Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment

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ABSTRACT: Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment Ovarian cancer is the most deadly US gynecologic malignancy with a mortality rate that exceeds 50% at 5 years. Ovarian cancer is characterized by early intraperitoneal metastasis and the development of a complex microenvironment which supports tumor cell growth, survival and spread. Understanding and eventually targeting this cancer-promoting tumor microenvironment offers the potential for powerful new therapeutic approaches. My ultimate goal is to become a world-class independent physician scientist studying the ovarian cancer microenvironment in order to develop new treatments and improve outcomes for women with ovarian cancer. This proposal describes important and innovative research which will lay the foundation for my future career in addition to providing the necessary skills and mentorship vital for my success. The ovarian tumor microenvironment (TME) is a diverse system of cellular and chemical components. The cellular TME includes tumor cells and non-malignant stromal cells. The chemical TME is marked by acidosis and hypoxia. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are multi-potent stromal cells within the cellular TME that can differentiate into multiple pro-tumorigenic stromal cell types including fibroblasts, myofibroblasts, and adipocytes. CA-MSCs are genotypically normal without malignant potential but are functionally different than normal tissue or bone marrow derived MSCs. Compared to normal MSCs, CA-MSCs demonstrate a unique molecular phenotype with very high expression of bone morphogenetic proteins (BMPs). Due to this unique phenotype, these CA-MSCs strongly promote ovarian cancer growth, enhance chemotherapy resistance and enrich the cancer stem cell-like population. How CA-MSCs develop their unique phenotype remains unclear. My preliminary data indicate that tumor secreted factors induce some of the molecular changes associated with CA-MSCs. Another potential mediator of the CA-MSC phenotype is hypoxia. Hypoxia is a hallmark of the chemical TME known to impact normal MSC function. In cancer, hypoxia influences tumor:stromal interactions and hypoxia is a key regulator of BMP expression—high levels of which characterize ovarian cancer CA-MSCs. Preliminary data indicates that hypoxia enhances the ability of tumor cells to induce a CA-MSC expression profile in normal MSCs. While the mechanism of this induction is unknown, given CA-MSCs are genetically normal yet maintain their unique phenotype across multiple passages, tumor-induced epigenetic regulation may be critical to the formation of the CA-MSC phenotype. Indeed, preliminary data indicates CA-MSCs exhibit significant hypomethylation compared to normal MSCs. In addition to influencing the formation of a CA-MSC, hypoxia may also critically regulate the function of CA- MSCs already established in the ovarian TME. My preliminary data suggests that hypoxia maintains the “stemness” of CA-MSCs slowing growth and maintaining differentiation capacity. Further, my data suggests that the hypoxia inducible factor pathway, the main hypoxia signaling pathway, is hyper-active in CA-MSCs compared to normal MSCs. Thus hypoxia may be a critical modulator of CA-MSCs within the ovarian TME. My main research goal is to understand how CA-MSCs obtain their unique phenotype and subsequently interact with and influence the function of the ovarian cancer microenvironment. To achieve this goal, I propose two specific aims: 1) Determine the ability of normal MSCs to acquire a CA-MSC-like phenotype 2) Determine the impact of hypoxia on established CA-MSCs within the tumor microenvironment. In aim 1, I hypothesize that tumor cell conditioning under hypoxia induces normal MSCs to become CA-MSCs. To test this I will perform cancer cell: normal MSC co-cultures under normoxia and hypoxia to determine if cancer cells can functionally turn a normal MSC into a CA-MSC. I will also explore differential DNA methylation as a mechanism for the creation of a CA-MSC. In aim 2, I focus on already established CA-MSCs. I hypothesize that hypoxia enhances the pro-tumorigenic effects of established CA-MSCs within the tumor microenvironment. To test this, I will utilize conditional HIF pathway knockout mice and CRISPER/CAS9 genome editing to assess the impact of hypoxia and HIF signaling on established CA-MSCs. In addition to furthering our understanding of CA-MSCs in ovarian cancer, the proposed research and training will cultivate expertise necessary for an independent career studying the ovarian TME. Through the support of Dr. Laird, I will master the assessment of genome-wide epigenetic modifications and the analysis of large scale “omics” data. Dr. Schipani will facilitate my education in hypoxia and HIF signaling. Through Dr. Schipani and Dr. Cho, I will learn to generate and manipulate transgenic mouse models. Dr. Buckanovich's ongoing mentorship will further my expertise in the function of the ovarian TME and, together with my mentoring committee, will help develop my leadership, team-building and communication skills. By the end of the training period, I will have developed a novel skill set which merges the expertise of multiple scientific leaders yielding a uniquely trained physician scientist ideal for the study of the ovarian cancer microenvironment.