Preethi Subramanian

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

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 Barretts 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 others expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.

Superoxide Dismutase 2 (Sod2) is Overexpressed at an Early Stage During Colorectal Carcinogenesis: A Putative Target for Celecoxib Chemoprevention

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.