Mechanisms regulating colon cancer initiating cell tumor formation and self renewal

Colon cancer is the second leading cause of cancer deaths in the United States and a mechanistic understanding of pathways regulating CRC initiation, recurrence and metastasis is therefore an important goal for cancer control. WNT and NOTCH signaling are important in maintaining intestinal homeostasis, however, their specific role in self renewal and maintaining tumor initiating capacity of CCIC has not been defined.CCIC form tumors that maintain properties of the primary CRCs from which they were derived, express cancer stem cell marks and characteristic CRC molecular markers whereas standard colon cancer lines do not. Furthermore, CCIC have the property of self-renewal. Hence they represent an advanced cell culture system to study pathways regulating self renewal and tumorgenesis as compared to standard colon cancer cells lines. In this study CCIC were derived from primary colon cancer metastatsis, characterized and used to elucidate the role of NOTCH signaling and to test new compounds that inhibit self renewal.This study demonstrates that NOTCH signaling is 10-30 fold higher in CCIC compared to commonly used colon cancer cell lines. Using small molecule inhibition and shRNA knockdown, this study shows that NOTCH signaling prevents CCIC apoptosis through repression of p27 and HATH1. NOTCH also plays critical roles in the intrinsic maintenance of CCIC self-renewal, and the repression of secretory cell lineage differentiation genes such as MUC2. This study describes a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest inhibition of NOTCH signaling is likely to be an important mechanism to improve CRC chemoprevention and chemotherapy.This study also investigates the effect of several chemotherapy agents on CCIC. We find that small molecule HDAC inhibitors specifically inhibit growth and self renewal of CCIC. This effect is specific to cancer cells and does not affect normal cells. HDAC inhibitors induce p21 expression and activate the intrinsic apoptotic pathway. Gene expression profiling and Ingenuity pathway analysis reveals that HDAC inhibitors activate tumor suppressor genes in CCIC that are epigenetically silenced. In summary this is the first study that describes self renewal pathways important in CCIC and investigates therapeutic agents to target CCIC and improve chemotherapy.