Abstract Over the past number of years, we and others have been found that high levels of circulating insulin contribute to the development and progression of a number of cancers through epidemiology and pre-clinical studies. Although hyperinsulinemia frequently occurs in individuals with obesity, and early type 2 diabetes, there are also “metabolically unhealthy” individuals with hyperinsulinemia who are classified as having a normal weight by traditional definitions. Hyperinsulinemia is therefore a marker of metabolic disease irrespective of body mass, and as such has been associated with an increased risk of triple negative breast cancer (TNBC). Through our work to date in this R01, we have found that hyperinsulinemia drives tumor growth directly by activating the insulin receptor signaling pathway, leading to increased expression of proteins associated with breast cancer metastasis, such as myc, vimentin and twist. Our pre-clinical results led to another collaborative project where we investigated the role of hyperinsulinemia in the racial disparities in breast cancer prognosis in self-identified Black and White US women. By renewing this R01, we aim to gain a greater understanding of the importance of the insulin receptor (IR) in mediating the effects of insulin in tumor cells, and molecular mechanisms involved. The IR exists as two isoforms, IR-A and IR-B that differ by one exon. In the setting of systemic metabolic diseases and cancer, there appears to be an isoform switch where tissues predominantly express IR-A. The isoform switching is hypothesized to contribute to cancer development and progression. Previous studies have found that IR-A isoform expression differs in breast cancers from women of different races, potentially making tumors more susceptible to the growth promoting effects of hyperinsulinemia. Much of our knowledge regarding the metabolic effects of IR-B and IR-A are based on human correlative studies, and studies of cell lines overexpressing IR-A or IR-B in vitro. In this research proposal, we will use patient derived xenograft (PDX) models with a range of IR-A / IR-B ratios to determine if hyperinsulinemia contributes to isoform switching in vivo using our hyperinsulinemic mice, and also to understand the downstream signaling pathways and gene expression changes resulting from expression of the different IR isoforms. Through our newly generated mouse models of tissue specific IR exon 11 deletion, we will examine how tumor cell IR-A expression contributes to TNBC development and progression. We will also delve into the mechanisms through which insulin receptor signaling regulates myc regulated genes. Our proposal will provide new insight into mechanisms through which hyperinsulinemia and IR isoform switching contribute to TNBC development and progression, and the downstream signaling mechanisms involved in hyperinsulinemia driven breast cancer progression. Understanding these mechanisms will help us to uncover ways to trea...