The goals of our project are to isolate prostate epithelial populations, including prostate stem cells, and to evaluate the tumorigenic and the metastatic capacity of such populations upon transformation by genetic mutations that are commonly associated with human prostate cancer. Here we describe our progress in characterizing a mouse model of prostate cancer initiated by prostate epithelial cell specific deletion of the tumor suppressors, Pten and Tp53. Loss of PTEN and loss of TP53 are common genetic aberrations occurring in prostate cancer. PTEN and TP53 contribute significantly to the regulation of adult stem/progenitor cells in somatic tissues, and we hypothesized that combined deletion of these tumor suppressors in prostate epithelium would provide insight into the pathophysiology of transformed prostate progenitor cells. Using the PB-Cre4; Ptenfl/fl;TP53fl/fl model of prostate cancer, we describe the histologic and metastatic properties of primary tumors, of transplanted primary tumor cells, and of clonal cell lines established from tumors. Adenocarcinoma was the major tumor type that developed in this mouse model, and in addition, basal carcinomas, prostatic urothelial carcinoma and late-developing sarcomatoid carcinomas were observed. We show here that tumor initiating cells with multi-lineage potential gave rise to orthotopic PIN (oPIN), adenocarcinomas and basal-phenotype carcinomas. CK8+ committed luminal epithelial cells were capable of undergoing epithelial to mesenchymal transition in vivo to sarcomatoid carcinomas that often produced cartilaginous and bone structures. Metastasis was rarely observed from primary tumors, even after secondary orthotopic transplantation, implying that additional selection must occur for metastatic colonization. Upon orthotopic tumor development from cell lines, multi-potential epithelial progenitor cells were highly metastatic to lung and lymph nodes while prostatic sarcomatoid carcinoma cells rarely metastasized. Combined loss of Pten and Tp53 in prostate epithelial progenitors results in lineage plasticity of transformed cells and heterogeneity of resultant tumors. TP53 contributes significantly to the regulation of stem cell self-renewal, and we hypothesized that Pten/TP53 play crucial roles in determining prostate cancer stem/progenitor cell properties. Clonogenic assays that isolate progenitor function in primary prostate epithelial cells were used to measure self-renewal, differentiation, and tumorigenic potential. Pten/TP53 null as compared to wild type protospheres showed increased self-renewal activity and modified lineage commitment. Pten/TP53 null progenitors also demonstrated increased dependence upon the AKT/mTORC1 and androgen receptor pathways for clonogenic and tumorigenic growth. Orthotopic transplantation of Pten/TP53 null cells derived from protospheres produced invasive PIN/adenocarcinoma, recapitulating the pathology seen in primary tumors. These data demonstrate roles for Pten/TP53 in prostate epithelial stem/progenitor cell function, and moreover, as seen in patients with castrate-resistant prostate cancer, suggest a crucial role for an AR-dependent axis in the clonogenic expansion of prostate cancer stem cells. The model system described here has proven to be unique among mouse models of prostate cancer in allowing robust serial transplantation of adenocarcinoma. This feature has allowed us to analyze the characteristics of tumor initiating populations of prostate adenocarcinoma. The characteristics analyzed have included androgen sensitivity and expression of stem cell markers. We have been able to directly show that adenocarcinoma is propagated by a castration-resistant luminal stem cell. We have begun using a novel cell culture system that has allowed the propagation of mouse and human luminal stem/progenitor cells in culture. Mouse prostate cancer luminal stem cells can be transplanted into immunocompromised mice to produce prostatic adenocarcinoma. We are using this procedure to derive primary cell cultures from human prostate cancer specimens. Such cell cultures will allow the ability to phenotype/ genotype and experimentally manipulate human prostate cancers. The inability to grow human prostate cancers has been a hurtle to accurate disease modeling in this field. Epithelial-mesenchymal transition (EMT) is implicated in various pathological processes within the prostate, including benign prostate hyperplasia (BPH) and prostate cancer progression. However, an ordered sequence of signaling events initiating carcinoma-associated EMT has not been established. In a model of TGFbeta-induced prostatic EMT, SLUG is the dominant regulator of EMT initiation in vitro and in vivo, and SLUG transcription is inhibited by KLF-4, which itself is degraded following TGFbeta treatment. Two mirR targets that regulate SLUG expression and that are degraded upon EMT transitions are miR-200 and mir-1. Mir-1 decreases significantly upon prostate cancer progression. We have analyzed several potential miR-1 targets and discovered that SRC is directly targeted by miR-1 for downregulation. Importantly, SRC is a known mediator of prostate cancer metastasis. These data suggest one mechanistic route through decreased expression of miR-1 leading to prostate cancer progression. Acquired resistance following treatment of progressive prostate cancer with androgen deprivation therapy is a significant clinical problem. An adaptive mechanism of resistance hypothesizes that castration tolerant stem/progenitor cells are precursors of castration resistant prostate cancer (CRPC) that subsequently develops as a result of mutation and selection. We have characterized primary Pten/Tp53 null prostate tumor cell fractions for differentiation potential, tumor-propagating cells (TPCs), and castration sensitivity. Although either basal or luminal cells can serve as cells of origin for prostate cancer, the identity of cells in primary tumors capable of autonomous tumor propagation and/or as precursors of CRPC have not been determined. Luminal-committed and bipotential progenitors were capable of autonomous tumor propagation. Importantly, luminal-committed and bipotential stem/progenitor cells survived castration with a high frequency and gave rise to adenocarcinoma upon transplantation into an intact host, modeling the development of castration resistance from intrinsically-castration tolerant progenitor cells. TP53 loss in the context of Pten loss, characteristic of metastatic prostate cancer, increased the presence of such luminal progenitors. These studies demonstrate the pathogenic potential of luminal-committed progenitor TPCs prior to acquired resistance and suggest that their early targeting in combination with androgen deprivation may be a therapeutic approach to decrease treatment-induced resistance. We are extending these studies by investigating the stem/progenitor cell populations in clinical samples of advanced prostate cancer. In addition, cell lines have been established from tumor-derived luminal progenitor cells. Screening of such lines in high throughput viability assays has identified Hsp90 inhibitors as active compounds in growth inhibition.
