The role of HOX genes in regulating stem cell population in normal and malignant colon tissue.

摘要

Colorectal cancer (CRCs) is a leading cause of cancer-related deaths in the United States. Stem cells (SCs) that reside at the base of normal colonic crypts are functional units of the colon and over-population of SCs leads to colon tumorigenesis. This study was designed to better investigate regulatory mechanisms by which SCs are maintained in the normal colon and to determine what triggers their dysregulation during colon cancer development. HOX genes that are master regulatory transcription factors controlling development and organogenesis are seen to be aberrantly expressed in several cancers, including CRCs. The primary objective is to investigate the role of HOX genes in colon cancer development and to determine if HOX genes have a potential role in regulating the SC population of normal and malignant colonic tissues. A unique gene expression signature for human colonic SCs was identified using two-color microarray as a first step towards understanding regulatory mechanisms of colonic SCs. Our microarray results indicated that developmental genes, in particular HOXA4, HOXA9 and HOXD10 were upregulated in the crypt bottom (SC-enriched region) relative to middle and top sub-sections of colonic crypts. Accordingly, I hypothesized that " HOXA4, HOXA9 and HOXD10 genes are differentially expressed in normal and malignant colonic stem cells and aberrant expression of HOXA4, HOXA9 and HOXD10 genes is necessary for the self-renewal ability of colon cancer stem cells".;The reason behind aberrant expression of HOX genes in CRCs is unknown. Real time PCR validated that HOXA4, HOXA9 and HOXD10 mRNAs are selectively expressed in the normal crypt bottom and are overexpressed in CRCs. Immunostaining showed that HOXA4, HOXA9 and HOXD10 proteins are co-expressed with the SC marker ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. HOXA4 and HOXA9 over-expression was also observed in colon CSCs that were isolated from SW480 and HT29 cancer cells. Thus, these findings showed that HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. The study also suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.;Because the mechanism by which HOX genes are contributing to the SC over population is unclear, functional studies were performed to investigate the role of HOX genes in regulating normal and malignant colonic SCs. Accordingly, siRNA-mediated knockdown of HOXA4 and HOXA9 in SW480 and HT29 cells was performed and the potential role of HOX genes in modulating proliferation and self-renewal ability of CSCs was evaluated. Our results revealed that proliferation of SW480 and HT29 cells transfected with either HOXA4 siRNA or HOXA9 siRNA was reduced compared to controls. It was also observed that HOXA4 and HOXA9 knockdown in SW480 and HT29 cells reduced gene expression of known SC markers i.e. ALDH1, CD166 and Lgr5. To further determine whether HOX has a potential role in modulating self-renewal ability of CSCs, sphere-forming assays were performed. The results of sphere-forming assays indicated that HOXA4 and HOXA9 siRNA transfected HT29 cells formed fewer colonospheres compared to controls. Not only was total number of colonospheres reduced for HT29 cells but also colonosphere size was significantly reduced. These results indicate that both numbers and proliferation ability of colon CSCs is reduced in HOXA4 and HOXA9 knockdown cells. Taken together, all findings from functional studies suggest that HOXA4 and HOXA9 play a regulatory role in normal and malignant colonic SCs and are critical for proliferation and self-renewal regulatory mechanisms.;The ultimate objective of this study is to find ways to design better treatments that are effective in targeting colon CSCs. Accordingly, a retinoid agent i.e. all trans retinoic acid (ATRA) was evaluated for its efficacy in eliminating colon CSCs. Treating colon cancer cells with ATRA down-regulates HOXA4 and HOXA9, decreases expression of the SC marker ALDH1A1, and reduces colonosphere formation. Thus, ATRA treatment in colon cancer may be very useful for modulating HOXA4 and HOXA9 gene expression and potentially targeting colon CSCs. Taken together, these results validate my hypothesis. The study suggests us that the strategies designed to target HOX genes may be a way to eradicate colon CSCs and lead to the development of more effective therapies for colon cancer.