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Posts Tagged ‘p53’


Lung Cancer Therapy

Curator: Larry H. Bernstein, MD, FCAP

 

Lung Cancer Targets

http://www.oncotherapynetwork.com/lung-cancer-targets

The enzyme ephrin receptor A2 (EphA2) normally blocks KRAS mutation-driven lung adenocarcinoma tumorigenesis, but a new study shows that EphA2 deletion mutation allows aggressive tumor growth—providing “important therapeutic targets” for this deadly form of lung cancer.

Using shRNA-mediated screening of 4,700 candidate human genes with tumor suppression potential, the team subsequently identified 16 genes in animal models that inhibit or slow KRAS- or TP53-driven tumorigenesis. The loss of EphA2 enhanced KRASG12D-driven lung adenocarcinoma carcinogenesis, they found.1

“We identified several tumor suppressors, including EphA2, loss of which promotes adenocarcinoma in the context of KRASG12D mutation,” the coauthors reported.1EphA2 loss promotes cell proliferation by activating ERK MAP kinase signaling and hedgehog signaling pathways, leading to tumorigenesis.”

A KRAS mutation is associated with tumorigenesis in 300 days, in animal models, noted senior author Inder Verma, PhD, Professor of Genetics and the Salk Institute’s Irwin and Joan Jacobs Chair in Exemplary Life Science.

“But without EphA2, the KRAS mutation leads to tumors in half the time, 120 to 150 days,” Dr. Verma noted. “This molecule EphA2 is having a huge effect on restraining cancer growth when KRAS is mutated.”

Up to 20% of all cancers harbor KRAS mutations, and these aberrations are particularly common in lung and colon cancers. EphA2 gene mutations were found in 54 of 230 patients whose lung adenocarcinoma tumor genomes were sequenced in the Cancer Genome Atlas Project, the coauthors noted.

“Oddly, among human lung cancer patients with EPhA2 mutations, around 8% of patients actually have high EphA2 expression,” cautioned coauthor Yifeng Xia, PhD, also at the Salk Institute. “So, in some instances, EphA2 is not suppressing tumors and may be context-dependent. Therefore, we need to carefully evaluate the molecule’s function when designing new therapeutics.”

EphA2 activation suppresses both cell signaling and cell proliferation, the team noted.  “We believe that the enzyme might serve as a potential drug target in KRAS-dependent lung adenocarcinoma,” explained lead study author Narayana Yeddula, PhD, a Salk research associate.

Salk Institute for Biological Studies. (2015). Molecular “brake” stifles human lung cancer.

 

Molecular “brake” stifles human lung cancer  

By testing over 4,000 genes in human tumors, a Salk team uncovered an enzyme responsible for suppressing a common and deadly lung cancer

 

 

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

Carminia Maria Della Corte1Claudio Bellevicine2Giovanni Vicidomini3Donata Vitagliano1Umberto Malapelle2, et al.

Clin Cancer Res Oct 15, 2015; 21: 4686   http://dx.doi.org:/10.1158/1078-0432.CCR-14-3319   

 

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.

Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines.

Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.

In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.

Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

This article is featured in Highlights of This Issue, p. 4497

Translational Relevance

The amplification of SMO in non–small cell lung cancer (NSCLC) resistant to EGFR-TKIs opens new possibilities of treatment for those patients who failed first-line EGFR-targeted therapies. The synergistic interaction of the Hedgehog (Hh) and MET pathways further support the rationale for a combined therapy with specific inhibitors. In addition, Hh pathway activation is essential for the acquisition of mesenchymal properties and, as such, for the aggressiveness of the disease. Also, in EGFR wild-type NSCLC models, inhibition of Hh, along with inhibition of EGF receptor (EGFR), can revert the resistance to anti-EGFR targeted drugs. In addition, inhibition of the Hh pathway sensitizes EGFR wild-type NSCLC to standard chemotherapy. These data encourage further evaluation of Hh inhibitors as novel therapeutic agents to overcome tyrosine kinase inhibitor (TKI) resistance and to revert epithelial-to-mesenchymal transition (EMT) in NSCLC.

Tyrosine kinase inhibitors (TKI) against the EGF receptor (EGFR) represent the first example of molecularly targeted agents developed in the treatment of non–small cell lung cancer (NSCLC) and are, currently, useful treatments after failure of first-line chemotherapy and, more importantly, for the first-line treatment of patients whose tumors have EGFR-activating gene mutations (1). However, after an initial response, all patients experience disease progression as a result of resistance occurrence. Recognized mechanisms of acquired resistance to anti-EGFR-TKIs in EGFR-mutated NSCLC are METgene amplification or the acquisition of secondary mutations such as the substitution of a threonine with a methionine (T790M) in exon 20 of the EGFR gene itself (2). However, these molecular changes are able to identify only a portion of patients with cancer defined as “non-responders” to EGFR-targeted agents. A number of molecular abnormalities in cancer cells may partly contribute to resistance to anti-EGFR agents (2, 3). Our group and others have shown that epithelial-to-mesenchymal transition (EMT) is a critical event in the metastatic switch and is generally associated with resistance to molecularly targeted agents in NSCLC models (4, 5). EMT is a process characterized by loss of polarity and dramatic remodeling of cell cytoskeleton through loss of epithelial cell junction proteins, such as E-cadherin, and gain of mesenchymal markers, such as vimentin (6). The clinical relevance of EMT and drug insensitivity comes from studies showing an association between epithelial markers and sensitivity to erlotinib in NSCLC cell lines, suggesting that EMT-type cells are resistant to erlotinib (7). In particular, recent data suggest that cancer cells with EMT phenotype demonstrate stem cell–like features and strategies reverting EMT could enhance the therapeutic efficacy of EGFR inhibitors (4, 5).

The Hedgehog (Hh) signaling cascade has emerged as an important mediator of cancer development and metastatic progression. The Hh signaling pathway is composed of the ligands sonic, Indian, and desert hedgehog (Shh, Ihh, Dhh, respectively) and the cell surface molecules Patched (PTCH) and Smoothened (SMO). In the absence of Hh ligands, PTCH causes suppression of SMO; however, upon ligand binding to PTCH, SMO protein leads to activation of the transcription factor GLI1, which in turn translocates into the nucleus, leading to the expression of Hh induced genes (8). The Hh signaling pathway is normally active in human embryogenesis and in tissue repair, as well as in cancer stem cell renewal and survival. This pathway is critical for lung development and its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated in bronchial epithelial cells exposed to cigarette smoke extraction. In particular, the activation of this pathway happens at an early stage of carcinogenesis when cells acquire the ability to growth in soft agar and as tumors when xenografted in immunocompromised mice. Treatment with Hh inhibitors at this stage can cause complete regression of tumors (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Reactivation of the Hh pathway with induction of EMT has been implicated in the carcinogenesis of several cancer types (15). Inhibition of the Hh pathway can reverse EMT and is associated with enhanced tumor sensitivity to cytotoxic agents (16). Recently, upregulation of the Hh pathway has been demonstrated in the NSCLC cell line A549, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype with increased cell motility and invasion (17).

The aim of the present work was to study the role of the Hh signaling pathway as mechanism of resistance to EGFR-TKIs in different models of NSCLC.

 

Activation of Hh signaling pathway in NSCLC cell lines with resistance to EGFR-TKIs

We established an in vitro model of acquired resistance to the EGFR-TKI gefitinib using the EGFR exon 19 deletion mutant (delE746-A750) HCC827 human NSCLC cell line by continuous culturing these cells in the presence of increasing doses of gefitinib. HCC827 cells, which were initially sensitive to gefitinib treatment (in vitro IC50 ∼ 80 nmol/L), became resistant (HCC827-GR cells) after 12 months of continuous treatment with IC50 > 20 μmol/L. This cell line was also cross-resistant to erlotinib and to the irreversible EGFR kinase inhibitor BIBW2992 (afatinib; data not shown). Sequencing of the EGFR gene in gefitinib-resistant HCC827-GR cells showed the absence of EGFRT790M mutation (data not shown). After the establishment of HCC827-GR cells, we characterized their resistant phenotype by protein expression analysis. While the activation of EGFR resulted efficiently inhibited by gefitinib treatment both in HCC827 and in HCC827-GR cells, phosphorylation of AKT and MAPK proteins persisted in HCC827-GR cells despite the inhibition of the upstream EGFR (Fig. 1A).

Figure 1.

Activation of Hh signaling pathway in NSCLC cell lines resistant to EGFR-TKIs. A, Western blot analysis of EGFR and of downstream signaling pathways in parental EGFR-mutated human lung adenocarcinoma HCC827 cells and in their gefitinib-resistant derivative (HCC827-GR). β-Actin was included as a loading control. B, Western blot analysis of Hh pathway, MET, and selected epithelial- and mesenchymal-related proteins in a panel of EGFR-TKI–sensitive (HCC827, H322, and Calu-3) and -resistant (HCC827-GR, H1299, Calu-3 ER, H460) NSCLC cell lines. β -Actin was included as a loading control. C, FISH analysis of gain in MET andSMO gene copy number in HCC827 and HCC827-GR. D, top, GLI-driven luciferase expression in HCC827 and HCC827-GR cells before and after depletion of GLI1 in both cell lines; bottom, evidence of GLI1 mRNA downregulation by siRNA. β-Actin was included as a loading control. E, MTT cell proliferation assays in HCC827-GR and PC9 cancer cell transfected with an empty vector or SMO expression plasmid with the indicated concentrations of gefitinib for 3 days. Bottom, Western blotting for evaluation of SMO after transfection.

HCC827-GR cells exhibited a mesenchymal phenotype with increased ability to invade, to migrate, and to grow in an anchorage-independent manner (Fig. 2A–C). Therefore, we next examined whether HCC827-GR cell line exhibits molecular changes known to occur during the EMT. Indeed, we found expression of vimentin and SLUG proteins and loss of E-cadherin protein expression in gefitinib-resistant cells as compared with gefitinib-sensitive cells (Fig. 1B). Although activation of the AXL kinase and NF-κB (20–22) have been described as known mechanisms of EGFR-TKI resistance, the analysis of their activation status resulted not significantly different among our cell lines. However, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

Figure 2.

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines. A, invasion assay. B, migration assay, C, anchorage-independent colony formation in soft agar. D, cell proliferation measured with the MTT assay in parental human lung adenocarcinoma HCC827 cells and in HCC827-GR derivative. The results are the average ± SD of 3 independent experiments, each done in triplicate.

Recently, expression of Shh and activation of the Hh pathway have been correlated to the TGFβ-induced EMT in A549 lung cancer cells (17). To investigate the expression profile of Hh signaling components in this in vitro model of acquired resistance to anti-EGFR–TKIs, we performed Western blot analysis for Shh, GLI1, 2, 3, SMO, and PTCH in HCC827-GR cells. While Shh levels did not differ between HCC827 and HCC827-GR cells, a significantly increased expression of SMO and GLI1 was found in HCC827-GR cells as compared with parental cells (Fig. 1B). No differences in the levels of GLI2 and 3 were observed (data not shown). Of interest, also PTCH protein levels resulted increased in HCC827-GR cells. This is of relevance, as PTCH is a target gene of GLI1 transcriptional activity and increased PTCH levels indicate activation of Hh signaling. We further analyzed expression and activation of MET, as a known mechanism of acquired resistance to anti-EGFR drugs in NSCLC. Indeed, MET phosphorylation resulted strongly activated in HCC827-GR cells (Fig. 1B). Analysis of the MET ligand levels, HGF, by ELISA assay, did not evidence any significant difference in conditioned media of our cells (data not shown). As previous studies have demonstrated MET gene amplification in NSCLC cell lines with acquired resistance to gefitinib (23), we evaluated MET gene copy number by FISH analysis and D-PCR in HCC827 and in HCC827-GR cell lines. The mean MET gene copy number was similar between gefitinib-sensitive and gefitinib-resistant HCC827 cell line (Fig. 1C).

Of interest, while we were working to these experiments, data on SMO gene amplification in EGFR-mutated NSCLC patients with acquired resistance to anti-EGFR targeted drugs were reported on rebiopsies performed at progression, revealing SMO amplification in 2 of 16 patients (12.5%; ref. 24). For this reason, we evaluated by FISH SMO gene copy number in HCC827-GR cells, in which the mean SMO gene copy number was 4-fold higher than that of parental HCC827 cells, indicating SMO gene amplification (Fig. 1C).

We further analyzed the expression and the activation of these molecules on a larger panel of EGFR-WT NSCLC cell lines, including NSCLC cells sensitive to EGFRTKIs, such as H322 and Calu-3 cells, NSCLC cell lines with intrinsic resistance to EGFR-TKIs, such as H1299 and H460 cells and Calu-3 ER (erlotinib-resistant) cells, which represents an in vitromodel of acquired resistance to erlotinib obtained from Calu-3 cells (refs. 4, 18; Supplementary Table S1). As shown in Fig. 1B, similarly to HCC827-GR cells, the Hh signaling pathway resulted in activation of these NSCLC models of intrinsic or acquired resistance to EGFR-TKI.

To further investigate the presence of specific mutations in the Hh pathway components, we sequenced DNA from our panel of NSCLC cell lines by Ion Torrent NGS; results indicated the absence of specific mutations in Hh-related genes (data not shown).

Because GLI1 is a transcription factor, we tested the functional significance of increased expression of this gene in the EGFR-sensitive and -resistant cell lines, using a GLI1-responsive promoter within a luciferase reporter expression vector (Fig. 1D). Analysis of luciferase activity of HCC827-GR cells revealed a 6- to 7-fold increase in GLI-responsive promoter activity as compared with HCC827 cells (P < 0.001), suggesting that transcriptional activity of GLI1 is significantly higher in gefitinib-resistant HCC827-GR cells. Furthermore, depletion of GLI1 protein expression by transfection with a GLI1-specific siRNA expression vector led to approximately 65% decrease in GLI1-driven promoter activity in HCC827-GR (P < 0.01; Fig. 1D). To determine whether SMO expression may promote resistance to gefitinib, 2 cell lines harboring the mutated EGFR gene, HCC827 and PC9 cells, and the sensitive EGFR-WT cell line Calu-3, were transiently transfected with an SMO expression plasmid. When treated with gefitinib, transfected cells exhibited a partial loss of sensitivity to the EGFR inhibition (Fig. 1E).

Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines

As previously mentioned, HCC827-GR cells acquired expression of vimentin and SLUG and loss of E-cadherin when compared with gefitinib-sensitive HCC827 cancer cells along with an increased ability to invade, migrate, and form colonies in semisolid medium (Fig. 2A–C). We next evaluated whether the Hh pathway activation was necessary for gefitinib acquired resistance by genetically or by pharmacologically inhibiting Hh components in the HCC827-GR cell line. Knockdown of GLI1 by a GLI1siRNA approach had a very little effect on HCC827-GR cells. However, when gefitinib treatment (1 μmol/L) was performed in HCC827-GR cells after GLI1 blockade, invasion, migration, and colony-forming capabilities were significantly inhibited (Fig. 2A–C). Next, we evaluated the effects of 2 small-molecule inhibitors of SMO, such as LDE225 and vismodegib. Treatment with LDE225 (1 μmol/L;Fig. 2A–D) or with vismodegib (1 μmol/L; data not shown) alone did not significantly affect the viability and the invasion and migration abilities of HCC827-GR cells. Combined treatment with gefitinib and LDE225 (1 μmol/L) or vismodegib (1 μmol/L) caused inhibition of these parameters in HCC827-GR cells (Fig. 2A–C).

Taken together, these data show that Hh activation is required for acquisition of gefitinib resistance in HCC827-GR cells.

As overexpression and activation of MET was found in HCC827-GR cells, we evaluated whether inhibition of MET phosphorylation by PHA-665752 could restore gefitinib sensitivity in this model. Although abrogation of MET signaling in combination with the inhibition of EGFR signaling marginally affected gefitinib sensitivity of HCC827-GR cells, surprisingly, inhibition of MET synergistically enhanced the effects of Hh inhibition in HCC827-GR cells (Fig. 2A–D) in terms of invasion, migration, colony-forming, and proliferation abilities, indicating a significant synergism between these 2 signaling pathways. The triple inhibition of EGFR, SMO, and MET did not result in any additional antiproliferative effects (data not shown).

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKI in EGFR-dependent NSCLC cell lines

To study the role of Hh pathway in the regulation of key signaling mediators downstream of the EGFR and to explore the interaction between Hh and MET pathways, we further characterized the effects of Hh inhibition alone and in combination with EGFR or MET inhibitor on the intracellular signaling by Western blotting. As illustrated in Fig. 3A, treatment of HCC827-GR cells with the SMO inhibitor LDE225, gefitinib or with the MET inhibitor PHA-665772, for 72 hours, did not affect total MAPK and AKT protein levels and activation. A marked decrease of the activated form of both proteins was observed only when LDE225 was combined with PHA-665772, at greater level than inhibition of EGFR and MET, suggesting that the Hh pathway cooperates with MET to the activation of both MAPK and AKT signaling pathways. In addition, vimentin expression, induced during the acquisition of gefitinib resistance, was significantly decreased after Hh inhibition, suggesting that the Hh pathway represents a key mediator of EMT in this model. The combination of MET and Hh inhibitors strongly induced cleavage of the 113-kDa PARP to the 89-kDa fragment, indicating an enhanced programmed cell death.

Figure 3.

Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKIs in HCC827-GR cells. A, Western blot analysis of Hh, MET, and EGFR activation and their downstream pathways activation following treatment with the indicated concentration LDE225 and PHA-556752 on HCC827-GR NSCLC cell line. β-Actin was included as a loading control. B, co-immunoprecipitation for the interaction between MET and SMO. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti- SMO (top) or anti-MET (bottom). The immunoprecipitates were subjected to Western blot analysis (WB) with indicated antibodies. Control immunoprecipitation was done using control mouse preimmune serum (PS). C, GLI-driven luciferase expression in HCC827-GR cells during treatment with gefitinib, LDE225, PHA-556752, or their combinations. D, co-immunoprecipitation for the interaction between SUFU and GLI1. Whole-cell extracts from HCC827 and HCC827-GR cells untreated or treated with LDE225 or/and PHA556752 were immunoprecipitated (IP) with anti-GLI1 (top) or anti-SUFU (bottom) antibodies. The immunoprecipitates were subjected to Western blot analysis with indicated antibodies. Control immunoprecipitation was done using control mouse PS.

Of interest, the inhibition of SMO by LDE225 also reduced the activated, phosphorylated form of MET (Fig. 3A), revealing an interaction between SMO and MET receptors. To address this issue, we hypothesized a direct interplay between both receptors. SMO immunoprecipitates from HCC827-GR cells showed greater MET binding than that from the parental HCC827 cells (Fig. 3B). As MET has been demonstrated to interact with HER3 to mediate resistance to EGFR inhibitors (25), we explored the expression of HER3 in SMO immunoprecipitates. Protein expression analysis did not show any association with HER3; similar results were obtained with EGFR protein expression analysis in the immunoprecipitates (data not shown).

The increased SMO/MET heterodimerization observed in HCC827-GR cells was partially reduced by the inhibition of SMO or MET with LDE225 or PHA-665752, respectively, and to a greater extent with the combined treatment (Fig. 3B). These results support the hypothesis that Hh and MET pathways interplay at level of their receptors.

To study whether the cooperation between these 2 pathways appears also at a downstream level, and considering that, as shown in Fig. 3A, MET inhibition partially reduces the levels of GLI1 and PTCH proteins, we analyzed luciferase expression of GLI1 reporter vector in HCC827-GR cells after treatment with LDE225, PHA-665752, or both. As shown in Fig. 3C, transcriptional activity of GLI1 resulted strongly decreased by the combined treatment. In particular, treatment with single-agent LDE225 did not abrogate the transcriptional activity of GLI1 suggesting a GLI1 noncanonical activation. In addition, single-agent PHA-665752 reduced GLI1-dependent signal, suggesting a role for MET in GLI1 regulation. To better investigate these findings, we hypothesized that MET can regulate GLI1 activity through its nuclear translocation. We, therefore, analyzed the binding ability of SUFU, a known cytoplasmic negative regulator of GLI1, following treatment of HCC827-GR cells with LDE225 and/or PHA-665752. Indeed, interaction between SUFU and GLI1 was markedly decreased in HCC827-GR cells as compared with HCC827 cells (Fig. 3D), which further confirmed the role of the activation of Hh pathway in this gefitinib-resistant NSCLC model. Furthermore, while combined treatment with LDE225 and PHA-665752 strongly increased the binding between GLI1 and SUFU, suggesting an inhibitory effect on GLI1 activity, also treatment with the MET inhibitor PHA-665752 alone favored the interaction of GLI1 with SUFU (Fig. 3D), indicating a role of MET on the activation of GLI1. This phenomenon could be a consequence of the decreased interplay between SMO and MET receptors or the effect of a direct regulation of GLI1 by MET.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts

We finally investigated the in vivo antitumor activity of Hh inhibition by LDE225, alone and in combination with gefitinib or with the MET inhibitor in nude mice bearing HCC827-GR cells. Treatment with gefitinib, as single agent, did not cause any change in tumor size as compared with control untreated mice, confirming that the in vitro model of gefitinib resistance is valid also in vivo. Treatment with LDE225 or with PHA-665752 as single agents caused a decrease in tumor size even stronger than that observed in vitro, suggesting a major role of these drugs on tumor microenvironment. However, combined treatments, such as LDE225 plus gefitinib or LDE225 plus PHA-665752, significantly suppressed HCC827-GR tumor growth with a major activity of LDE225 plus PHA-665752 combination. Indeed at 21 days from the starting of treatment, the mean tumor volumes in mice bearing HCC827-GR tumor xenografts and treated with LDE225 plus gefitinib or with LDE225 plus PHA-665752 were 24% and 2%, respectively, as compared with control untreated mice (Fig. 4A). Figure 4B shows changes in tumor size from baseline in the 6 groups of treatment. A total of eight mice for each treatment group were considered. Combined treatment of LDE225 plus gefitinib caused objective responses in 5 of 8 mice (62.5%). Of interest, the most active treatment combination was LDE225 plus PHA-665752 with complete responses in 8 of 8 mice (100%).

Figure 4.

Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts. A, athymic nude mice were injected subcutaneously into the dorsal flank with 107 HCC827-GR cancer cells. After 7 to 10 days (average tumor size, 75 mm3), mice were treated as indicated in Materials and Methods for 3 weeks. HCC827-GR xenografted mice received only vehicle (control group), gefitinib (100 mg/kg daily orally by gavage), LDE225 (20 mg/kg intraperitoneally three times a week), PHA-665752 (25 mg/kg intraperitoneally twice a week), or their combination. Data represent the average (±SD). The Student t test was used to compare tumor sizes among different treatment groups at day 21 following the start of treatment. B, waterfall plot representing the change in tumor size from baseline in the 6 groups of treatment. A total of 8 mice for each treatment group were evaluated. C, effects of combined LDE225 and PHA-665752 on expression of MET, PTCH, and vimentin. Tissues were stained with hematoxylin and eosin (H&E). Representative section from each condition.

We then studied the effects of gefitinib, LDE225, PHA-665752, and their combinations on the expression of PTCH, MET, and vimentin in tumor xenografts biopsies from mice of each group of treatment (Fig. 4C and Supplementary Table S2). We measured PTCH expression, as it represents a direct marker of Hh activation. While vimentin staining was particularly intense in control and gefitinib-treated tumors, treatment with LDE225 alone and in combination with PHA-665752 significantly reduced the intensity of the staining further confirming the role of Hh inhibition on the reversal of mesenchymal phenotype. Of interest, MET immunostaining resulted in a consistent nuclear positivity: this particular localization has been described as a marker of poor outcome and tendency to a mesenchymal phenotype (26). Although the combination of LDE225 and gefitinib resulted in a significant reduction of tumor growth with a concomitant reduction in staining intensity of vimentin, the combination of LDE225 and PHA-665752 was the most effective treatment, with 8 of 8 (100%) mice having a complete response in their tumors. In fact, histologic evaluations of these tumors found only fibrosis and no viable cancer cells. According to Western blot analysis of protein extracts harvested from the HCC827-GR xenograft tumors, the levels of phospho-EGFR, phospho-MET, and GLI1 resulted in a decrease after treatment with the respective inhibitor. Interestingly, the combined treatment with LDE225 and PHA-665752 resulted in a stronger inhibition of phospho-MAPK and phospho-AKT (Supplementary Fig. S1).

Role of the Hh pathway in mediating resistance to EGFR inhibitors in EGFR-WT NSCLC

As shown in Fig. 1B, although H1299, H460, and Calu-3 ER lacked SMO amplification (data not shown), these cells displayed Hh pathway activation. We further conducted luciferase expression analysis that showed a 8- to 9-fold increase in GLI1-dependent promoter activity in these lines as compared with EGFR inhibitor–sensitive H322 and Calu-3 cells, suggesting that transcriptional activity of GLI1 is higher in EGFR-TKI–resistant EGFR-WT NSCLC lines (Supplementary Fig. S2A). Similar to HCC827-GR cells, these cells showed also activation of MET. However, as reported in previous studies (4), MET inhibition alone or in combination with EGFR inhibition or with SMO inhibition resulted ineffective in inhibiting cancer cell proliferation and survival (data not shown).

We therefore tested the effects of Hh inhibition, by silencing GLI1 or by using LDE225, alone and/or in combination with erlotinib. Although knockdown of GLI1 or treatment with LDE225 (1 μmol/L) did not significantly affect NSCLC cell viability, combined treatment with erlotinib restored sensitivity to erlotinib (Supplementary Fig. S2B).

In addition, H1299, Calu-3 ER, and H460 cells exhibited significantly higher invasive and migratory abilities than H322 and Calu-3 cells and inhibition of Hh pathway significantly reduced these abilities. Collectively, these results suggest that Hh pathway activation mediates the acquisition of mesenchymal properties in EGFR-WT lung adenocarcinoma cells with erlotinib resistance (Supplementary Fig. S2B–S2D).

We next evaluated the effects of LDE225 alone and/or in combination with erlotinib on the activation of downstream pathways. Erlotinib treatment result was unable to decrease the phosphorylation levels of AKT and MAPK in H1299 and Calu-3 ER cells (Fig. 5A). However, when LDE225 was combined with erlotinib, a strong inhibition of AKT and MAPK activation was observed in these EGFR inhibitor–resistant cells (Fig. 5A). Furthermore, flow cytometric analysis revealed that combined treatment with both erlotinib and LDE225 significantly enhanced the apoptotic cell percentage to 65% and 70% (P < 0.001) in H1299 and Calu-3 ER cells, respectively (Fig. 5B), confirmed by the induction of PARP cleavage after the combined treatment (Fig. 5A). These findings suggest that Hh pathway drives proliferation and survival signals in NSCLC cells in which EGFR is blocked by erlotinib, and only the inhibition of both pathways can induce strong antiproliferative and proapoptotic effects. The in vitro synergism between EGFR and SMO was confirmed alsoin vivo. Combination of erlotinib and LDE225 significantly suppressed growth of Calu-3 ER xenografted tumors in nude mice (Supplementary Fig. S1F).

Figure 5.

Activation of Hh signaling pathway mediates resistance to EGFR-TKI in EGFR-WT NSCLC cell lines. A, Western blot analysis of EGFR and its downstream pathways activation, including PARP cleaved form, following treatment with the indicated concentration LDE225 and erlotinib on Calu-3, Calu-3 ER, and H1299 NSCLC cell line. β-Actin was included as a loading control. B, apoptosis was evaluated as described in Supplementary Materials and Methods with annexin V staining in Calu-3, Calu-3-GR, and H1299 cancer cells, which were treated with the indicated concentration LDE225 and erlotinib. Columns, mean of 3 identical wells of a single representative experiment; bars, top 95% confidence interval; ***, P < 0.001 for comparisons between cells treated with drug combination and cells treated with single agent.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy

To extend our preclinical observations, we further investigated the effects of Hh pathway inhibition on sensitivity of EGFR-WT NSCLC cells to standard chemotherapy used in this setting and mostly represented by cisplatin.

To investigate the role of the Hh pathway in mediating resistance also to chemotherapy, we evaluated the efficacy of cisplatin and Hh inhibition treatment alone or in combination on the colony-forming ability in semisolid medium of H1299 and H460 cell lines (Fig. 6). Treatment with cisplatin alone resulted in a dose-dependent inhibition of colony formation with an IC50 value of 13 and 11 μmol/L for H1299 and H460 cells, respectively. However, when combined with LDE225, the treatment resulted in a significant synergistic antiproliferative effect in both NSCLC cell lines (Fig. 6). Together, these results indicate that treatment of EGFR-WT NSCLC cells with Hh inhibitors could improve sensitivity of NSCLCs to standard chemotherapy.

Figure 6.

Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy. Anchorage-independent colony formation in soft agar in human lung adenocarcinoma H1299 and H460. The results are the average ± SD of 3 independent experiments, each done in triplicate. For defining the effect of the combined drug treatments, any potentiation was estimated by multiplying the percentage of cells remaining by each individual agent. The synergistic index was calculated as previously described (19). In the following equations, A and B are the effects of each individual agent and AB is the effect of the combination. Subadditivity was defined as %AB/(%A%B) < 0.9; additivity was defined as %AB/(%A%B) = 0.9–1.0; and supra-additivity was defined as %AB/(%A%B) > 1.0.

Discussion

Resistance to currently available anticancer drugs represents a major clinical challenge for the treatment of patients with advanced NSCLC. Our previous works (4, 18) reported that whereas EGFR-TKI–sensitive NSCLC cell lines express the well-established epithelial markers, cancer cell lines with intrinsic or acquired resistance to anti-EGFR drugs express mesenchymal characteristics, including the expression of vimentin and a fibroblastic scattered morphology. This transition plays a critical role in tumor invasion, metastatic dissemination, and the acquisition of resistance to therapies such as EGFR inhibitors. Among the various molecular pathways, the Hh signaling cascade has emerged as an important mediator of cancer development and progression (8). The Hh signaling pathway is active in human embryogenesis and tissue repair in cancer stem cell renewal and survival and is critical for lung development. Its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated by cigarette smoke extraction exposure in bronchial epithelial cells (12). In particular, the activation of this pathway correlated with the ability to growth in soft agar and in mice as xenograft and treatment with Hh inhibitors showed regression of tumors at this stage (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).

Recently, alterations of the SMO gene (mutation, amplification, mRNA overexpression) were found in 12.2% of tumors of The Cancer Genome Atlas (TCGA) lung adenocarcinomas by whole-exome sequencing (27). The incidence of SMO mutations was 2.6% and SMO gene amplifications were found in 5% of cases. SMO mutations and amplification strongly correlated with SHH gene dysregulation (P < 0.0001). In a small case report series, 3 patients with NSCLC with Hh pathway activation had been treated with the SMO inhibitor LDE225 with a significant reduction in tumor burden, suggesting that Hh pathway alterations occur in NSCLC and could be an actionable and valuable therapeutic target (27). Recently, upregulation of Shh, both at the mRNA and at the protein levels, was demonstrated in the A549 NSCLC cell line, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype (17, 28, 29) and mediated increased cell motility, invasion, and tumor cell aggressiveness (30, 31).

In the present study, SMO gene amplification has been identified for the first time as a novel mechanism of acquired resistance to EGFR-TKI in EGFR-mutant HCC827-GR NSCLC cells. These data are in agreement with the results of a cohort of patients with EGFR-mutant NSCLC that were treated with EGFR-TKIs (24). Giannikopoulus and colleagues have demonstrated the presence of SMO gene amplification in tumor biopsies taken at occurrence of resistance to EGFR-TKIs in 2 of 16 patients (24). In both cases, theMET gene was also amplified. In this respect, although the MET gene was not amplified in HCC827-GR cells, we found a significant functional and structural interaction between MET and Hh pathways in these cells. In fact, the combined inhibition of both SMO and MET exerted a significant antiproliferative and proapoptotic effect in this model, demonstrated by tumor regressions with complete response in 100% of HCC827-GR tumors xenografted in nude mice.

Several MET inhibitors have been evaluated in phase II/III clinical studies in patients with NSCLC, with controversial results. Most probably, blocking MET receptor alone is not enough to revert the resistant phenotype, as it is implicated in several intracellular interactions, and the best way to overcome resistance to anti-EGFR-TKIs is a combined approach, with Hh pathway inhibitors.

In the context of EMT, Zhang and colleagues demonstrated that AXL activation drives resistance in erlotinib-resistant subclones derived from HCC827, independently from MET activation in the same subclone, and that its inhibition is sufficient to restore erlotinib sensitivity by inhibiting downstream signal MAPK, AKT, and NF-κB (21). In addition, Bivona and colleagues described in 3 HCC827 erlotinib-resistant subclones increased RELA phosphorylation, a marker of NF-κB activation, in the absence of MET upregulation, and demonstrated that NF-κB inhibition enhanced erlotinib sensitivity, independently from AKT or MAPK inhibition (22). Differently, we detected Hh and MET hyperactivation in our resistance model HCC827-GR without a clear increase in AXL and NF-κB activation.

Although the level of activation of AXL and NF-κB did not result in contribution to resistance in our model, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.

In a preclinical model, the evolution of resistance can depend strictly from the selective activation of specific pathways, whereas different mechanisms can occur simultaneously in patients with NSCLC, due to tumor heterogeneity. Thus, all data regarding EFGFR-TKIs resistance have to be considered equally valid.

We further extended the evaluation of the Hh pathway to NSCLC cell lines harboring the wild-type EGFR gene and demonstrated that Hh is selectively activated in NSCLC cells with intrinsic or acquired resistance to EGFR inhibition and occurred in the context of EMT.

To further validate these data, we blocked SMO or downregulated GLI1 RNA expression in NSCLC cells that had undergone EMT, and this resulted in resensitization of NSCLC cells to erlotinib and loss of vimentin expression, indicating an mesenchymal-to-epithelial transition promoted by the combined inhibition of EGFR and Hh. Inhibition of the Hh pathway alone was not sufficient to reverse drug resistance but required concomitant EGFR inhibition to block AKT and MAPK activation and to restore apoptosis, indicating that the prosurvival PI3K/AKT pathway and the mitogenic RAS/RAF/MEK/MAPK pathways likely represent the level of interaction of EGFR and Hh signals.

In EGFR-WT NSCLC models, the role of MET amplification/activation is less clear, and in our experience, its inhibition did not increase the antitumor activity of SMO inhibitors.

In addition, Hh inhibition contributed to increase the response to cisplatin treatment which is the standard chemotherapeutic option used in EGFR-WT NSCLC patients and in EGFR-mutated patients after progression on first-line EGFR-TKI, thus representing a valid contribution to achieve a better disease control in those patients without oncogenic activation or after progression on molecularly targeted agents.

Collectively, the results of the present study provide experimental evidence that activation of the Hh pathway, through SMO amplification, is a potential novel mechanism of acquired resistance in EGFR-mutated NSCLC patients that occurs concomitantly with MET activation, and the combined inhibition of these 2 pathways exerts a significant antitumor activity. In light of these results, screening of SMO alteration is strongly recommended in EGFR-mutated NSCLC patients with acquired resistance to EGFR-TKIs at first progression.

 

Early-stage lung cancer patients considered to be high risk for surgery can achieve good clinical outcomes with surgical resection, according to a new study.

Pembrolizumab, a PD-1 inhibitor, demonstrated better overall survival and progression-free survival vs docetaxel in non–small-cell lung cancer patients.

A study looking at trends from 1985 to 2005 found that overall survival has increased in Medicare patients with small-cell lung cancer, and that treatment with chemotherapy is associated with improved survival.

Patients with ALK-rearranged non–small-cell lung cancer and brain metastases survive longer when treated with radiotherapy and tyrosine kinase inhibitors.

– See more at: http://www.cancernetwork.com/lung-cancer

 

Pembrolizumab Offers Survival Benefit in NSCLC

http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc

The maker of pembrolizumab, a programmed death 1 (PD-1) inhibitor (Keytruda; Merck), announced phase II/III trial results showing that the drug resulted in better overall survival (OS) and progression-free survival (PFS) compared with docetaxel in patients with non–small-cell lung cancer (NSCLC). The study included only patients who had failed prior systemic therapy and whose tumors expressed programmed death ligand 1 (PD-L1). – See more at: http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc#sthash.NUIqYKmi.dpuf

“The results from this trial provide part of a growing body of evidence supporting the potential of Keytruda in the treatment of NSCLC,” said Merck’s president, Roger M. Perlmutter, MD, PhD, in a press release.

The KEYNOTE-010 trial results have not yet been presented or published. The study compared two doses of pembrolizumab (2 mg/kg and 10 mg/kg) with docetaxel in 1,034 patients. All had progressed following treatment with platinum-containing systemic therapy, and all had tumors expressing PD-L1.

According to Merck’s release, pembrolizumab was associated with longer OS, in both the 2-mg/kg and 10-mg/kg dose groups, compared with docetaxel. This survival benefit was seen both in a subgroup of patients with PD-L1 expression tumor proportion scores of 50% or higher, as well as in all enrolled patients (all had a score of 1% or higher).

Both doses also resulted in longer PFS vs docetaxel in the 50% or higher group; this was not statistically significant in the full cohort of patients.

Pembrolizumab received an accelerated approval from the US Food and Drug Administration (FDA) in early October (at the 2-mg/kg dose level). The FDA noted in a press release that the most common side effects in a safety cohort of 550 patients included fatigue, dyspnea, and decreased appetite.

Earlier this year, results of a phase I study of pembrolizumab yielded promising survival outcomes. The median OS in that cohort of 495 patients was 12.0 months, and there was an overall response rate to the drug of 19.4%; this was higher in patients who had not received any previous treatment.

Pembrolizumab is not the first immunotherapy agent approved for the treatment of NSCLC. The FDA granted approval to nivolumab (Opdivo; Bristol-Myers Squibb) for the treatment of metastatic squamous NSCLC in March, and the indication was expanded to advanced non-squamous NSCLC in October.

– See more at: http://www.cancernetwork.com/lung-cancer/pembrolizumab-offers-survival-benefit-nsclc#sthash.NUIqYKmi.dpuf

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Drug-resistance Mechanism in Tumor Cells

Curator: Larry H. Bernstein, MD, FCAP

Targeting the RNA-binding protein that promotes resistance could lead to better cancer therapies.

Anne Trafton | MIT News Office

http://news.mit.edu/2015/finding-drug-resistance-mechanism-tumor-cells-1022

P53, which helps healthy cells prevent genetic mutations, is missing from about half of all tumors. Researchers have found that a backup system takes over when p53 is disabled and encourages cancer cells to continue dividing. In the background of this illustration are crystal structures of p53 DNA-binding domains.

About half of all tumors are missing a gene called p53, which helps healthy cells prevent genetic mutations. Many of these tumors develop resistance to chemotherapy drugs that kill cells by damaging their DNA.

MIT cancer biologists have now discovered how this happens: A backup system that takes over when p53 is disabled encourages cancer cells to continue dividing even when they have suffered extensive DNA damage. The researchers also discovered that an RNA-binding protein called hnRNPA0 is a key player in this pathway.

“I would argue that this particular RNA-binding protein is really what makes tumor cells resistant to being killed by chemotherapy when p53 is not around,” says Michael Yaffe, the David H. Koch Professor in Science, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the study, which appears in the Oct. 22 issue of Cancer Cell.

The findings suggest that shutting off this backup system could make p53-deficient tumors much more susceptible to chemotherapy. It may also be possible to predict which patients are most likely to benefit from chemotherapy and which will not, by measuring how active this system is in patients’ tumors.

Rewired for resistance

In healthy cells, p53 oversees the cell division process, halting division if necessary to repair damaged DNA. If the damage is too great, p53 induces the cell to undergo programmed cell death.

In many cancer cells, if p53 is lost, cells undergo a rewiring process in which a backup system, known as the MK2 pathway, takes over part of p53’s function. The MK2 pathway allows cells to repair DNA damage and continue dividing, but does not force cells to undergo cell suicide if the damage is too great. This allows cancer cells to continue growing unchecked after chemotherapy treatment.

“It only rescues the bad parts of p53’s function, but it doesn’t rescue the part of p53’s function that you would want, which is killing the tumor cells,” says Yaffe, who first discovered this backup system in 2013.

In the new study, the researchers delved further into the pathway and found that the MK2 protein exerts control by activating the hnRNPA0 RNA-binding protein.

RNA-binding proteins are proteins that bind to RNA and help control many aspects of gene expression. For example, some RNA-binding proteins bind to messenger RNA (mRNA), which carries genetic information copied from DNA. This binding stabilizes the mRNA and helps it stick around longer so the protein it codes for will be produced in larger quantities.

“RNA-binding proteins, as a class, are becoming more appreciated as something that’s important for response to cancer therapy. But the mechanistic details of how those function at the molecular level are not known at all, apart from this one,” says Ian Cannell, a research scientist at the Koch Institute and the lead author of the Cancer Cell paper.

In this paper, Cannell found that hnRNPA0 takes charge at two different checkpoints in the cell division process. In healthy cells, these checkpoints allow the cell to pause to repair genetic abnormalities that may have been introduced during the copying of chromosomes.

One of these checkpoints, known as G2/M, is controlled by a protein called Gadd45, which is normally activated by p53. In lung cancer cells without p53, hnRNPA0 stabilizes mRNA coding for Gadd45. At another checkpoint called G1/S, p53 normally turns on a protein called p21. When p53 is missing, hnRNPA0 stabilizes mRNA for a protein called p27, a backup to p21. Together, Gadd45 and p27 help cancer cells to pause the cell cycle and repair DNA so they can continue dividing.

Personalized medicine

The researchers also found that measuring the levels of mRNA for Gadd45 and p27 could help predict patients’ response to chemotherapy. In a clinical trial of patients with stage 2 lung tumors, they found that patients who responded best had low levels of both of those mRNAs. Those with high levels did not benefit from chemotherapy.

“You could measure the RNAs that this pathway controls, in patient samples, and use that as a surrogate for the presence or absence of this pathway,” Yaffe says. “In this trial, it was very good at predicting which patients responded to chemotherapy and which patients didn’t.”

“The most exciting thing about this study is that it not only fills in gaps in our understanding of how p53-deficient lung cancer cells become resistant to chemotherapy, it also identifies actionable events to target and could help us to identify which patients will respond best to cisplatin, which is a very toxic and harsh drug,” says Daniel Durocher, a senior investigator at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital in Toronto, who was not part of the research team.

The MK2 pathway could also be a good target for new drugs that could make tumors more susceptible to DNA-damaging chemotherapy drugs. Yaffe’s lab is now testing potential drugs in mice, including nanoparticle-based sponges that would soak up all of the RNA binding protein so it could no longer promote cell survival.

Aurelian Udristioiu comment:

A mutation causes the p53 gene to lose any of its functions anfd this will inevitably lead to carcinogenesis by letting the cell grow indefinitely, without any regulation. The p53 gene has been mapped in chromosome 17. In the cell, p53 protein binds DNA, stimulating another gene to produce the protein p21 that interact with cycle cell in division, stimulating a protein of stop division (cdk2).
When p21 forms a complex with cdk2, the cell cannot pass through to the next stage of cell division, and remains arrested in G1.
The p53 protein product of a TP53 mutant gene cannot bind damaged DNA in an effective way, and as a consequence, the p21 protein is not made available to act as the stop signal for the cell cycle/cell division. Therefore, cells divide uncontrollably and form tumors.
Not surprisingly, there is an increased frequency in the amplification of the ubiquitin ligases protein (MDM2) involved in the mechanism for the down regulation of p53 activity through ubiquitin-dependent proteosomal degradation of p53.
P53 has been shown to promote hematopietic stem cells (HSCs) quiescence and self-renewal with recent studies showing that deficiency of p53 likely promotes acute myeloid leukemia (AML) by eliminating its ability to limit aberrant self-renewal in hematopoietic progenitors.
Was demonstrate that the human p53 promoter is trans-activated by high c-Myc expression and repressed by high max-expression. In examining the relative levels of c-Myc and p53 in human Burkitt’s lymphomas and otherB-lymphoid lines, were found that there is a correlation between the levels of c-Myc protein and p53 mRNA expression.
In particular, the cells that express very low levels of c-Myc protein also express low levels of p53mRNA, while the cells that express high levels of c-Myc tend to express high levels of p53 mRNA .
[] Roy B, Beamon J, Balint E, Reisman D. Transactivation of the Human p53 Tumor Suppressor Gene by c-Myc/Max Contributes to Elevated Mutant p53 Expression in Some Tumors. Molecular, 2010 ].
In experimental models, disrupting the MDM2–p53 interaction restored p53 function and sensitized tumors to chemotherapy or radiotherapy.
This strategy could be particularly beneficial in treating cancers that do not harbor TP53 mutations. For example in hematologic malignancies, such as multiple myeloma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and Hodgkin’s disease, the induction of p53 – using a small MDM2-inhibitor molecule, nutlin-3 – can induce the apoptosis of malignant cells.
Nutlins are a group of cis-imidazoline analogs, first identified by Vassilev et al [2004], which have a high binding potency and selectivity for MDM2. Crystallization data have shown that nutlin-3 mimics the three residues of the helical region of the trans-activation domain of p53 (Phe19, Trp23 and Leu26), which are conserved across species and critical for binding to MDM2.
Nutlin-3 displaces p53 by competing for MDM2 binding. It has also been found that nutlin-3 potently induces apoptosis in cell lines derived from hematologic malignancies, including AML, myeloma, ALL, and
B-cell CLL. [Secchiero P, Voltan R, Iasio GM, Melloni . The oncogene DEK promotes leukemic cell survival and is down regulated by both Nutlin-3 and chlorambucil in B-chronic lymphocytic leukemic cells. Clin Cancer Res 2010; 16: 1824–1833].
A large cohort study of primary CLL, done on over 100 patients, examined the samples from the patients for a response to MDM2 inhibition. The study found direct correlation between wild-type TP53 status and MDM2 inhibitor-induced (nutlin-3 and MI-219) cytotoxicity across various CLL subtypes.
However, a large number of patients with cancer did produce p53-reactive T cells [Van der Burg SH, Cock K, Menon AG, Franken KL. Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with respected primary colorectal cancer. Eur. J. Immunol 2001; 31: 146–155].
The results from these studies served as a good reason to attempt the vaccination of patients using p53-derived peptides, and a several clinical trials are currently in progress. The most advanced work used a long synthetic peptide mixture derived from p53 (p53-SLP; ISA Pharmaceuticals, Bilthoven, the Netherlands].
The vaccine is delivered in the adjuvant setting and induces T helper type cells. However, the response may not be potent enough to result in clinical benefit as a mono-therapy: This indicated that these p53-specific T-helper responses are not polarized. Therefore, approaches are being investigated to promote a stronger and more correctly polarized response using new clinicla trials.

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Curator: Ritu Saxena, Ph.D.

Melanoma

Melanoma represents approximately 4% of human skin cancers, yet accounts for approximately 80% of deaths from cutaneous neoplasms. It remains one of the most common types of cancer among young adults. Melanoma is recognized as the most common fatal skin cancer with its incidence rising to 15 fold in the past 40 years in the United States. Melanoma develops from the malignant transformation of melanocytes, the pigment-producing cells that reside in the basal epidermal layer in human skin. (Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36 ; Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6).  Classic clinical signs of melanoma include change in color, recent enlargement, nodularity, irregular borders, and bleeding. Cardinal signs of melanoma are sometimes referred to by the mnemonic ABCDEs (asymmetry, border irregularity, color, diameter, elevation) (Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824).

Clinical characteristics

Melanoma primarily affects fair-haired and fair-skinned individuals, and those who burn easily or have a history of severe sunburn are at higher risk than their darkly pigmented, age-matched controls. The exact mechanism and wavelengths of UV light that are the most critical remain controversial, but both UV-A (wavelength 320–400 nm) and UV-B (290–320 nm) have been implicated (Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112). Case-control studies have identified several risk factors in populations susceptible to developing melanoma. MacKie RM et al (1989) stated that the relative risk of cutaneous melanoma is estimated from the four strongest risk factors identified by conditional logistic regression. These factors are

  • total number of benign pigmented naevi above 2 mm diameter;
  • freckling tendency;
  • number of clinically atypical naevi (over 5 mm diameter and having an irregular edge, irregular pigmentation, or inflammation); and
  • a history of severe sunburn at any time in life.

Use of this risk-factor chart should enable preventive advice for and surveillance of those at greatest risk (MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90).

Cutaneous melanoma can be subdivided into several subtypes, primarily based on anatomic location and patterns of growth (Table 1).

Image

Table 1: Clinical Classification of Melanoma (Chudnovsky Y, et al, 2005)

The genetics of melanoma

As in many cancers, both genetic predisposition and exposure to environmental agents are risk factors for melanoma development. Many studies conducted over several decades on benign and malignant melanocytic lesions as well as melanoma cell lines have implicated numerous genes in melanoma development and progression.

Image

Table 2: Genes involved in Melanoma (Chudnovsky Y, et al, 2005)

Apart from the risk factors such as skin pigmentation, freckling, and so on, another significant risk factor is ‘strong family history of melanoma’. Older case-control studies of patients with familial atypical mole-melanoma (FAMM) syndrome suggested an elevated risk of ∼434-to 1000-fold over the general population (Greene MH, et al, Ann Intern Med, Apr 1985;102(4):458-65). A more recent meta-analysis of family history found that the presence of at least one first-degree relative with melanoma increases the risk by 2.24-fold (Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59). Genetic studies of melanoma-prone families have given important clues regarding melanoma susceptibility loci.

CDKN2A, the familial melanoma locus

CDKN2A is located at chromosome 9p21 and is composed of 4 exons (E) – 1α, 1β, 2, and 3. LOH or mutations at this locus cosegregated with melanoma susceptibility in familial melanoma kindred and 9p21 mutations have been observed in different cancer cell lines. The locus encodes two tumor suppressors via alternate reading frames, INK4 (p16INK4a) and ARF (p14ARF). INK4A and ARF encode alternative first exons, 1α and 1β respectively and different promoters. INK4A is translated from the splice product of E1α, E2, and E3, while ARF is translated from the splice product of E1β, E2, and E3. Second exons of the two proteins are shared and two translated proteins share no amino acid homology.

INK4A is the founding member of the INK4 (Inhibitor of cyclin-dependent kinase 4) family of proteins and inhibits the G1 cyclin-dependent kinases (CDKs) 4/6, which phosphorylate and inactivate the retinoblastoma protein (RB), thereby allowing for S-phase entry. Thus, loss of INK4K function promotes RB inactivation through hyperphosphorylation, resulting in unconstrained cell cycle progression.

ARF (Alternative Reading Frame) protein of the locus inhibits HDM2-mediated ubiquitination and subsequent degradation of p53. Thus, loss of ARF inactivates another tumor suppressor, p53. The loss of p53 impairs mechanisms that normally target genetically damaged cells for cell cycle arrest and/or apoptosis, which leads to proliferation of damaged cells. Loss of CDKN2A therefore contributes to tumorigenesis by disruption of both the pRB and p53 pathways.

figure 1

Figure 1:  Genetic encoding and mechanism of action of INK4A and ARF.

(Chudnovsky Y, et al, 2005)

RAF and RAS pathways

A genetic hallmark of melanoma is the presence of activating mutations in the oncogenes BRAF and NRAS, which are present in 70% and 15% of melanomas, respectively, and lead to constitutive activation of mitogen-activated protein kinase (MAPK) pathway signaling. However, molecules that inhibit MAPK pathway–associated kinases, like BRAF and MEK, have shown only limited efficacy in the treatment of metastatic melanoma. Thus, a deeper understanding of the cross talk between signaling networks and the complexity of melanoma progression should lead to more effective therapy.

NRAS mutations activate both effector pathways, Raf-MEK-ERK and PI3K-Akt in melanoma. The Raf-MEK-ERK pathway may also be activated via mutations in the BRAF gene. In a subset of melanomas, the ERK kinases have been shown to be constitutively active even in the absence of NRAS or BRAF mutations. The PI3K-Akt pathway may be activated through loss or mutation of the tumor suppressor gene PTEN, occurring in 30–50% of melanomas, or through gene amplification of the AKT3 isoform. Activation of ERK and/or Akt3 promotes the development of melanoma by various mechanisms, including stimulation of cell proliferation and enhanced resistance to apoptosis.

JCI0524808.f3

Figure 2: Schematic of the canonical Ras effector pathways Raf-MEK-ERK and PI3K-Akt in melanoma.

Curtin et al (2005) compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and NRAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. Significant differences were observed in number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Eighty-one percent of the melanomas on skin without sun-induced damaged had mutations in BRAF or NRAS. Melanomas with wild-type BRAF or NRAS frequently had increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), downstream components of the RAS-BRAF pathway. Thus, the genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or NRAS. (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47).

Genetic Heterogeneity of Melanoma

Melanoma exhibits molecular heterogeneity with markedly distinct biological and clinical behaviors. Lentigo maligna melanomas, for example, are indolent tumors that develop over decades on chronically sun-exposed area such as the face. Acral lentigenous melanoma, or the other hand, develops on sun-protected regions, tend to be more aggressive. Also, transcription profiling has provided distinct molecular subclasses of melanoma. It is also speculated that alterations at the DNA and RNA and the non-random nature of chromosomal aberrations may segregate melanoma tumors into subtypes with distinct clinical behaviors.

The melanoma gene atlas

Whole-genome screening technologies such as spectral karyotype analysis and array-CGH have identified many recurrent nonrandom chromosomal structural alterations, particularly in chromosomes 1, 6, 7, 9, 10, and 11 (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47); however, in most cases, no known or validated targets have been linked to these alterations.

In A systematic high-resolution genomic analysis of melanocytic genomes, array-CGH profiles of 120 melanocytic lesions, including 32 melanoma cell lines, 10 benign melanocytic nevi, and 78 melanomas (primary and metastatic) by Chin et al (2006) revealed a level of genomic complexity not previously appreciated. In total, 435 distinct copy number aberrations (CNAs) were defined among the metastatic lesions, including 163 recurrent, high-amplitude events. These include all previously described large and focal events (e.g., 1q gain, 6p gain/6q loss, 7 gain, 9p loss, and 10 loss). Genomic complexity observed in primary and benign nevi melanoma is significantly less than that observed in metastatic melanoma (Figure 3)  (Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182).

Genetic heterogeneity Melanoma

Figure 3: Genome comparisons of melanocyte lesions (Chin L, et al, 2006)

Thus, genomic profiling of various melanoma progression types could reveal important information regarding genetic events those likely drive as metastasis and possibly, reveal provide cues regarding therapy targeted against melanoma.

Reference:

  1. Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36
  2. Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6
  3. Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824
  4. Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112
  5. MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90)
  6. Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59)
  7. Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47
  8. Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182

Related articles on Melanoma on this Open Access Online Scientific Journal, include the following: 

Thymosin alpha1 and melanoma Author/Editor- Tilda Barliya, Ph.D.

A New Therapy for Melanoma Reporter- Larry H Bernstein, M.D.

Melanoma: Molecule in Immune System Could Help Treat Dangerous Skin Cancer Reporter: Prabodh Kandala, Ph.D.

Why Braf inhibitors fail to treat melanoma. Reporter: Prabodh Kandala, Ph.D.

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Author and Curator: Ritu Saxena, Ph.D.

Introduction: Mitochondrial fission & fusion

Mitochondria, double membranous and semi-autonomous organelles, are known to convert energy into forms that are usable to the cell. Apart from being sites of cellular respiration, multiple roles of mitochondria have been emphasized in processes such as cell division, growth and cell death. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. Mitochondria have been discussed in several posts published in the Pharmaceutical Intelligence blog.

Mitochondria do not exist as lone organelles, but are part of a dynamic network that continuously undergoes fusion and fission in response to various metabolic and environmental stimuli. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid. Mitochondrial fusion and fission within a cell is speculated to be involved in several functions including mtDNA DNA protection, alteration of cellular energetics, and regulation of cell division.

Proteins involved in mitochondrial fission & fusion

Multiple mitochondrial membrane GTPases that regulate mitochondrial networking have recently been identified. They are classified as fission and fusion proteins:

Fusion proteins: Members of dynamin family of protein, mitofusin 1 (Mfn-1) and mitofusin 2 (Mfn-2), are involved in fusion between mitochondria by tethering adjacent mitochondria. These proteins have two transmembrane segments that anchor them in the mitochondrial outer membrane. Mutations in Mitofusin proteins gives rise to fragmented mitochondria, but this can be reversed by mutations in mammalian Drp1. Mitochondrial inner membranes are fused by dynamin family members called Opa1.

Fission proteins: Another member of the dynamin family of proteins, dynamin-related protein 1 (Drp-1) mediates fission of mitochondria. Drp-1 is activated by phosphorylation. Drp-1 proteins are largely cytosolic, but cycle on and off of mitochondria as needed for fission. Fission is a complex process and involves a series of well-defined stages and proteins. Cytosolic Drp-1 is activated by calcineurin or other cytosolic signaling proteins after which it translocates to the mitochondrial tubules where it assembles into foci through its interaction with another protein, hFis1. Once Drp-1 rings assemble on the constricted spots, outer membrane of mitochondria undergoes fission through GTP hydrolysis. Drp-1 is now left bound to one of the newly formed mitochondrial ends after which it slowly disassembles before returning to the cytoplasm.

Control of mitochondrial fission & fusion

  • Mitochondrial fission and fusion are controlled by several regulatory mechanisms. Few of which are mentioned as follows:
  • Drp-1 activation by Cdk1/Cyclin B mediated phosphorylation during mitosis – triggers fission
  • Drp-1 inactivation by cAMP-dependent protein kinase (PKA) in quiescent cells- prevents fission
  • Drp-1 activation after reversal of PKA phosphorylation by Calcineurin- triggers fission
  • Ubiquination of fission and fusion proteins by E3 ubiquitin ligase- alters fission
  • Sumoylation of fission proteins – regulates fission

Imparied mitochondrial fission leads to loss of mtDNA

Mitochondrial fission plays an important role in mitochondrial and cellular homeostasis. It was reported by Parone et al (2008) that preventing mitochondrial fission by down-regulating expression of Drp-1 lead to loss of mtDNA and mitochondrial dysfunction. An increase in cellular reactive oxygen species (ROS) was observed. Other cellular implications included depletion of cellular ATP, inhibition of cell proliferation and autophagy. The observations were made in HeLa cells.

MicroRNA regulation of mitochondrial fission

Although several factors have been attributed to the regulation of mitochondrial fission, the mechanism still remains poorly understood. Recently, regulation of mitochondrial fission via miRNAs has become a topic of interest. Following miRNAs have been found to be involved in mitochondrial fission:

  • miR-484:  Wang et al (2012) demonstrated that miR-484 was able to regulate mitochondrial fission by suppressing the translation of a fission protein Fis1, leading to inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. The authors showed that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. Underlying mechanism involved transactivation of miR-484 by a transcription factor, Foxo3a and miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation.
  • miR-499: miR-499 was reported by Wang et al (2011) to be able to directly target both the α- and β-isoforms of the calcineurin catalytic subunit. Suppression of calcineurin-mediated dephosphorylation of  Drp-1 lead to inhibition of the fission machinery ultimately resulting in the inhibition of cardiomyocyte apoptosis. miR-499 levels, by altering mitochondrial fusion were able affect the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. Modulation of miR-499 expression could provide a therapeutic approach for myocardial infarction treatment.
  • miR-30: It was reported by Li et al (2010) that miR-30 family members were able to inhibit mitochondrial fission and also the resulting apoptosis. While exploring the underlying molecular mechanism, the authors identified that miR-30 family members can suppress p53 expression. When cell received apoptotic stimulation, p53 was found to transcriptionally activate the fission protein, Drp-1. Drp-1 was able to induce mitochondrial fission. miR-30 family members were observed to inhibit mitochondrial fission through attenuation of p53 expression and its downstream target Drp-1.

Mitochondrial fission & fusion as a therapeutic target

Since alteration of mitochondrial fission and fusion have been reported to affect various cellular processes including apoptosis, proliferation, ATP consumption, the proteins involved in the process of fission and fusion might be harnessed as therapeutic target.

Mentioned below is a description of research where dynamics of the mitochondrial organelle has been utilized as a therapeutic target:

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

A recent article published by Rehman et al (2012) in the FASEB journal drew much attention after interesting observations were made in the mitochondria of lung adenocarcinoma cells. The mitochondrial network of these cells exhibited both impaired fusion and enhanced fission. It was also found that the fragmented phenotype in multiple lung adenocarcinoma cell lines was associated with both a down-regulation of the fusion protein, Mfn-2 and an upregulation of expression of fission protein, Drp-1. The imbalance of Drp-1/Mfn-2 expression in human lung cancer cell lines was reported to promote a state of mitochondrial fission. Similar increase in Drp-1 and decrease in Mfn-2 was observed in the tissue samples from patients compared to adjacent healthy lung. Authors used complementary approaches of Mfn-2 overexpression, Drp-1 inhibition, or Drp-1 knockdown and were able to observe reduction of cancer cell proliferation and an increase spontaneous apoptosis. Thus, the study identified mitochondrial fission and Drp-1 activation as a novel therapeutic target in lung cancer.

Image

Reference:

Research articles-

http://www.ncbi.nlm.nih.gov/pubmed/20556877

http://www.ncbi.nlm.nih.gov/pubmed?term=18806874

http://www.ncbi.nlm.nih.gov/pubmed/22510686

http://www.ncbi.nlm.nih.gov/pubmed/21186368

http://www.ncbi.nlm.nih.gov/pubmed?term=20062521

http://www.ncbi.nlm.nih.gov/pubmed?term=22321727

News brief:

http://www.uchospitals.edu/news/2012/20120221-mitochondria.html

http://news.uchicago.edu/article/2012/02/23/energy-network-within-cells-may-be-new-target-cancer-therapy

http://www.doctortipster.com/7881-mitochondria-could-represent-a-new-target-for-cancer-therapy-according-to-new-study.html

Related reading:

Reviewer: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Author and Curator: Larry H Bernstein, MD, FACP https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Reporter and Editor: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Author and Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/09/10/%CE%B2-integrin-emerges-as-an-important-player-in-mitochondrial-dysfunction-associated-gastric-cancer/

Author: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Author and Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

Reporter: Venkat S. Karra, PhD

https://pharmaceuticalintelligence.com/2012/08/14/detecting-potential-toxicity-in-mitochondria/

Reporter: Aviva Lev-Ari, PhD, RN https://pharmaceuticalintelligence.com/2012/08/01/mitochondrial-mechanisms-of-disease-in-diabetes-mellitus/

Author and Curator: Ritu Saxena, PhD; Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

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Author and Curator: Ritu Saxena, Ph.D.

 

Introduction

Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer

Bcl2

Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.

p53

p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.

HIF-1a

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.

PTEN

Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.

C-Src

C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.

Reference:

Muntané J and la Mata MD. Nitric oxide and cancer. World J Hepatol. 2010 Sep 27;2(9):337-44. http://www.ncbi.nlm.nih.gov/pubmed/21161018

Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9. http://www.ncbi.nlm.nih.gov/pubmed/22425962

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.http://www.ncbi.nlm.nih.gov/pubmed/20167555

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34. http://www.ncbi.nlm.nih.gov/pubmed/11518674

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Author and Curator: Ritu Saxena, Ph.D.

A recent post by Dr. Margaret Baker entitled “Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes” talks about how the ENCODE project is revealing new insights into the functions of non-coding region of the human genome previously labeled as “junk DNA”. MicroRNA or miRNA, which as stated by Dr. Baker, “are among the non-gene encoding sequences in the genome and have been shown to play a major post-transcriptional role in expression of multiple genes.”

The post has touched upon several aspects of miRNA including origin, function, and mechanism of action. This commentary is an extension of Dr. Baker’s post, expanding upon the mechanism of action of miRNAs along with their role in potential disease therapy.

microRNA: Revisiting the past

MicroRNA were not discovered long back, infact, it was in 1998 when the presence of the non-coding RNAs that could be involved in switching ‘on’ and ‘off’ of certain genes. In the last decade, 2006 Nobel Prize for medicine or physiology was awarded to scientists Andrew Fire and Craig Mello for their discovery of this new role of RNA molecules.

A breakthrough research was published in the September 2010 issue of Nature journal, stating that mammalian microRNAs predominantly act by decreasing the levels of target mRNA. Mammalian microRNAs predominantly act to decrease target mRNA levels. miRNAs were initially thought to repress protein output without changes in the corresponding mRNA levels. Guo et al challenged the previous notion of ‘translational repression’ and concluded on the basis of their experimental results that ‘mRNA-destabilization’ scenario for the major part is responsible for the repression in protein expression via miRNAs. Authors utilized the method of ‘ribosome profiling’ to measure the overall effects of miRNA on protein production and then compared these to simultaneously measured effects on mRNA levels. Ribosome profiling prepares maps that exact positions of ribosomes on transcripts after nucleases chew upon the exposed part of transcripts that are not covered by ribosomes. MiR-1 and miR-155 were introduced into the HeLa-cell line. Both of these miRNAs are not  normally expressed in HeLa cells. Another miRNA used was mir-223 which is expressed in significant amounts in neutrophils. The reason for choosing the set of these miRNAs was that they had already been shown to repress protein levels via proteomics research. It was deciphered that miRNA-mediated repression was similar regardless of target expression level and further stated that “for both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for lowered mRNA levels accounted for most for most (>/=84%) of the decreased protein production.” These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

Authors concluded that the discovery “will apply broadly to the vast majority of miRNA targeting interactions. If indeed general, this conclusion will be welcome news to biologists wanting to measure the ultimate impact of miRNAs on their direct regulatory targets.”

Since then and even before the paper was published, several other miRNAs and their roles have been discovered. Information on miRNAs has been consolidated in a database that can be accessed online at http://www.mirbase.org/

microRNA: From bench to bedside

Scientific community had speculated the role of non-coding RNAs in disease treatment right after their discovery. One such study demonstrating the utilization of microRNA for Cancer treatment was published in the September 2010 issue of the journal Nature Medicine. miR-380-5p represses p53 to control cellular survival and is associated with poor outcome inMYCN-amplified neuroblastoma

The p53 gene is known as a tumor suppressor gene and its inactivation has been associated in some cancers such as neuroblastoma. The study reported that microRNA-380 (miR-380) was able to repress the expression of p53 gene in cancer patients causing uninhibited cell survival and proliferation. The research group was able to decrease the tumor size in vivo in a mouse model of the neuroblastoma by delivering miR-380 antagonist. The researchers also observed that the inhibition of endogenous miR-380 in embryonic stem or neuroblastoma cells resulted in induction of p53, and extensive apoptotic cell death.

Thus, the success of miR antagonist for decreasing tumor size speaks of the effectiveness of miR as a potential therapeutic target for cancer treatment.

In conclusion, as stated by Dr. Baker in her post, “the miRNA data for tissues and specific cell types involved in disease pathology form a new approach to either detecting or possibly correcting gene (coding or non-coding) dysregulation. miRNA mimics and anti-miRNA agents are being developed as new therapeutic modalities.”

Reference:

Pharmaceutical Intelligence post, Author, Dr. Margaret Baker: Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

https://pharmaceuticalintelligence.com/2012/09/24/junk-dna-codes-for-valuable-mirnas/

 

Research articles: Mammalian microRNAs predominantly act to decrease target mRNA levels

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome inMYCN-amplified neuroblastoma

Expert reviews- miRNA and Cancer treatment

 

News briefs: http://ygoy.com/2010/10/02/new-treatment-for-junk-dna-induced-cancers-discovered/

http://www.evolutionnews.org/2010/10/micrornas–once_dismissed_as_j038861.html

 

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