PROJECT SUMMARY Colorectal cancer (CRC) metastasis is a major cause of mortality, yet there is a distinct lack of therapies targeting metastasis. Among the major modifiable external factors known to affect CRC risk are diet and obesity; however, unlike cancer initiation, how pro-obesity high fat diets (HFD) can impact cancer metastasis remains an essential question. Our understanding of the biology of metastatic cells is significantly impeded by a lack of in-vivo CRC models that recapitulate metastatic disease. This proposal leverages our orthotopic transplantation model of CRC, where genetically-engineered CRC organoids harboring common mutations found in human CRC are transplanted via colonoscopy into recipient host colons, forming tumors that later metastasize to the liver. Using this system, we have begun to study how CRC with different driver mutations such as APC, KRAS and p53, metastasize and adapt to the liver microenvironment in diet induced obesity. We have observed that diet induced obesity increases CRC metastasis to the liver. Our initial single cell RNA-Sequencing analyses of the primary colon and liver metastatic tumors have identified tumor intrinsic and extrinsic changes in response to an obesogenic HFD. Tumor intrinsic responses to HFD include increased YAP1 signaling and tumor-specific “revival stem cell” or revSC like populations. In our AIM1 of mentored K99 phase, using CRISPR mediated knock out models and untargeted metabolite profiling, we will identify the role of Hippo/YAP1 signaling in tumor metastasis and metabolic adaptations in pro-obesity HFD. In our AIM2 (K99 phase), leveraging reporter mouse models and single cell RNA-Sequencing, we will identify the contribution of revSCs in metastatic seeding, and the role of YAP1 regenerative signaling in mediating revSC like phenotypes in obesity. In our AIM3 of independent R00 phase, we will identify tumor extrinsic niche factors that contribute to increased metastasis in HFD. In our preliminary single cell profiling of the tumor niche in HFD, we observe evolution of specific Spp1 and Cxcl1 expressing cancer associated fibroblasts (CAFs) in the colon and liver metastatic niche. AIM3 will specifically characterize the origin, role and interactions of these CAF populations. The K99 training will mainly focus on cancer stem cell niche and modeling, single cell analyses, and tumor metabolism. This will be accomplished by training with the primary mentor, Dr Omer Yilmaz, with expertise in intestinal stem cells and their niche, colorectal cancer and gastrointestinal pathology, and mentoring team Dr Alex Shalek (single cell and computational analysis), Drs Alpaslan Tasdogan and Matthew Vander Heiden (tumor metabolism) and Dr Jacqueline Lees (cancer stem cells). This will be supplemented with conference, coursework and workshops in Metabolomics, Metastasis and Bioinformatics, and mentorship and teaching exercises to prepare for future academic career in the independent R00 phase. Successful completion of this study will identify mechanisms integral to initiate and maintain metastasis, revealing targetable vulnerabilities as well as provide insights to guide dietary interventions.

Project Narrative Colorectal cancer is one of the major causes of all cancer related deaths and a critical challenge in colorectal cancer outcome is lack of therapies designed to combat metastatic disease. Diet and obesity are among the major lifestyle choices that can affect colorectal cancer risk, however not much is known about how pro-obesity diets can affect cancer metastasis. This proposal investigates the molecular mechanisms underlying how pro- obesity high fat diets impact colorectal cancer metastasis, revealing therapeutic vulnerabilities and enabling evidence-based effective dietary recommendations.