The prostate contains classes of epithelial cells with variable differentiation status that may serve as targets for tumor initiation (8-10). Androgen cycling experiments suggest that stem cell activity is present in the prostate (11). These cells are thought to reside in the basal layer of the prostate because these cells are androgen-independent and preferentially retained in castrated rodents. Stem cells in the basal layer give rise to transit-amplifying epithelial cells (TACs) that differentiate to produce terminally differentiated secretory luminal cells (12).The most common form of human prostate cancer expresses luminal cell-specific markers (cytokeratins 8 and 18 and prostate-specific antigen) but low levels of the basal cell marker (p63), suggesting that the disease originates in terminally differentiated luminal cells (12,13). Some studies suggest that the disease is derived from high-grade prostatic intraepithelial neoplasia (PIN), which is believed to arise from intermediate epithelial cells (14). Other studies demonstrate that the majority of androgen-independent tumors express genes such as bcl-2 that are characteristic of the basal͞stem cell compartment in normal prostate tissue (8). Prostate cancers may arise from stem cells that undergo aberrant proliferation and differentiation to produce a heterogeneous population of cells expressing luminal-, basal-, and intermediate cell-specific genes (14, 15).The tissue recombination procedure developed by Cunha and Lung (16) uses fragments dissected from rat or mouse urogenital sinus mesenchyme (UGSM) in combination with adult epithelial tissue to regenerate prostate-like tissues. We developed a dissociated cell system to facilitate the study of unique prostate cell subpopulations to demonstrate that a subpopulation of murine prostate cells possesses stem-like capacity for de novo tubule regeneration (17). We find that Sca-1 (stem cell antigen-1), a glycosylphosphatidylinositol-linked cell surface protein known to enrich for somatic stem cells in other tissues (18,19), can be used to enrich for prostate-regenerating cells (PRCs). Sca-1 ϩ cell fractions contain an increased percentage of cells with immature cell properties, including replication quiescence, androgen independence, and multilineage differentiation potential. Perturbations of the PTEN͞AKT signaling axis in these cells result in the initiation of prostate tumorigenesis, and cancer progression is associated with a concomitant increase in Sca-1 ϩ cells. These studies suggest that Sca-1-enriched PRCs possess multiple stem͞progenitor cell properties and can serve as targets for the initiation of prostate tumorigenesis. Materials and Methods Infection of Dissociated Prostate Cells with Lentivirus and ProstateRegeneration. ␤-Actin GFP transgenic mice were purchased from The Jackson Laboratory [C57BL͞6-TgN(ACTbEGFP)1Osb]. Dissociated prostate cells were prepared from 6-to 10-week-old C57BL͞6 and ␤-actin GFP transgenic mice as described in ref.

Murine prostate stem cells express integrin ␣6, which modulates survival, proliferation, and differentiation signaling through its interaction with the extracellular protein laminin. When plated in vitro in laminin containing Matrigel medium, 1 of 500 -1,000 murine prostate cells can grow and form clonogenic spheroid structures that we term prostate spheres. Prostate spheres can be serially passaged individually or in bulk to generate daughter spheres with similar composition, demonstrating that sphere-forming cells are capable of self-renewal. Spheres spontaneously undergo lineage specification for basal and transit-amplifying cell types. P63-expressing cells localized to the outer layers of prostate spheres possess higher self-renewal capacity, whereas cells toward the center display a more differentiated transitamplifying phenotype, as demonstrated by the expression of the prostate stem cell antigen. When dihydrotestosterone is added to the medium, the androgen receptor is stabilized, is imported to the nucleus, and drives differentiation to a luminal cell-like phenotype. A fraction of sphere cells returned to an in vivo environment can undergo differentiation and morphogenesis to form prostate tubular structures with defined basal and luminal layers accompanied by prostatic secretions. This study demonstrates self-renewal and multilineage differentiation from single adult prostate stem/ progenitor cells in a specific in vitro microenvironment.

Chromosomal rearrangements involving erythroblast transformation specific (ETS) family transcription factors were recently defined as the most common genetic alterations in human prostate cancer. Despite their prevalence, it is unclear what quantitative role they play in either initiation or progression of the disease. Using a lentiviral transduction and dissociated cell prostate regeneration approach, we find that acutely increased expression of ETS proteins in adult murine prostate epithelial cells is sufficient to induce the formation of epithelial hyperplasia and focal prostatic intraepithelial neoplasia (PIN) lesions, but not progression to carcinoma. However, combined expression of ERG with additional genetic alternations associated with human prostate cancer can lead to aggressive disease. Although ERG overexpression does not cooperate with loss of the tumor suppressor p53, it does collaborate with alterations in PI3K signaling, such as Pten knockdown or AKT up-regulation, to produce a well-differentiated adenocarcinoma. Most striking is our finding that overexpression of androgen receptor (AR) does not give rise to any hyperplastic lesions, but when combined with high levels of ERG, it promotes the development of a more poorly differentiated, invasive adenocarcinoma. These findings suggest that in human prostate cancer, the most potent function of ETS gene fusions may be to synergize with alternative genetic events and provide different pathways for carcinoma production and invasive behavior. Our results provide direct evidence for selective cooperating events in ERG-induced prostate tumorigenesis and offer a rational basis for combined therapeutic interventions against multiple oncogenic pathways in prostate cancer.AKT ͉ androgen receptor ͉ ERG ͉ prostate cancer ͉ PTEN H uman cancer is a multigene disorder in which several somatic mutations in cancer predisposition genes are required in a stepwise manner (1). The first genetic alteration usually confers a selective growth advantage and subsequently results in clonal expansion to initiate the neoplastic process. But the cells must accumulate several other rate-limiting mutations over a long period to become malignant (2). For prostate cancer (PCa), various oncogenes and tumor suppressor genes, including NKX3.1, PTEN, p27, and p53, and androgen receptor (AR) signaling have been implicated in the disease progression (3). However, the key initiating steps in prostate carcinogenesis remain uncertain, and most genetic alterations that can collaborate to drive tumor progression have yet to be elucidated.Chromosomal translocations play critical roles in hematological malignancies (4). Several lines of evidence in epithelial cancers of distinct tissue origins suggest that acquired chromosomal rearrangements could also be the common genetic events in many, if not all, epithelial cancers (5). Recent studies showed that 50-80% of patients with PCa were found to harbor fusion transcripts between an androgen-regulated gene, TMPRSS2, and members of the erythroblast tr...

We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin + cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model. [Cancer Res 2009;69(22):8555-62]