Project Summary There is an urgent need to develop new therapeutic options for carcinogenesis. The goal of this proposal is to help fulfill this need by shedding new light on the molecular pathways driving tumorigenesis. Recently published data reveals pro-oncogenic activity of heat shock factor 1 (HSF1), the master regulator of the heat shock response, and indicate that AMP-activated protein kinase (AMPK) phosphorylates S121 of HSF1, inactivating it and thereby disrupting malignancy. Whether HSF1 reciprocally impacts AMPK has yet to be explored. Preliminary data indicate that HSF1 physically interacts with and inhibits AMPK independently of HSF1 transcriptional activity at steady state. Genetic deletion of HSF1 suppresses cellular lipogenesis, cholesterol synthesis, protein cholesteroylation (important for the stability and activity of the target protein), and fat mass in mixed background mice through AMPK. Overexpression of HSF1 promotes a lipogenic phenotype and tumor growth. These results suggest that HSF1 transcriptionally independently inhibits AMPK to promote lipogenic phenotype of tumor growth. The proposed project addresses this hypothesis by pursuing two Specific Aims. Aim 1: To elucidate the detailed mechanism of HSF1-mediated AMPK suppression. First, a high-resolution library of HSF1 peptides will be used to study AMPK inhibition in in vitro and in vivo models. Second, a biophysical method will be used to determine the impact of HSF1 on AMPK conformation. Aim 2: To test the hypothesis that HSF1 supports lipid biosynthesis in tumor growth. First, how exactly HSF1 regulates AMPK-mediated lipogenesis, cholesterol synthesis, and protein cholesteroylation will be determined. Second, the mechanism will be studied in inbred mice with AMPK suppression. Third, the transcriptionally independent activity of HSF1 and its associated AMPK-regulated lipid and cholesterol metabolism will be studied in a human melanoma xenograft model. This proposal is significant because it will provide a deeper understanding of proteostasis in tumor progression, investigate a novel therapeutic target for cancer treatment, and identify a gene expression signature that informs clinical progression and outcomes. The project is conceptually and technically innovative as it will: i) examine the role of HSF1 as a novel inhibitor for AMPK, ii) investigate protein cholesteroylation by HSF1 and AMPK, and iii) develop endogenous HSF1 as a novel prognostic indicator for cancer patient survival. The proposal will provide a stepping stone to enable the candidate to establish an independent research career and ultimately become a leader in the field of carcinogenesis, contributing to breakthrough discoveries that lead to improved treatment strategies.