Abstract 1338: Citrobacter rodentium -induced autophagy protects cancer stem cells to facilitate tumor development and progression in the colons of Apc1638N/+ mice

Background: Colon cancer stem cells (CCSCs) play crucial roles in tumorigenesis, chemotherapy resistance, tumor recurrence and cancer metastasis. Autophagy, a lysosomal degradation and recycling process implicated in cancer progression and therapy resistance, plays an important role in host defense against enteric pathogen, Citrobacter rodentium (CR), and controls CR-induced infectious colitis. However, the role of CR infection in autophagy induction and CCSCs-driven neoplastic transformation has not been studied. Aim: To examine whether CR-induced autophagy protects CCSCs to promote colon cancer development and progression. Methods: Both male and female Apc1638N/+ mice from NCI repository were either infected with CR (109CFUs) and additionally given AOM (@10mg/Kg bw) or treated with a combination of AOM+DSS (7 days @ 3%). ApcMin/+ and BLT1-/-;ApcMin/+ mice were used as additional models mimicking FAP in humans. Apc++ mice were used as controls. Standard histologic, biochemical and molecular approaches were implemented. Results: CR infection induced significant autophagy in Apc++ mice with formation of autophagosomes and increases in LC3B and Beclin-1 staining at 12-27 days post-infection. While the frequency of tumor development in the colons of uninfected mice was lower, CR infection-induced tumor incidence in the colon was associated with increases in Ki-67, β-catenin, EZH2 and CCSC marker Dclk1/AcTub levels, respectively. In response to AOM treatment of CR-infected mice, we discovered colons to respond better compared to small intestine, with increases in Ki-67 and EZH2 staining although increases in Dclk1/AcTub levels were less pronounced compared to AOM-treated alone. When Apc1638N/+ mice were subjected to AOM/DSS treatment, colonic tumors exhibited more pronounced changes in Ki-67, EZH2 and Dclk1/AcTub staining than tumors of the small intestine. These tumors were also highly inflamed as was revealed by infiltration of F4/80+ macrophages and CD3+ lymphocytes. Interestingly, CD3 co-localized with β-catenin in these tumors, indicating contribution of Wnt signaling in the tumorigenic process. Both intestinal and colonic tumors also stained positive for migrating cancer stem cell markers CD110 and CDCP1, respectively. Colonic tumors additionally exhibited stromal positivity, suggesting local spread. Further investigation of tumors from AOM/DSS-treated Apc1638N/+ mice revealed significant autophagy wherein Dclk1/AcTub+ cells co-localized with Atg7 and p6