Mutual Regulation of Expression Between MUC4 and CDx2 in Colon Cancer Cells

Abstract

Background:Mucins are a family of large glycoproteins that have been implicated in colorectal carcinogenesis. Our group has explored the role of MUC4, a transmembrane mucin, which is generally upregulated in most cancers (pancreas, lung etc), during colorectal carcinogenesis and noted that MUC4 is paradoxically downregulated in colorectal cancers. The progressive downregulation occurs since early stages of carcinogenesis (premalignant mucosa). However, the mechanism remains unclear. CDX2 is a caudal-related homeobox gene that has been known to be dysregulated in colon carcinogenesis. Intriguingly, a recent report implicated CDX2 in MUC4 regulation in Barrett's esophagus. We, therefore, wanted to elucidate the role of CDX2 in the MUC4 loss in colon carcinogenesis Materials and Methods:HT29 cells were incubated in standard conditions and transiently transfected with CDX2 siRNA using Lipofectamine. Separately, HT29 cells were stably transfected withMUC4 shRNA and suitable control vector (CV). MUC4 shRNA transfected HT29 cells and CV transfected HT29 cells were treated with PI3K inhibitor LY294002. Protein lysates and RNA were obtained from the transfected cells and western blotting and qRT-PCR performed to assess the expression of MUC4, CDX2 and pAKT (S473). Results:In HT29 cells, a modest decrease in CDX2 expression by SiRNA (34% decreased CDX2 expression) concomitantly reduced MUC4 expression by 20%. Conversely, MUC4 shRNA transfection of HT29 cells (75% reduced MUC4 expression; p=0.05) resulted in 80% reduction in CDX2 mRNA expression and 60% reduction (p<0.02) in CDX2 protein levels. Since PI3K-AKT signaling cascade is known to modulate CDX2 levels, we assessed this pathway in MUC4 shRNA transfected HT29 cells, and demonstrated 50% increase (p<0.02) in Serine 473 phosphyrated AKT, which was mitigated by PI3K inhibitor, LY294002. However, despite the fact that MUC4 knockdown activated AKT, there was no effect of LY294002 treatment on the expression of CDX2 in MUC4 shRNA transfected HT29 cells, indicating that MUC4 modulates CDX2 expression via PI3K/pAKT independent manner. Conclusions: We report, herein, for the first time, that the dysregulation of two putative tumor suppressor genes, MUC4 and CDX2, is intimately related in colon cancer. Not only do CDX2 and MUC4 mirror each other's expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.