PROJECT SUMMARY/ABSTRACT Advanced age is the single most significant risk factor for cancer. Although there are many plausible age-related mechanisms that mediate the increased risk for cancer in the elderly, functional evidence is lacking for the majority of these mechanisms. This proposal will focus on the role of the aged microenvironment, particularly senescence, in promoting the formation of colorectal cancer (CRC), which is a common age-related cancer in the U.S. It is well established that the majority of CRCs arise through the serial accumulation of mutations and epigenetic alterations in oncogenes and tumor suppressor genes in colon epithelial stem cells. However, our group and others have found cells with cancer-causing DNA alterations (e.g. oncogenic KRAS and TP53) in the histologically normal colon mucosa of people, which suggests that the DNA alterations alone are not sufficient to drive the complete CRC formation process. Our group has recently shown that a senescent normal colon tissue microenvironment can promote the neoplastic behavior of colon epithelial cells ex vivo. We found increased senescent fibroblasts in the colons of the elderly and of people at high risk for CRC. We found that certain senescence associated secretory phenotype (SASP) factors produced by senescent fibroblasts can induce cancer hallmark behaviors through the activation of oncogenic signaling pathways. Based on our preliminary data we hypothesize that senescent fibroblasts in older people and the accompanying specific Senescence Associated Secretory Phenotype (SASP) secreted proteins promote the tumorigenesis of colon epithelial cells that have acquired age-related oncogenic mutations, which we have defined as cancer-initiating cells. To provide direct functional evidence to test this hypothesis, we propose the following Specific Aims: AIM 1A. To determine whether senescent fibroblasts can promote the survival and/or transformation of colon cancer initiating cells in ex vivo and in vivo organoid model systems. AIM 1B. To determine the necessary and sufficient role of specific cancer-associated SASP factors produced by the senescent fibroblasts in cancer formation. AIM 2. To determine whether a senescent tissue microenvironment is necessary for the survival and clonal expansion of mutant APC and mutant KRAS cancer-initiating cells using a well characterized CRC mouse model, the inducible Villin-Cre;Apcflx/flx; KrasLSL-G12D (AK) mice, with two approaches: AIM 2A: a pharmacological approach using the senomorphic drug rapamycin and the senolytic drug treatment DNQ (dasatinib and quercetin) AIM 2B: a genetic approach using the p16-3MR transgenic model, which allows the inducible ablation of senescent cells.

PROJECT NARRATIVE Aging is the single most significant risk factor for cancer. Although there are many plausible age-related mechanisms that mediate the increased risk for cancer in the elderly, functional evidence is lacking for the majority of these mechanisms. This proposal will focus on the role of the aged microenvironment, particularly senescence, in promoting the formation of colorectal cancer (CRC), a common age-related cancer in the U.S. Our group has recently shown that a senescent normal colon tissue microenvironment, which includes fibroblasts, immune cells, stromal proteins, and secreted proteins, can promote the neoplastic behavior of colon cells. Based on our preliminary data, we will study the effects of senescent fibroblasts that accumulate in elderly people and their secreted proteins to promote the tumorigenesis of colon epithelial cells that have acquired age-related oncogenic mutations.