MYC Regulates α6 Integrin Subunit Expression and Splicing Under Its Pro-Proliferative ITGA6A Form in Colorectal Cancer Cells

Abstract

The α6 integrin subunit (ITGA6) pre-mRNA undergoes alternative splicing to form two splicing variants, named ITGA6A and ITGA6B. In primary human colorectal cancer cells, the levels of both ITGA6 and β4 integrin subunit (ITGB4) subunits of the α6β4 integrin are increased. We previously found that the upregulation of ITGA6 is a direct consequence of the increase of the pro-proliferative ITGA6A variant. However, the mechanisms that control ITGA6 expression and splicing into the ITGA6A variant over ITGA6B in colorectal cancer cells remain poorly understood. Here, we show that the promoter activity of the ITGA6 gene is regulated by MYC. Pharmacological inhibition of MYC activity with the MYC inhibitor (MYCi) 10058-F4 or knockdown of MYC expression by short hairpin RNA (shRNA) both lead to a decrease in ITGA6 and ITGA6A levels in colorectal cancer cells, while overexpression of MYC enhances ITGA6 promoter activity. We also found that MYC inhibition decreases the epithelial splicing regulatory protein 2 (ESRP2) splicing factor at both the mRNA and protein levels. Chromatin immunoprecipitation revealed that the proximal promoter sequences of ITGA6 and ESRP2 were occupied by MYC and actively transcribed in colorectal cancer cells. Furthermore, expression studies in primary colorectal tumors and corresponding resection margins confirmed that the up-regulation of the ITGA6A subunit can be correlated with the increase in MYC and ESRP2. Taken together, our results demonstrate that the proto-oncogene MYC can regulate the promoter activation and splicing of the ITGA6 integrin gene through ESRP2 to favor the production of the pro-proliferative ITGA6A variant in colorectal cancer cells.

Integrin α6β4 in Colorectal Cancer: Expression, Regulation, Functional Alterations and Use as a Biomarker

“…It is interesting to note that the cells of the crypt proliferative compartment, which predominantly express α6A/ITGA6A [42,43], are also the cells that express MYC in the normal colon [9]. Experimental studies using a normal intestinal epithelial crypt cell line showed that MYC regulates ITGA6A expression [49]. These data suggest that the MYC-dependent regulation of ITGA6A is a phenomenon occurring in the normal intestine, which appears to be exploited throughout the neoplastic process.…”

“…Considering the fact that MYC expression is up-regulated in a large proportion of CRC [47,48] and that data indicating that MYC controls the expression of many genes in CRC cells including several key integrin subunits [40], which comprise ITGB4 [25], MYC represents a potential regulator of ITGA6 transcription. Pharmacological inhibition of MYC activity as well as molecular manipulations of intracellular MYC levels has shown a direct concordance between MYC levels and ITGA6 expression in intestinal cells while chromatin immunoprecipitation assays have confirmed the functionality of the MYC binding site on the ITGA6 promoter in the intestinal context [49].…”

“…Alternative splicing of ITGA6 has been relatively well studied and at least 10 splicing factors have been identified under various contexts but not in CRC cells. Among the various factors tested, the epithelial splicing regulatory protein 2 (ESPR2) was identified to be the main splicing factor responsible for ITGA6A expression in CRC cells [49]. It is noteworthy that ESPR1 was found to be responsible for ITGA6 splicing in breast cancer cells [50] but in contrast to ESPR2 that was found to be up-regulated by MYC, ESPR1 was not modulated in CRC cells by this factor [49].…”

“…Among the various factors tested, the epithelial splicing regulatory protein 2 (ESPR2) was identified to be the main splicing factor responsible for ITGA6A expression in CRC cells [49]. It is noteworthy that ESPR1 was found to be responsible for ITGA6 splicing in breast cancer cells [50] but in contrast to ESPR2 that was found to be up-regulated by MYC, ESPR1 was not modulated in CRC cells by this factor [49]. Further identification of MYC as a direct activator of ESPR2 active transcription indicates that MYC regulates both ITGA6 and ESPR2 expression resulting in the specific up regulation of ITGA6A in CRC cells [30].…”

“…Indeed, colorectal cancer cell lines express 5-10 times more ITGA6A and MYC that their normal counterparts [49] while the integrin α6Aβ4 was found to promote proliferation in colorectal cancer cells [44]. Specific knockdown expression of ITGA6A in various CRC cell lines was also accompanied by a reduction in the capacity of these cells to develop tumours [44].…”