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. ' 2009 UICC

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-451was 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-451in 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 miR451down-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 miR451and MIF expression in tumoral gastric biopsies. Conclusions: These findings support the role of miR-451as a regulator of cancer proliferation and opennew perspectives for the development of effective therapies for chemoradioresistant cancers.

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 selfrenewal, 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.

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.