PROJECT SUMMARY/ABSTRACT Resistance to anti-cancer therapies largely explains the abysmal 5-year survival rate of patients with advanced colon cancer. Traditional chemotherapy regimens have been designed to efficiently stop proliferation and initiate apoptosis in cancer cells, but have failed to appreciate the pro-chemoresistance signals emanating from cells surrounding the tumor. We have identified a novel cellular component of the tumor microenvironment: the enteric glial cells (EGC). We and others have shown over the last 15 years that EGC are potent inducers of barrier function and healing in a healthy colon. Recently we have demonstrated that the EGC network substantially infiltrates human colon adenocarcinomas and promotes cancer stem cell tumor-forming abilities via a paracrine PGE2-EP4 pathway. Nevertheless, whether EGC impact colon cancer resistance to chemotherapy remains unknown. Our preliminary studies indicate that EGC protect cancer stem cells against apoptosis induced by chemotherapeutic drugs, allowing for enhanced tumor formation and growth despite the chemotherapy treatment. We also have evidence that this is (1) dependent on activation of the MRN-ATM pathway - a central player in DNA repair- in cancer cells and (2) exacerbated by EGC activation with chemotherapy. Using mass spectrometry analyses, we have identified FSTL3 as a novel EGC-derived factor and generated preliminary results implicating FSTL3 in EGC chemoprotective effects. Therefore, we propose to test the hypothesis that “in response to chemotherapeutic drugs, EGC release larger amounts of FSTL3 in the tumor microenvironment, which enhances cancer stem cell chemoresistance and allows for tumor formation and growth by promoting DNA repair driven by the MRN-ATM pathway”. Specific Aim 1 will determine whether EGC promote cancer stem cell resistance to chemotherapies via the release of FSTL3. Specific Aim 2 will test whether EGC protective effects are mediated by increased DNA repair as a result of activation of the MRN-ATM pathway. Specific Aim 3 will determine whether blocking FSTL3 production in EGC sensitizes colon tumors to chemotherapies in vivo. Studies will use translationally relevant primary cultures of human EGC and cancer cells, 3D co-culture platforms, orthotopic co-engraftment in immunodeficient mice, murine models of colon carcinogenesis, transgenic mice allowing for chemogenetic activation of EGC (GFAP-hM3Dq) and inducible gene targeting in EGC (GFAP- CreERT2), in addition to cutting-edge molecular profiling using single cell RNA seq and mass spectrometry studies to identify the pro-chemoresistance factor(s) (and in particular FSTL3) and pathway(s) involved. These studies will not only improve our understanding of the cellular and molecular mechanisms driving colon cancer chemoresistance but will also demonstrate the therapeutic potential of developing strategies combining targeted therapies against EGC-derived FSTL3 and traditional chemotherapy regimens.

PROJECT NARRATIVE/PUBLIC HEALTH RELEVANCE STATEMENT The abysmal survival rate of patients with advanced colon cancer is attributable to resistance to chemotherapies that have been designed to efficiently stop proliferation and initiate apoptosis in cancer cells, but fail to appreciate the pro-chemoresistance signals emanating from cells surrounding the tumor. Proposed studies will explore the impact of enteric glial cells, which form a dense network infiltrating colon adenocarcinomas, and yet have been understudied in colon carcinogenesis, and in particular colon cancer resistance to chemotherapies. Therefore, the outcomes of this project will not only improve our understanding of the cellular and molecular mechanisms driving colon cancer chemoresistance but may also demonstrate the therapeutic potential of developing strategies combining targeted therapies against enteric glial cell-derived chemoprotective ligands and traditional chemotherapy regimens.