Milena S. Nicoloso

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies ( n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

PURPOSE: The epithelial-to-mesenchymal transition (EMT) is a cell development-regulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFR therapy. EXPERIMENTAL DESIGN: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFR and ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3'-untranslated region/ERRFI-1/luciferse reporters. RESULTS: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFR inhibitors-induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3'-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. CONCLUSIONS: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFR therapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.