Metastases to bone continue to be the major cause of Prostate Cancer (PCa) morbidity and mortality, contributing to the death of more than 28,000 men in the United States every year. Our P01 project continues to focus on how Tumor Associated Macrophages (TAMs) promote PCa metastasis and growth in the bone microenvironment. TAMs have been implicated in invasiveness and tumor metastasis. Work during the current grant period has demonstrated that TAMs promote metastasis by inducing epithelial PCa cells (ePCa) to undergo a stable EMT to become mesenchymal PCa cells (mPCa) in a Snail, Slug, and TGF? independent manner in vitro and in vivo. TAMs induce high expression of Zeb1 and repress the Ovo-family of transcription factors to stabilize the mesenchymal phenotype. We have discovered a novel function of the two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancer. We demonstrate that the expression of the OVO- TFs in mesenchymal PCa cells attenuates their metastatic potential by controlling MET through a regulatory feedback loop with the EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). The purpose of this renewal application is to test our hypothesis that TAM-induced EMT plays a central role in the establishment of PCa bone metastasis. The following aims are planned:
Aim 1 : Define the M2-TAM-induced signaling pathways utilized by the Ovo family of transcription factors that lead to EMT transformation in PCa cells.
In Aim 1 A we will delineate the mechanism by which the chemokines CCL28, 18, and 16 induce Ovo TF down-regulation / ZEB1 upregulation.
In Aim 1 B we will delineate the downstream signal transduction pathways controlling EMT via the interaction of the Ovo family of transcription factors and Zeb1 in preclinical models.
In Aim 1 C we will study the expression of Ovo-family and Zeb1 driven signal transduction molecules in PCa cells from patients with PCa.
Aim 2 : Delineate the functional phenotypes of epithelial (ePCa) and mesenchymal PCa (mPCa) cells as they interact with TAMs.
In Aim 2 A we will compare and contrast changes in gene expression as the different PCa cell phenotypes interact with TAMs of prostate and bone in ex vivo models.
In Aim 2 B we will further study the functional characteristics of ePCa and mPCa cells in vitro utilizing invasion and motility assays and in vivo by following intracardiac or orthotopic injections.
Aim 3 : Study the EMT/MET plasticity of PCa cells in the bone microenvironment.
In Aim 3 A we will characterize PCa cells plasticity in primary PCas and in patients with preclinical and clinical bone metastases utilizing our PCa Tissue Microarrays (TMAs) to understand the relationship between phenotype and proliferative capacity.
In Aim 3 B we will analyze CTCs and DTCs obtained from PCa patients with different stages of PCa to identify and measure plasticity.
These studies will significantly alter our understanding of how PCa cells metastasize and proliferate in the bone marrow microenvironment, opening new avenues towards the development of prognostic markers and therapy. Based on our preliminary data, we believe that in the primary tumor, PCa cells originally have an epithelial phenotype. As they proliferate, they secrete CCL2 to attract monocytes that differentiate into M2- TAMs (current project). The TAMs induce EMT in the PCa cells and these EMT-PCa cells have the ability to invade and metastasize to bone (current project and proposed mechanistic studies). When they reach the bone marrow microenvironment, they take up residence and eventually undergo MET (proposed studies). These cells then proliferate, attract TAMs, and repeat the EMT/MET cycle to induce secondary metastases.
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