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.

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.

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.

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.

PON1-108 TT and PON1-192 RR genotypes are more frequently encountered in Greek PCOS than non-PCOS women, and are associated with hyperandrogenaemia.

OBJECTIVE: To investigate the frequencies of three paraoxonase (PON)1 polymorphisms in Greek polycystic ovary syndrome (PCOS) and non-PCOS women, and their genotypes association with hyperandrogenaemia and insulin resistance. DESIGN: Case-control genetic association study. SETTING: University Hospital Endocrine Unit. PATIENTS: A total of 142 PCOS cases (NIH criteria) and 112 controls. MAIN OUTCOME MEASURE: Genotyping of the c.-108C>T (PON1-108), the c.163T>A (PON1-55) and the c.575A>G (PON1-192) polymorphisms and measurement of baseline androgen and insulin resistance profile. RESULTS: The PON1-108 TT and PON1-192 RR genotypes were more frequently encountered in the PCOS than in the control group. The PON1-192 R allele frequency was greater in the PCOS than in the control group. Comparing the PCOS and the control groups, statistical significances favoured a recessive and a dominant genetic model, respectively, for the single PON1-108 T and PON1-192 R alleles. Free Androgen Index (FAI) levels were higher in patients with PON1-108 TT, whereas Testosterone, FAI and Dehydroepiandrosterone sulphate (DHEAS) levels were higher in patients with PON1-192 RR than patients with the wild or the heterozygous genotype. CONCLUSIONS: The decreased PON1 activity-associated PON1-108 TT and the PON1-192 RR genotypes are more frequently found in Greek PCOS women and are associated with hyperandrogenaemia. Hyperandrogenaemia must depend also on other genetic factors because the same genotypes were not associated with hyperandrogenaemia in the control group. Through identification of the involved polymorphisms, women with PCOS could potentially have a better therapeutic screening.

The rs10830963 variant of melatonin receptor MTNR1B is associated with increased risk for gestational diabetes mellitus in a Greek population.

OBJECTIVE: To investigate the association between Gestational Diabetes Mellitus (GDM) and the variants rs10830963 and rs1387153 in the MTNR1B locus in a sample of the Greek population. DESIGN: One hundred seventy-five unrelated pregnant Greek women (77 with GDM and 98 non-diabetic control subjects) were enrolled and the SNaPshot method was employed in order to investigate the association between GDM and the variants rs10830963 and rs1387153 in the MTNR1B locus. Pregnant women were screened for GDM at the 26th week with the 75 g glucose oral glucose tolerance test according to the American Diabetes Association criteria. RESULTS: The GG genotype and the G-allele of the rs10830963 (C/G) variant was found to be positively associated with a significantly increased risk for GDM (p = 0.047 and p = 0.012, respectively). No differences in fasting glucose and insulin levels were found between GDM patients with and without the studied variants. The MTNR1B locus (rs10830963 C/G) seems to predispose for GDM in Greek pregnant women. CONCLUSIONS: Our study confirms the association of GDM with the rs10830963 (C/G) variant in a sample of the Greek population. Population based whole genome screening studies and larger studies with detailed phenotypic data in patients with GDM are needed to address the clinical significance of this finding.