INVESTIGATION OF A PUTATIVE PROSTATE STEM CELL NICHE

Background: Despite recent advances in the treatment of castration-resistant prostate cancer using agents such as abiraterone and enzalutamide that target the androgen receptor signaling pathway, most patients with advanced prostate cancer will eventually develop resistance to these therapies. In approximately 30% of cases, treatment resistance is associated with the emergence of neuroendocrine cells. However, remarkably little is known about the precise function and properties of neuroendocrine cells, either in the normal prostate or during prostate tumorigenesis.Objective: This proposal will investigate the hypothesis that neuroendocrine cells comprise an important component of a niche (supportive microenvironment) for prostate stem/progenitor cells during development, and also provide paracrine support for the activity of tumor-propagating cells (putative cancer stem cells). In preliminary studies, we have found that neuroendocrine cells in the normal mouse prostate are highly localized to the proximal region of prostatic ducts, which is enriched for stem/progenitor cells. We have also confirmed the enrichment of stem/progenitor activity within the proximal region using a novel three-dimensional culture system that we have developed for growth of prostate epithelial organoids. Furthermore, we have developed a mouse model of treatment-emergent neuroendocrine prostate cancer that can be used to test the putative niche activities of neuroendocrine cells in disease progression. Taken together, these findings support the rationale as well as feasibility of our proposed studies of the stem cell niche function of neuroendocrine cells.Specific Aims: To investigate our hypothesis, our proposed studies have two linked specific aims:(1) Analysis of neuroendocrine cells as a prostate stem cell niche by performing functional analyses of neuroendocrine cells in assays for stem/progenitor activity. We will use isolated lineage-marked neuroendocrine cells as well as neuroendocrine cell lines to investigate their ability to enhance stem/progenitor properties in organoid culture and renal grafting assays.(2) Investigation of neuroendocrine cells as a potential niche for cancer stem cells using a mouse model for treatment-emergent neuroendocrine prostate cancer. We will determine whether isolated lineage-marked neuroendocrine cells can enhance the tumor-propagating ability of adenocarcinoma cells in organoid culture and renal grafting assays, and we will perform bioinformatic analyses to identify candidate pathways and biological processes that may be affected by paracrine signals from neuroendocrine cells.Study Design: The studies of this application are conceptually innovative by providing a novel perspective for investigating the significance of neuroendocrine differentiation in castration-resistant prostate cancer. In Aim 1, we will assess the paracrine effects of purified lineage-marked neuroendocrine cells to enhance stem/progenitor activity in organoid culture assays using distal and proximal prostate epithelial cells, as well as in assays using CARNs (castration-resistant Nkx3.1-expressing cells), a luminal stem/progenitor population, and LSCs (Lin- Sca-1+CD49fhigh cells), a basal stem/progenitor population. These organoid culture assays will be complemented by classical assays that involve tissue recombination with embryonic urogenital mesenchyme and grafting under the kidney capsule.For studies of neuroendocrine function in prostate tumors in Aim 2, we will use tamoxifen-inducible Nkx3.1CreERT2/+; Ptenflox/flox; Trp53flox/flox (NPP53) mice as a model of treatment-emergent neuroendocrine prostate cancer. We will determine whether luminal adenocarcinoma cells from this model will have increased tumor-propagating activities when co-cultured with neuroendocrine cells in organoid formation assays, or in renal grafting assays. Organoids and/or grafts that show a positive response to neuroendocrine cells will then be used to generate expression profiles for bioinformatic identification of candidate pathways that might be responsive to neuroendocrine activity.Impact: Our proposed studies of neuroendocrine function in modulating stem cell properties should provide important new insights into the molecular basis for the emergence of highly castration-resistant phenotypes that lead to lethality, and ultimately may have implications for the design of new therapeutic regimens for castration-resistant prostate cancer. Therefore, these studies should have significant implications for the overarching challenge of developing effective treatments and addressing mechanisms of resistance for men with high risk or metastatic prostate cancer, and they will have a strong impact on the focus areas of mechanisms of resistance and response as well as tumor and microenvironment biology.