CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

Abstract

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.[Supplemental material is available for this article.]Notwithstanding the considerable advancements in our understanding of the molecular genetic basis of cancer, in the majority of cancer-associated genomic regions, the responsible protein-coding genes have not been identified yet. The discovery of short (19-22 nt), noncoding RNAs (ncRNAs)-called microRNAs (miRNAs) (Ambros 2001)-not only revealed a novel mechanism of gene regulation but also led to the identification of miRNAs directly involved in cancer development (Spizzo et al. 2009). It is therefore plausible that as-yet-unidentified members of the broader category of ncRNA mapping to cancer-associated genomic regions play ratelimiting roles in tumor initiation and/or progression (Rinn and Chang 2012). For instance, we previously reported that highly conserved genomic regions (ultraconserved regions, or UCRs) (Bejerano et al. 2004) are frequently transcribed as long (>200 bp) ncRNAs (lncRNAs) in both normal and tumor tissues (Calin et al. 2007). Furthermore, germline mutations, as well as single nucleotide polymorphisms (SNPs) in ultraconserved ncRNAs, were found to occur more frequently in patients with colon cancer and chronic leukemia than in the general population (Wojcik et al. 2010).The rs6983267 SNP, mapping to the 8q24.21 chromosomal region, has been consistently associated with an increased risk of colorectal cancer (CRC) (Haiman et al. 2007): The G allele was associated with greater predisposition to CRC than the T allele (odds ratios of 1.27 and 1.47 for heterozygotes and homozygotes, respectively; P = 1.27 3 10 À14 ) (Tomlinson et al. 2007). The increased cancer risk from this SNP variant was also observed in other cancer types, including prostate, ovarian, and inflammatory breast cancer (Ghoussaini et al. 2008;Bertucci et al. 2012). Despite the consistent association between rs6983267 and cancer risk, the underlying molecular and cellular mechanisms remain largely unknown. The genomic region spanning rs6983267 was found to contain DNA (Pom...

Long non-coding RNAs in colorectal cancer: implications for pathogenesis and clinical application

“…In the case of CRC, the administration of specific siRNAs targeting the oncogenic HOTAIR, CCAT2, and uc.73a transcripts was shown to decrease the invasion or proliferation of colon cancer cells. 20,58,62 Although the results presented in these studies are only preliminary in vitro data, these reports suggest that RNAi-based therapy strategies are amenable to further development, particularly for cellular therapies.…”

“…17 The expression level of the lncRNA colon cancerassociated transcript 2 (CCAT2) in microsatellitestable (MSS) CRC patients was higher than that in microsatellite instable-high (MSI-H) CRC patients, and cancer cells transduced with CCAT2-containing retrovirus exhibited numerical and structural chromosomal changes, illustrating that CCAT2 can contribute to the MSS phenotype by inducing chromosomal instability. 58 Additionally, CCAT2 itself has a role in CRC pathogenesis through its direct involvement in the regulatory network. CCAT2 participates in loop formation between the genomic locus rs6983267 and the MYC promoter and works with the enhancer element to activate the transcription of the MYC oncogene.…”

“…Moreover, CCAT2 enhances WNT activity by binding to TCF7L2, a pivotal transcription factor in the WNT signaling pathway, and facilitates MYC function, thereby enhancing cancer cell invasion and metastasis. 58 Intriguingly, CCAT2 levels can then be reduced by WNT signaling under TCF7L2 knockdown conditions, suggesting that a feedback loop may exist between CCAT2 and WNT signaling. 58 An attractive therapeutic strategy might involve attenuating CCAT2 activity and blocking the function of the downstream oncogenes in the WNT signaling pathway.…”

“…Although the underlying mechanism is generally unknown, it has been proposed that the expression of certain lncRNAs in CRC can be initiated or modulated by genetic (eg, SNPs in BA318C17.1, 66 sequence mutation of MALAT-1 67 ), epigenetic (eg, MIR211-dependent regulation of LOC285194, histone deacetylationinduced hypoxia-mediated downregulation of lncRNA-LET/NPTN-IT1 68 ), and transcriptional (eg, TCF7L2-dependent regulation of CCAT2, 58 TP53-dependent regulation of PVT1 69 ) regulatory factors and may allow for the exact execution of malignant transformation to some extent. 58,67,68 Intriguingly, in addition to being affected by MIR211, 64 LOC285194 was also found to be regulated by genetic deletion (copy number variation) and TP53, 64,65 suggesting that the expression of specific lncRNAs may be regulated by several regulatory mechanisms.…”

“…71 The G allele of rs6983267, in addition to being related to a significantly increased risk of CRC, 72 results in more CCAT2 transcripts than the T allele, which further affects the regulation of MYC by CCAT2; thus different rs6983267 alleles can affect CCAT2 expression and function. 58 Studies examining other cancer types have shown that SNPs in the key regulatory regions of an lncRNA can alter its structural motifs 73 and may consequently affect its expression and function in malignancies, 74 additionally contributing to cancer risk. 75,76 SNPs in the cancer-associated lncRNAs could potentially have pivotal roles in colorectal tumorigenesis and progression.…”