Regulation of prostate neuroendocrine cell differentiation and tumorigenesis

Neuroendocrine (NE) cells represent one of three lineages present in the normal prostatic epithelium. The features and functions of NE cells remain ill-defined. The development of neuroendocrine differentiation (NED) in conventional adenocarcinoma of the human prostate (CaP) is associated with more aggressive disease, but the underlying mediators and mechanisms are poorly understood. This thesis combines a transgenic mouse model of metastatic prostate cancer arising in NE cells, with functional genomics and computational approaches to characterize the biological properties of this lineage, and to identify the role of the bHLH transcription factor mAsh1 in NE specification/differentiation.; Transcriptional regulatory elements from the mouse cryptdin-2 (CR2) gene were used to direct expression of SV40 large T antigen to a subset of prostatic NE cells. Primary tumors and metastases were isolated from CR2-TAg transgenic mice and gene expression characterized using GeneChips, real-time quantitative RT-PCR of laser-capture microdissected NE cells, and multilabel immunohistochemistry. Candidate mediators and biomarkers of NE differentiation and tumorigenesis identified in the CR2-TAg model, including mAsh1 are also expressed in foci of NED in human CaP. To further characterize the pathways that control NE cell proliferation and differentiation, I established Prostate NE Cancer (PNEC) cell lines from CR2-TAg prostate tumors and metastases. PNECs form neurosphere-like structures when grown in suspension, neurite-like processes when cultured as attached monolayers, and tumors when injected into Balb/c nude mouse recipients. Hierarchical clustering and multidimensional scaling of a 580 gene dataset culled from GeneChip analyses of cultured cell lines at different passages and as xenografted tumors, indicate that PNECs express consistent features ex vivo and share a remarkable degree of similarity with CR2-TAg primary prostate NE tumors. RNAi knockdown of mAsh1 in a cloned prostate-derived PNEC line, GeneChip and qRT PCR profiling of treated and control cell populations, together with a computational analysis that combined phylogenetic data with down-regulated genes to identify mAsh1 regulatory sequences revealed 12 motifs enriched in this gene set. Adcy9, Hes6, Iapp1, Ndrg4, c-Myb, and Mesdc2 (all down-regulated by mAsh1 RNAi) are enriched for the E-box motif CAGGTG, providing evidence that it represents a true mAsh1 binding site. The presence of MAZ and MAZR sites suggests a connection between mAsh1 and c-Myc related pathways. Finding a c-Myb binding site and observing that c-Myb itself is down-regulated by mAsh1 RNAi, suggest that mAsh1 and c-Myb are in the same pathway. These studies indicate that PNECs will be useful for genetic and/or pharmacologic tests of the roles of various factors in NE differentiation and growth.