Noxa upregulation and 5-gene apoptotic biomarker panel in colorectal cancer.

BACKGROUND: NOXA and MCL1 are involved in the intrinsic pathway of apoptosis, where Noxa selectively binds to MCL1 and prevents it from inhibiting apoptosis. Both factors are considered as potential tumour biomarkers, while MCL1 has attracted interest as target in cancer. The purpose of this study was to investigate the expression of NOXA and MCL1 in 160 CRC tumour samples, to investigate their significance, also in combination with IAPs, DR5 expression and KRAS gene mutations in CRC. MATERIALS AND METHODS: Fresh frozen colorectal tissue was obtained from patients undergoing surgery for CRC. Real-time quantitative PCR was performed for the determination of mRNA expression levels. Protein expression was determined immunohistochemically. Differences in the mRNA expression profile were evaluated with the nonparametric Wilcoxon signed ranks test. Statistical analysis was performed with the use of Mann-Whitney U test and receiver-operating characteristic (ROC) curve. RESULTS: NOXA was found to be overexpressed in CRC tumours (P < .0001), even from early stage. Moreover, NOXA/MCL1 mRNA expression was significantly elevated in tumour samples compared to normal pairs (P < .0001). ROC curve analysis showed that both NOXA expression and its combination with Mcl1 expression have fair discriminatory value between CRC and normal colorectal tissue. Combinatorial ROC analysis revealed the most significant discriminatory value of NOXA, MCL1 with cIAP1 and cIAP2 (AUC = 0.834, P < .0001) as a 5-gene panel of markers. CONCLUSION: Noxa, Mcl1, DR5, cIAP1 and cIAP2 mRNA expressions are significantly deregulated in CRC and could provide a panel of markers with significant discriminatory value between CRC and normal colorectal tissue.

IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis.

BACKGROUND: High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs. METHODS: In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols. RESULTS: It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression. CONCLUSIONS: Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology.

Tumor heterogeneity revealed by KRAS, BRAF, and PIK3CA pyrosequencing: KRAS and PIK3CA intratumor mutation profile differences and their therapeutic implications.

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.

Design and Synthesis of Novel 2-Acetamido, 6-Carboxamide Substituted Benzothiazoles as Potential BRAFV600E Inhibitors - In vitro Evaluation of their Antiproliferative Activity.

The oncogenic BRAFV600E kinase leads to abnormal activation of the MAPK signaling pathway and thus, uncontrolled cellular proliferation and cancer development. Based on our previous virtual screening studies which issued 2-acetamido-1,3 benzothiazole-6-carboxamide scaffold as active pharmacophore displaying selectivity against the mutated BRAF, eleven new substituted benzothiazole derivatives were designed and synthesized by coupling of 2-acetamidobenzo[d]thiazole-6-carboxylic acid with the appropriate amines in an effort to provide even more efficient inhibitors and tackle drug resistance often developed during cancer treatment. All derived compounds bore the benzothiazole scaffold substituted at position-2 by an acetamido moiety and at position-6 by a carboxamide functionality, the NH moiety of which was further linked through an alkylene linker to a sulfonamido (or amino) aryl (or alkyl) functionality or a phenylene linker to a sulfonamido aromatic (or non-aromatic) terminal pharmacophore in the order -C6 H4 -NHSO2 -R or reversely -C6 H4 -SO2 N(H)-R. These analogs were subsequently biologically evaluated as potential BRAFV600E inhibitors and antiproliferative agents in several colorectal cancer and melanoma cell lines. In all assays applied, one analog, namely 2-acetamido-N-[3-(pyridin-2-ylamino)propyl]benzo[d]thiazole-6-carboxamide (22), provided promising results in view of its use in drug development.

BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study.

BACKGROUND: Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described. METHODS: Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. RESULTS: Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFß-1 pathway to provide cells with additional transforming properties. CONCLUSION: This study discriminates oncogene-specific cell migration and invasion pathways mediated by Rho GTPases in colon cancer cells and reveals potential new oncogene-specific characteristics for targeted therapeutics.

The expression of ELK transcription factors in adult DRG: Novel isoforms, antisense transcripts and upregulation by nerve damage.

ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged.

BRAF paradox breakers PLX8394, PLX7904 are more effective against BRAFV600Ε CRC cells compared with the BRAF inhibitor PLX4720 and shown by detailed pathway analysis.

PLX7904 and PLX8394 are novel BRAFV600E inhibitors-BRAFi that are designed to evade the paradoxical MAPK activation, a trait for the name "paradox breakers"-PB. Current FDA approved inhibitors (Vemurafenib, Dabrafenib, Encorafenib) although improved progression-free survival of mtBRAF melanoma patients suffer from this treatment related side effect. mtBRAF Colorectal Cancer (CRC) is resistant to the approved BRAF inhibitors, although combinatorial treatment co-targeting BRAF and EGFR/MEK is offering a promising prospect. In an effort to explore the potential of the novel BRAF inhibitors-PB to impede CRC cell proliferation, they were tested on RKO, HT29 and Colo-205 cells, bearing the BRAFV600E mutation. This study shows that the BRAF paradox breakers PLX7904 and PLX8394 cause a more prolonged MAPK pathway inhibition and achieve a stronger blockage of proliferation and reduced viability than PLX4720, the sister compound of Vemurafenib. In some treatment conditions, cells can undergo apoptosis. Genomic analysis on the more resistant RKO cells treated with PLX7904, PLX8394 and PLX4720 showed similar gene expression pattern, but the alterations imposed by the PB were more intense. Bioinformatic analysis resulted in a short list of genes representing potential master regulators of the cellular response to BRAF inhibitors' treatments. From our results, it is clear that the BRAF paradox breakers present a notable differential regulation of major pathways, like MAPK signalling, apoptosis, cell cycle, or developmental signalling pathways. Combinatorial treatments of BRAFi with Mcl-1 and Notch modulators show a better effect than mono-treatments. Additional pathways could be further exploited in novel efficient combinatorial treatment protocols with BRAFi.

Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic.

Epithelial-mesenchymal transition (EMT) is a key step during embryogenesis. Accumulating evidence suggests a critical role in cancer progression, through which tissue epithelial cancers invade and metastasise. Cell characteristics are highly affected during EMT, resulting in altered cell-cell and cell-matrix interactions, cell motility and invasiveness. Nevertheless, the demonstration of this process in human cancer has been proven difficult and controversial. Besides the fact that the acquisition of mesenchymal characteristics is not a prerequisite for cell migration/invasion, it is a transient event that concerns only few cells in a tumour mass. The induction of EMT depends on the tumour type and its genetic alterations as well as on its interaction with the extracellular matrix. In parallel, trials for EMT identification in clinical samples lack of a widely accepted methodology, nomenclature and reliable markers. This review summarizes the main EMT characteristics and proposes methodologies for better analysis in vitro. It also highlights recent studies identifying cells with EMT characteristics in human cancer and proposes certain markers to identify them in tumour samples. Finally, it cites the recent literature concerning the mechanisms of drug resistance related to EMT in the context of anti-tumour therapies and proposes related new targets for therapy.

Gene expression profile associated with oncogenic ras-induced senescence, cell death, and transforming properties in human cells.

We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics.

TRAIL receptor upregulation and the implication of KRAS/BRAF mutations in human colon cancer tumors.

TRAIL raises hopes as a promising anti-tumor agent due to its selectivity toward cancer cells. Higher expression of its pro-death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) attenuates higher sensitivity to TRAIL-induced apoptosis, and represents a marker for better cancer prognosis and treatment. Since receptor availability can be analogous to ligand efficacy, we performed RT-PCR analysis of DR4 and DR5 in 51 colon cancer biopsy specimens and respective normal mucosa, while 11 of these tumors were determined immunohistochemically for protein expression. Transcriptional analysis showed that DR4 and DR5 were significantly upregulated in 37 and 47% of the tumor samples respectively, while both DR4 and DR5 were coinstantaneously upregulated in 31% of the samples analyzed. Positive transcriptional regulation of DRs was recorded as early as Dukes' A stage. Furthermore, protein expression analysis yielded results comparable to DR4 and DR5 increased mRNA levels. Possible contributing events to DR upregulation involve presence of frequent oncogenic mutations in the MAPK pathway, and was investigated by direct sequencing in all 51 tumors. Samples (6/8) hosting either a KRAS(G12V) or BRAF(V600E) mutation, significantly amplified the upregulated expression of DR4 and DR5, showing strong inter-relation between overexpression and presence of oncogenic KRAS/ BRAF mutations. In the light of recent data concerning TRAIL receptor distribution, we contribute further by presenting DR5 as the most frequently upregulated DR in colon cancer. Furthermore, oncogenic mutations may directly or indirectly enhance DR expression, potentially sensitizing these tumors to TRAIL-based therapies.

TATA box-binding protein-associated factor 12 is important for RAS-induced transformation properties of colorectal cancer cells.

Activating mutations in the RAS proto-oncogene result in constant stimulation of its downstream pathways, further leading to tumorigenesis. Transcription factor IID (TFIID) can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. To investigate potential links between the regulation of TFIID function and the RAS-induced carcinogenesis, we monitored the expression of the TATA box-binding protein and its associated factors (TAF) in human colon carcinoma cells. We primarily identified TAF12 levels as being up-regulated in cell lines bearing natural RAS mutations or stably overexpressing a mutated RAS isoform via a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-dependent pathway. We further showed by electrophoretic mobility shift assays and chromatin immunoprecipitation that the ETS1 protein was interacting with an ETS-binding site on the TAF12 promoter and was regulating TAF12 expression. The binding was enhanced in extracts from oncogenic RAS-transformed cells, pointing to a role in the RAS-mediated regulation of TAF12 expression. Reduction of TAF12 levels by small interfering RNA treatment induced a destabilization of the TFIID complex, enhanced E-cadherin mRNA and protein levels, and reduced migration and adhesion properties of RAS-transformed cells with epithelial to mesenchymal transition. Overall, our study indicates the importance of TAF12 in the process of RAS-induced transformation properties of human colon cells and epithelial to mesenchymal transition, most notably those related to increased motility, by regulating specifically expression of genes such as E-cadherin.

Fra-1 regulates vimentin during Ha-RAS-induced epithelial mesenchymal transition in human colon carcinoma cells.

The process of epithelial mesenchymal transition, whereby cells acquire molecular alterations and fibroblastic features, is a fundamental process of embryogenesis and cancer invasion/metastasis. The mechanisms responsible for epithelial mesenchymal transition remain elusive. Human tumors frequently establish constitutively activated RAS signaling, which contributes to the malignant phenotype. In an effort to dissect distinct RAS isoform specific functions, we previously established human colon cell lines stably overexpressing activated Harvey-RAS (Ha-RAS) and Kirsten-RAS (Ki-RAS). Using these, we observed that only oncogenic Ha-RAS overexpression resulted in morphologic and molecular changes suggestive of epithelial to mesenchymal transition. We showed that vimentin, a key molecule of epithelial mesenchymal transition, was differentially regulated between Ha-RAS and Ki-RAS leading to a Ha-RAS specific induction of a migratory phenotype and eventually epithelial to mesenchymal transition. We demonstrated that the AP-1 sites in vimentin promoter could be involved in this regulation. A potential role of FRA-1 was suggested in the regulation of vimentin during the Ha-RAS-induced epithelial to mesenchymal transition, in association with colon cell migration. Our results therefore propose that in colon cells, the induction of epithelial mesenchymal transition by oncogenic Ha-RAS could occur through the overexpression of proteins like FRA-1 and vimentin.

Culture of primary epithelial adenoma cells from familial adenomatous polyposis patients.

BACKGROUND: Colorectal tumors arise from unregulated cell proliferation of the intestinal epithelium through a multistep process the first step usually being premalignant adenomas. Familial adenomatous polyposis patients carry a germ line mutation in the APC gene leading to the development of thousands of polyps, which, if left untreated, lead to cancer. The goal of this study was the establishment of conditions for the culture of epithelial cells composing an adenomatic structure. MATERIALS AND METHODS: All colorectal specimens were obtained from FAP patients within 1-2 hours of surgery. Cells were cultured by standard procedures. Characterization was carried out by immunostaining with pancytokeratin, vimentin and desmoplakin antibodies. RESULTS: A culture protocol that gave rise to epithelial cell growth with high efficiency and efficacy was established. Successful subculturing of the cell sheets took place only when dispase prepared in Ca2+ and Mg2+ free medium, was used to digest polyp tissue taken from FAP patients. By using immunostaining these cells were characterized as epithelial. CONCLUSION: The protocol we developed here provides a means of preparing cell cultures from human colorectal adenomas, which aid in the research of the transition from adenoma to carcinoma.

Quercetin enhances TRAIL-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts.

Cytokines such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in colon cancer cells through engagement of death receptors. Nevertheless, evading apoptosis induced by anticancer drugs characterizes many types of cancers. This results in the need for combination therapy. In this study, we have investigated whether the flavonoid quercetin could sensitize human colon adenocarcinoma cell lines to TRAIL-induced apoptosis. We report that quercetin enhanced TRAIL-induced apoptosis by causing the redistribution of DR4 and DR5 into lipid rafts. Nystatin, a cholesterol-sequestering agent, prevented quercetin-induced clustering of death receptors and sensitization to TRAIL-induced apoptosis in colon adenocarcinoma cells. In addition, our experiments show that quercetin, in combination with TRAIL, triggered the mitochondrial-dependent death pathway, as shown by Bid cleavage and the release of cytochrome c to the cytosol. Together, our findings propose that quercetin, through its ability to redistribute death receptors at the cell surface, facilitates death-inducing signaling complex formation and activation of caspases in response to death receptor stimulation. Based on these results, this study provides a challenging approach to enhance the efficiency of TRAIL-based therapies.

Death receptor 5 (DR5) and a 5-gene apoptotic biomarker panel with significant differential diagnostic potential in colorectal cancer.

High expression of Inhibitor of apoptosis proteins (IAPs) has been related to colorectal cancer (CRC) progression, resistance to treatment and poor prognosis. TRAIL (TNF-related apoptosis-inducing ligand) through its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) can selectively induce cancer cell apoptosis. The mRNA expression of DR4, DR5, c-IAP1, c-IAP2, XIAP and BIRC5/Survivin genes was examined in 100 paired (cancerous-normal) colorectal tissue specimens by real-time PCR, 50 of which were KRAS wild-type and 50 KRAS-mutant. DR5, XIAP and BIRC5/Survivin genes are significantly up-regulated (p < 0.0001, p = 0.012 and p = 0.0003, respectively), whereas c-IAP1 and c-IAP2 genes are significantly down-regulated at mRNA and protein levels in CRC (p < 0.0001 for both). ROC analyses showed that DR5, cIAP1 and cIAP2 expression has discriminatory value between CRC and normal tissue (AUC = 0.700, p < 0.0001 for DR5; AUC = 0.628, p = 0.011 for cIAP1; AUC = 0.673, p < 0.0001 for cIAP2). Combinatorial ROC analysis revealed the marginally fair discriminatory value of 5 genes as a panel (AUC = 0.685, p < 0.0001). Kaplan-Meier survival curves revealed significant association of cIAP2 down-regulation in CRC with lower overall survival probability of CRC patients (p = 0.0098). DR5, BIRC5/Survivin, XIAP, c-IAP1 and c-IAP2 mRNA expression are significantly deregulated in CRC and could provide a panel of markers with significant discriminatory value between CRC and normal colorectal tissue.

BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells.

Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.

EZH2 regulates cofilin activity and colon cancer cell migration by targeting ITGA2 gene.

Reorganization of cytoskeleton via actin remodeling is a basic step of cell locomotion. Although cell migration of normal and cancer cells can be stimulated by a variety of intra- and extra-cellular factors, all paths ultimate on the regulation of cofilin activity. Cofilin is a small actin-binding protein able to bind both forms of actin, globular and filament, and is regulated by phosphorylation at Serine 3. Following phosphorylation at serine 3 cofilin is inactive, therefore cannot bind actin molecules and cytoskeleton remodeling is impaired. The histone methyltransferase EZH2 is frequently over expressed in many tumour types including colorectal cancer (CRC). EZH2 over activity, which results in epigenetic gene-silencing, has been associated with many tumour properties including invasion, angiogenesis and metastasis but little is known about the underneath molecular mechanisms. Herein, we report that EZH2 is able to control cofilin activity and consequently cell locomotion of CRC cell lines through a non-conventional novel axis that involves integrin signaling. Indeed, we show how genetic and pharmacological inhibition (DZNep and GSK343) of EZH2 function produces hyper phosphorylation of cofilin and reduces cell migration. We previously demonstrated by chromatin immuno-precipitation that Integrin alpha 2 (ITGα2) expression is regulated by EZH2. In the present study we provide evidence that in EZH2-silenced cells the signaling activity of the de-repressed ITGα2 is able to increase cofilin phosphorylation, which in turn reduces cell migration. This study also proposes novel mechanisms that might provide new anti-metastatic strategies for CRC treatment based on the inhibition of the epigenetic factor EZH2 and/or its target gene.

BRAF vs RAS oncogenes: are mutations of the same pathway equal? Differential signalling and therapeutic implications.

As the increased knowledge of tumour heterogeneity and genetic alterations progresses, it exemplifies the need for further personalized medicine in modern cancer management. Here, the similarities but also the differential effects of RAS and BRAF oncogenic signalling are examined and further implications in personalized cancer diagnosis and therapy are discussed. Redundant mechanisms mediated by the two oncogenes as well as differential regulation of signalling pathways and gene expression by RAS as compared to BRAF are addressed. The implications of RAS vs BRAF differential functions, in relevant tumour types including colorectal cancer, melanoma, lung cancer are discussed. Current therapeutic findings and future viewpoints concerning the exploitation of RAS-BRAF-pathway alterations for the development of novel therapeutics and efficient rational combinations, as well as companion tests for relevant markers of response will be evaluated. The concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance posed a major therapy hindrance.

Epigenetic regulation of miR-21 in colorectal cancer: ITGB4 as a novel miR-21 target and a three-gene network (miR-21-ITGΒ4-PDCD4) as predictor of metastatic tumor potential.

Previous studies have uncovered several transcription factors that determine biological alterations in tumor cells to execute the invasion-metastasis cascade, including the epithelial-mesenchymal transition (EMT). We sought to investigate the role of miR-21 in colorectal cancer regulation. For this purpose, miR-21 expression was quantified in a panel of colorectal cancer cell lines and clinical specimens. High expression was found in cell lines with EMT properties and in the vast majority of human tumor specimens. We demonstrate in a cell-specific manner the occupancy of MIR-21 gene promoter by AP-1 and ETS1 transcription factors and, for the first time, the pattern of histone posttranslational modifications necessary for miR-21 overexpression. We also show that Integrin-ß4 (ITGß4), exclusively expressed in polarized epithelial cells, is a novel miR-21 target gene and plays a role in the regulation of EMT, since it is remarkably de-repressed after transient miR-21 silencing and downregulated after miR-21 overexpression. miR-21-dependent change of ITGß4 expression significantly affects cell migration properties of colon cancer cells. Finally, in a subgroup of tumor specimens, ROC curve analysis performed on quantitative PCR data sets for miR-21, ITGß4, and PDCD4 shows that the combination of high miR-21 with low ITGß4 and PDCD4 expression is able to predict presence of metastasis. In conclusion, miR-21 is a key player in oncogenic EMT, its overexpression is controlled by the cooperation of genetic and epigenetic alterations, and its levels, along with ITGß4 and PDCD4 expression, could be exploited as a prognostic tool for CRC metastasis.

The TRAIL of oncogenes to apoptosis.

Despite the significant advances in clinical research, surgical resection, radiotherapy and chemotherapy are still used as the primary method for cancer treatment. As compared to conventional therapies that often induce systemic toxicity and eventually contribute to tumor resistance, the TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that selectively triggers apoptosis in various cancer cells by interacting with its proapoptotic receptors DR4 and KILLER/DR5, while sparing the normal surrounding tissue. The intensive studies of TRAIL signaling pathways over the past decade have provided clues for understanding the molecular mechanisms of TRAIL-induced apoptosis in carcinogenesis and identified an array of therapeutic responses elicited by TRAIL and its receptor agonists. Analysis of its activity at the molecular level has shown that TRAIL improves survival either as monotherapies or combinatorial therapies with other mediators of apoptosis or anticancer chemotherapy. Combinatorial treatments amplify the activities of anticancer agents and widen the therapeutic window by overcoming tumor resistance to apoptosis and driving cancer cells to self-destruction. Although TRAIL sensitivity varies widely depending on the cell type, nontransformed cells are largely resistant to death mediated by TRAIL Death Receptors (DRs). Genetic alterations in cancer can contribute in tumor progression and often play an important role in evasion of apoptosis by tumor cells. Remarkably, RAS, MYC and HER2 oncogenes have been shown to sensitise tumor cells to TRAIL induced cell death. Here, we summarise the cross-talk of oncogenic and apoptotic pathways and how they can be exploited toward efficient combinatorial therapeutic protocols.