration of the experimental protocol. Differentiation of ESCs (wild-type [WT] and 2 Jmjd3−/− ESCs clones) on OP9 feeder cells was analyzed. B, Left, Representative fluorescence activated cell sorting (FACS) plots showing Brachyury expression of ESC-derived cells. Right, Quantification of FACS analyses (n=3). C, Left, Representative FACS plots showing fetal liver kinase 1 (Flk1) and vascular endothelial-cadherin expression on ESC-derived cells. Right, Quantification of FACS analyses in Flk1+ cells (n=3).

Figure 3. Jmjd3 is required for embryonic stem cells (ESCs) differentiation to the endothelial and cardiac lineage. A, mRNA expression of endothelial markers at day 7 of endothelial differentiation (n=3). B, Platelet endothelial cell adhesion molecule (Pecam)-1 staining of wild-type (WT) and Jmjd3−/− ESCs at day 8 of endothelial differentiation. Phalloidin is used to stain F-actin. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 20 pm. C, Gene expression of cardiac progenitor markers at day 3 of cardiac differentiation. D, Number of beating embryoid bodies (EBs) at day 10 of cardiac differentiation (n=8). E, Gene expression of cardiac markers at day 7 of cardiac differentiation (n=6). F, α-Myosin heavy chain staining of WT and Jmjd3−/− ESCs at day 9 of cardiac differentiation. Nuclei are stained with Hoechst (blue). Scale bar, 20 pm. *P<0.05, **P<0.01, and ***P<0.001

Online Figure I. Generation and characterization of Jmjd3 ESCs  (A) Targeting strategy to generate Jmjd3 mutant ESCs by homologous recombination. Primers used for PCR are shown. (B) Genotyping of Jmjd3 ESCs by using 2 different primers. (C) Oct4 and Nanog staining in WT and Jmjd3−’− ESCs. Scale bar indicates 10µm. (D) Expression of Oct4 and Nanog in WT and Jmjd3 ESCs. Data are presented as fold changes compared with day 0 WT ESCs. N=6. (E) Tunel staining (green) of WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (F) Growth curves of WT and Jmjd3 ESCs. N=6-8. (G) H3K27me3 staining in WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (H) ChIP assay of undifferentiated WT and Jmjd3 ESCs for H3K27me3. ChIP enrichments are normalized to Histone H3 density and represented as fold change relative to WT. N=3. Data represent mean ± SEM

Online Figure II. Jmjd3 ESCs show an impaired mesoderm differentiation. (A) Microarray gene expression heat map depicting expression of representative pluripotency and lineage markers 4 days after differentiation in Jmjd3 ESCs versus WI ESCs. Coloring illustrates log2 fold changes between Jmjd3 ESCs and WI ESCs. Green and red colors represent down-regulation and up-regulation, respectively. (B) Gene ontology analysis for more than 2-fold altered genes in Jmjd3 ESCs compared to WI ESCs 4 days after differentiation. Red and green colors represent down-regulation and up-regulation, respectively.

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