Ruth Zarate

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes.In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor.Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Epigenetic regulation of microRNA expression in colorectal cancer

In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor‐supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down‐regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa‐miR‐9, hsa‐miR‐129 and hsa‐miR‐137) in 3 CRC cell lines. Expression of hsa‐miR‐9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa‐miR‐9‐1, hsa‐miR‐129‐2 and hsa‐miR‐137 CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa‐miR‐9‐1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.

microRNA-451 Regulates Macrophage Migration Inhibitory Factor Production and Proliferation of Gastrointestinal Cancer Cells

Purpose: microRNAs (miRNA) are small RNAs that function as post-transcriptional regulators of gene expression. Recent evidence has shown that some miRNAs can act as oncogenes or tumor suppressors. This study was conducted to evaluate the potential association of miRNA expression with clinical outcome in patients with gastric cancer. Experimental Design: Expression of 250 human mature miRNAs was measured by real-time PCR on paraffin-embedded tumor samples of 21 patients with gastric cancer stage III uniformly treated with surgical resection followed by chemoradiation. We identified the miRNAs correlated with disease-free and overall survival times, and the results were evaluated including 24 other patients. In vitro cell proliferation and radiosensitivity studies were done to support clinical data. Results: The results revealed that down-regulation of miR-451 was associated with worse prognosis. miR-451 was detected by in situ hybridization in epithelial cells and showed decreased expression in gastric and colorectal cancer versus nontumoral tissues. Overexpression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. Microarray and bioinformatic analysis identified the novel oncogene macrophage migration inhibitory factor (MIF) as a potential target of miR-451. In fact, overexpression of miR-451 down-regulated mRNA and protein levels of MIF and decreased expression of reporter genes with MIF target sequences. Moreover, we found a significant inverse correlation between miR-451 and MIF expression in tumoral gastric biopsies. Conclusions: These findings support the role of miR-451 as a regulator of cancer proliferation and open new perspectives for the development of effective therapies for chemoradioresistant cancers.

MicroRNA‐451 Is Involved in the Self‐renewal, Tumorigenicity, and Chemoresistance of Colorectal Cancer Stem Cells

Many antitumor therapies affect rapidly dividing cells. However, tumor proliferation may be driven by cancer stem cells (CSCs), which divide slowly and are relatively resistant to cytotoxic drugs. Thus, many tumors may progress because CSCs are not sensitive to the treatment. In this work, we searched for target genes whose expression is involved in proliferation and chemoresistance of CSCs. Both of these processes could be controlled simultaneously by cell regulators such as microRNAs (miRNAs). Therefore, colonospheres with properties of CSCs were obtained from different colon carcinoma cells, and miRNA profiling was performed. The results showed that miR‐451 was downregulated in colonspheres versus parental cells. Surprisingly, expression of miR‐451 caused a decrease in self‐renewal, tumorigenicity, and chemoresistance to irinotecan of colonspheres. We identified cyclooxygenase‐2 (COX‐2) as an indirect miR‐451 target gene involved in sphere growth. Our results indicate that miR‐451 downregulation allows the expression of the direct target gene macrophage migration inhibitory factor, involved in the expression of COX‐2. In turn, COX‐2 allows Wnt activation, which is essential for CSC growth. Furthermore, miR‐451 restoration decreases expression of the ATP‐binding cassette drug transporter ABCB1 and results in irinotecan sensitization. These findings correlate well with the lower expression of miR‐451 observed in patients who did not respond to irinotecan‐based first‐line therapy compared with patients who did. Our data suggest that miR‐451 is a novel candidate to circumvent recurrence and drug resistance in colorectal cancer and could be used as a marker to predict response to irinotecan in patients with colon carcinoma. STEM CELLS 2011;1661–1671

miR-192/miR-215 Influence 5-Fluorouracil Resistance through Cell Cycle-Mediated Mechanisms Complementary to Its Post-transcriptional Thymidilate Synthase Regulation

Thymidylate synthase (TYMS) is a target of the most widely used chemotherapeutic agents against gastrointestinal malignancies, the fluoropyrimidine-based therapy. TYMS expression levels have been identified as predictive biomarkers for 5-fluoruracil (FU) response in colorectal cancer, but their clinical utility remains controversial. The complexity of fluoropyrimidine response must require more mechanisms that currently have not been completely elucidated. In this context, microRNAs (miRNA) may play a role in modulating chemosensitivity. By carrying out an in silico analysis coupled to experimental validation, we detected that miR-192 and miR-215 target TYMS expression in colorectal cancer cell lines. However, downregulation of TYMS by these miRNAs does not sensitize colorectal cancer cell lines to FU treatment. The overexpression of miR-192/215 significantly reduces cell proliferation by targeting cell cycle progression. This effect was partially associated with p53 status, because reduction of cell proliferation and cell cycle arrest was associated with p21 and p27 induction. The decrease of S-phase cells by these miRNAs mitigates the effects of S phase–specific drugs and suggests that other mechanisms different from TYMS overexpression are essential to direct FU resistance. Finally, ectopic expression of miR-192/215 might have stronger impact to predict FU response than TYMS inhibition. Prospective studies to elucidate the role of these miRNAs as predictive biomarkers to FU are necessary. Mol Cancer Ther; 9(8); 2265–75. ©2010 AACR.

Fc gamma receptor polymorphisms as predictive markers of Cetuximab efficacy in epidermal growth factor receptor downstream-mutated metastatic colorectal cancer

BACKGROUND The immunoglobulin G1 (IgG(1)) monoclonal antibody (MoAb) Cetuximab is active in metastatic colorectal cancer (mCRC) as first or subsequent lines of therapy. Efficacy seems restricted to KRAS wild-type tumours. IgG(1) may also induce antibody dependent cell mediated citotoxicity (ADCC) by recruitment of immune effector cells. ADCC is influenced by Fc gamma receptor (FcγR) polymorphisms. We investigated the association of FcγR polymorphisms and disease control rate (DCR) in mCRC patients treated with chemotherapy plus Cetuximab. PATIENTS AND METHODS Tumour tissues from 106 patients were screened for KRAS codon 12 and 13 mutations using a sensitive multiplex assay (DxS, Manchester, United Kingdom). NRAS (codons: 12, 13 and 61), PI3K (exon 20) and BRAF (exon 15) were analysed by direct sequencing. Fcγ RIIa and Fcγ RIIIa polymorphisms were genotyped by TaqMan assays. RESULTS DCR was significantly higher in KRAS wild-type tumours (61% versus 39%, p = 0.049). In epidermal growth factor receptor (EGFR) downstream-mutated mCRC patients, those harbouring an FcγRIIa H/H genotype had a higher DCR than alternative genotypes (67% versus 33%, p = 0.017). By multivariate analysis, FcγRIIa-131H/H remained significantly correlated with DCR (p = 0.008). CONCLUSION FcγR polymorphisms may play a role in the clinical efficacy of Cetuximab in EGFR downstream mutated mCRC patients. Further research into Cetuximab immune-based mechanisms in KRAS-mutated patients seems warranted.

Oxaliplatin, irinotecan and capecitabine as first-line therapy in metastatic colorectal cancer (mCRC): a dose-finding study and pharmacogenomic analysis

Background:A dose-finding study was performed to evaluate the dose-limiting toxicity (DLT), maximum-tolerated dose (MTD) and the recommended dose (RD) of escalating the doses of capecitabine and fixed doses of irinotecan and oxaliplatin on a biweekly schedule for metastatic colorectal cancer patients (mCRC). A pharmacogenomic analysis was performed to investigate the association between SNPs and treatment outcome.Methods:Eighty-seven chemotherapy-naïve mCRC patients were recruited through a two-step study design; 27 were included in the dose-finding study and 60 in the pharmacogenomic analysis. Oxaliplatin (85 mg m-2) and CPT-11 (150 mg m-2), both on day 1, and capecitabine doses ranging from 850 to 1500 mg m-2 bid on days 1–7 were explored. Peripheral blood samples were used to genotype 13 SNPs in 10 genes related to drug metabolism or efficacy. Univariate and multivariate Cox analysis was performed to examine associations between SNPs, ORR and PFS.Results:The capecitabine RD was 1000 mg m−2 bid. Diarrhoea and neutropenia were the DLTs. After a median follow-up of 52.5 months, the median PFS and OS were 12 (95% CI; 10.6–13.4) and 27 months (95% CI; 17.2–36.8), respectively.The GSTP1-G genotype, the Köhne low-risk category and use of a consolidation approach strongly correlated with decreased risk of progression. Patients with all favourable variables showed a median PFS of 42 months vs 3.4 months in the group with all adverse factors. A superior clinical response was obtained in patients with one GSTP1-G allele as compared with GSTP1-AA carriers (P=0.004).Conclusion:First-line therapy with oxaliplatin, irinotecan and capecitabine is efficient and well-tolerated. The GSTP1 polymorphism A>G status was significantly associated with ORR and PFS in mCRC treated with this triplet therapy.

Identification of colorectal cancer metastasis markers by an angiogenesis-related cytokine-antibody array

AIM To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer (mCRC) with the aim of identifying prognostic markers. METHODS The expression of 44 angiogenesis-secreted factors was measured by a novel cytokine antibody array methodology. The study evaluated vascular endothelial growth factor (VEGF) and its soluble vascular endothelial growth factor receptor (sVEGFR)-1 protein levels by enzyme immunoassay (EIA) in a panel of 16 CRC cell lines. mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and vascular endothelial growth factor receptor (VEGFR)-2 expression was analyzed by flow cytometry. RESULTS Metastasis-derived CRC cell lines expressed a distinctive molecular profile as compared with those isolated from a primary tumor site. Metastatic CRC cell lines were characterized by higher expression of angiogenin-2 (Ang-2), macrophage chemoattractant proteins-3/4 (MCP-3/4), matrix metalloproteinase-1 (MMP-1), and the chemokines interferon γ inducible T cell α chemoattractant protein (I-TAC), monocyte chemoattractant protein I-309, and interleukins interleukin (IL)-2 and IL-1α, as compared to primary tumor cell lines. In contrast, primary CRC cell lines expressed higher levels of interferon γ (IFN-γ), insulin-like growth factor-1 (IGF-1), IL-6, leptin, epidermal growth factor (EGF), placental growth factor (PlGF), thrombopoietin, transforming growth factor β1 (TGF-β1) and VEGF-D, as compared with the metastatic cell lines. VEGF expression does not significantly differ according to the CRC cellular origin in normoxia. Severe hypoxia induced VEGF expression up-regulation but contrary to expectations, metastatic CRC cell lines did not respond as much as primary cell lines to the hypoxic stimulus. In CRC primary-derived cell lines, we observed a two-fold increase in VEGF expression between normoxia and hypoxia as compared to metastatic cell lines. CRC cell lines express a similar pattern of VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉) despite variability in VEGF expression, where the major transcript was VEGF₁₂₁. No relevant expression of VEGFR-2 was found in CRC cell lines, as compared to that of human umbilical vein endothelial cells and sVEGFR-1 expression did not depend on the CRC cellular origin. CONCLUSION A distinct angiogenesis-related expression pattern characterizes metastatic CRC cell lines. Factors other than VEGF appear as prognostic markers and intervention targets in the metastatic CRC setting.

Proteomic analysis in cancer research: potential application in clinical use

SummaryThe ultimate goal of cancer proteomics is to adapt proteomic technologies for routine use in clinical laboratories for the purpose of diagnostic and prognostic classification of disease states, as well as in evaluating drug toxicity and efficacy. The novel technologies allows researchers to facilitate the comprehensive analyses of genomes, transcriptomes, and proteomes in health and disease. The information that is expected from such technologies may soon exert a dramatic change in cancer research and impact dramatically on the care of cancer patients. Analysis of tumor-specific proteomic profiles may also allow better understanding of tumor development and the identification of novel targets for cancer therapy. The localization of gene products, which is often difficult to deduce from the sequence, can be determined experimentally. Mechanisms, such as regulation of protein function by proteolysis, recycling, and isolation in cell compartments, affect gene products, not genes. Finally, protein-protein interactions and the molecular composition of cellular structures can be determined only at the protein level. The biological variability among patient samples as well as the great dynamic range of biomarker concentrations are currently the main challenges facing efforts to deduce diagnostic patterns that are unique to specific disease states. While several strategies exist to address this problem, we have tried to offer a wide perspective about the current possibilities.

MiRNAs and LincRNAs: Could they be considered as biomarkers in colorectal cancer?

Recent advances in the field of RNA research have provided compelling evidence implicating microRNA (miRNA) and long non-coding RNA molecules in many diverse and substantial biological processes, including transcriptional and post-transcriptional regulation of gene expression, genomic imprinting, and modulation of protein activity. Thus, studies of non-coding RNA (ncRNA) may contribute to the discovery of possible biomarkers in human cancers. Considering that the response to chemotherapy can differ amongst individuals, researchers have begun to isolate and identify the genes responsible. Identification of targets of this ncRNA associated with cancer can suggest that networks of these linked to oncogenes or tumor suppressors play pivotal roles in cancer development. Moreover, these ncRNA are attractive drug targets since they may be differentially expressed in malignant versus normal cells and regulate expression of critical proteins in the cell. This review focuses on ncRNAs that are differently expressed in malignant tissue, and discusses some of challenges derived from their use as potential biomarkers of tumor properties.