Carrie D. House, Ph.D. - US grants

RESEARCH PROJECT-OBESITY IN CANCER ? Project Summary The overall objective of this study is to define the role of obesity in activating the NF-?B pathway in ovarian cancer tumor-initiating cells (TICs) to facilitate tumor growth and chemoresistance. Ovarian cancer is the most lethal gynecological malignancy in the United States and although most patients initially respond to platinum- based chemotherapy, over 70% of advanced stage tumors relapse leading to high morbidity and mortality. One risk factor for the development of ovarian cancer is obesity, a condition associated with disease progression and poor outcomes after initial treatment. Studies show that a high fat diet and obesity activate the NF-?B signaling pathway to trigger inflammation. NF-?B is a ubiquitous signaling pathway whose target genes encode proteins that regulate immune response, cell survival, proliferation, adhesion, and interaction with the microenvironment. NF-?B is aberrantly activated in ovarian cancer and promotes a TIC phenotype necessary for chemotherapy resistance. It is unknown what factor(s) trigger NF-?B activation in ovarian cancer cells. This proposal investigates the hypothesis that adipocytes (fat cells that comprise adipose tissue) secrete factors that activate NF-?B pathways in ovarian cancer cells to drive tumor growth and chemotherapy resistance. Aim 1 will test whether obesity enriches for ovarian cancer TICs through the secretion of adipokines. This will be investigated through co-culture experiments of ovarian cancer cells with patient-derived adipocytes to measure adipokine secretion, gene expression changes, enrichment of cancer cells with TIC features, and tumor growth in vivo. Aim 2 will determine how inflammatory stimuli lead to differential NF-?B activation in ovarian cancer cells. NF-?B signaling cascades will be examined after exposure to inflammatory cytokines and adipokines prominent in the ovarian tumor microenvironment. NF-?B reporter activity, DNA binding, transcription factor activation, and pharmacological inhibition will be used to investigate NF-?B proteins critical for inflammation-induced signaling and tumor formation. Aim 3 will discover why chemotherapy resistance of ovarian cancer cells is enhanced in the obese setting. Chemotherapy sensitivity and changes in pathways known to mediate drug resistance (NF- kB, AKT, STAT3) will be evaluated in ovarian cancer cells cultured with adipocytes. A novel therapeutic that targets obesity, inhibits NF-?B, and effectively eliminates TICs in vitro will be tested in vivo for its ability to prevent chemotherapy resistance and trigger tumor regression in an obese mouse model of ovarian cancer. Results of this proposal will advance the ovarian cancer field by providing mechanistic insight into the role of obesity in disease progression and chemotherapy resistance. Completion of the proposal aims will provide novel information about ovarian cancer biology and the role of adipocytes and NF-?B in promoting TICs. Knowledge gained from these studies will lead to more effective preventive and therapeutic strategies for cancers in which obesity is a risk factor.

? DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the role of the NF-kB pathway in ovarian cancer tumor-initiating cells (TICs) and to ascertain mechanisms required for successful repopulation of tumors following chemotherapy. Ovarian cancer is the most lethal gynecological malignancy in the United States and although most patients initially respond to platinum-based chemotherapy, over 70% of advanced stage tumors relapse leading to high morbidity and mortality. Thus, there is an urgent need to clarify processes that support therapy resistance and tumor regrowth, and the purpose of this research is to identify mechanisms that can be targeted to prevent tumor recurrence. NF-kB activity is associated with increased aggressiveness, enhanced metastatic potential and poor outcome in ovarian cancer. NF-kB is a ubiquitous signaling pathway whose target genes encode proteins that regulate immune response, cell survival, proliferation, adhesion, and interaction with the microenvironment. This proposal investigates the hypothesis that NF-kB pathways support subpopulations of ovarian cancer cells that can resist chemotherapy and drive tumor recurrence. The proposed project will address several questions relevant to the biology of ovarian cancer relapse. First, which functions of NF-kB support TICs that comprise a small percentage of the total cells in the tumor, and non-TICs that make up the bulk of the tumor? Are non- TICs necessary for efficient repopulation of the tumor? Does NF-kB activation integrate with MAPK/ERK to support non-TICs? Do transcriptional regulators that maintain stem cell phenotypes control NF-kB expression and activity? This proposal seeks to understand the coordination of multiple pathways in TICs and non-TICs necessary for ovarian cancer relapse. To begin answering these questions I first developed a culture system to enrich for TICs and compared NF-kB and MAPK/ERK activity in these cells with cells grown in non-TIC enriching conditions (adherent monolayer). My preliminary studies suggest classical NF-kB and MAPK/ERK activity support non-TICs whereas alternative NF-kB activity supports TICs. I will genetically interrupt the components leading to classical and alternative NF-kB activity in established cell lines and primary patient cell lines, and measure chemoresistance and tumor repopulation in vivo using xenograft mouse models. These studies will be extended in sorted TIC and non-TIC populations. Sorting will be accomplished using a novel reporter that responds to activity of stem cell transcription factors rather than traditional cell surface markers that ar context dependent. Investigation of pathways specifically activated in TICs or non-TICs will elucidate the potential cooperation of these different cell types in successful repopulation of tumors. Lastly, I will investigate novel mechanisms leading to alternative NF-kB activation in TICs to provide insights into new targets for prevention of secondary disease. During the K99 phase I will receive extensive training using mouse models to measure chemoresistance and tumor relapse and I will develop a reliable reporter for identifying and isolating ovarian cancer TICs. At the end of the K99 phase I will possess the tools necessary to be an independent investigator with expertise in xenograft mouse models and ovarian TIC biology. This new training will be applied to my overall study design during the R00 phase of this award, which aims to address significant gaps in our current understanding of ovarian cancer relapse. The expertise gained during the K99 phase, together with my molecular biology and genomics background, will allow me to develop an integrated research program designed to investigate novel mechanisms of ovarian cancer persistence and relapse, leading ultimately to new therapies to prolong the lives of women with this disease. Results of this proposal will advance the ovarian cancer field by providing mechanistic insight into ovarian cancer recurrence and the role of specific cell subtypes. Completion of the proposed aims will guide the development of alternative therapeutic strategies for patients with ovarian cancer and may highlight mechanisms relevant to other cancer types.