The authors show that miR-34a regulates progenitor and metastatic properties of prostate cancer cells, and they identify CD44 as a relevant target of the microRNA. The inhibition of metastasis observed after systemic delivery of miR-34a suggests that it could be used as a potential therapeutic agent in prostate cancer. Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis1. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs2,3,4,5, and dysregulation of miRNAs has been implicated in tumorigenesis6. CSCs in many tumors—including cancers of the breast7, pancreas8, head and neck9, colon10,11, small intestine12, liver13, stomach14, bladder15 and ovary16—have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic17 and tumor-initiating and metastatic18,19 capacities are enriched in the CD44+ cell population, but whether miRNAs regulate CD44+ prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target20,21,22,23,24, was underexpressed in CD44+ prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44+ prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44− prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44+ prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

Cancer cell molecular mimicry of stem cells (SC) imbues neoplastic cells with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to show oncogenic potential. We have recently reported that short-hairpin RNA-mediated knockdown of the embryonic stem cell (ESC) self-renewal gene NANOG significantly reduced the clonogenic and tumorigenic capabilities of various cancer cells. In this study, we sought to test the potential pro-tumorigenic functions of NANOG, particularly, in prostate cancer (PCa). Using qRT–PCR, we first confirmed that PCa cells expressed NANOG mRNA primarily from the NANOGP8 locus on chromosome 15q14. We then constructed a lentiviral promoter reporter in which the −3.8-kb NANOGP8 genomic fragment was used to drive the expression of green fluorescence protein (GFP). We observed that NANOGP8-GFP⁺ PCa cells showed cancer stem cell (CSC) characteristics such as enhanced clonal growth and tumor regenerative capacity. To further investigate the functions and mechanisms of NANOG in tumorigenesis, we established tetracycline-inducible NANOG-overexpressing cancer cell lines, including both PCa (Du145 and LNCaP) and breast (MCF-7) cancer cells. NANOG induction promoted drug resistance in MCF-7 cells, tumor regeneration in Du145 cells and, most importantly, castration-resistant tumor development in LNCaP cells. These pro-tumorigenic effects of NANOG were associated with key molecular changes, including an upregulation of molecules such as CXCR4, IGFBP5, CD133 and ALDH1. The present gain-of-function studies, coupled with our recent loss-of-function work, establish the integral role for NANOG in neoplastic processes and shed light on its mechanisms of action.

The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

Primary keratinocytes exhibit three typical clonal morphologies represented by holoclones, meroclones, and paraclones, with holoclones containing self-renewing stem cells, and meroclones and paraclones containing more mature and differentiated cells. Interestingly, long-term–cultured human epithelial cancer cells in clonal cultures also form holoclones, meroclones, and paraclones, and tumor cell holoclones have been hypothesized to harbor stem-like cells or cancer stem cells. However, the key question of whether tumor cell holoclones genuinely contain tumor-initiating cells has not been directly addressed. Here, using PC3 human prostate carcinoma cells as a model, we provide direct experimental evidence that tumor cell holoclones contain stem-like cells that can initiate serially transplantable tumors. Importantly, holoclones derived from either cultured PC3 cells or holoclone-initiated tumors can be serially passaged and regenerate all three types of clones. In contrast, meroclones and paraclones cannot be continuously propagated and fail to initiate tumor development. Phenotypic characterizations reveal high levels of CD44, α₂β₁ integrin, and β-catenin expression in holoclones, whereas meroclones and paraclones show markedly reduced expression of these stem cell markers. The present results have important implications in understanding morphologic heterogeneities and tumorigenic hierarchies in human epithelial cancer cells. [Cancer Res 2008;68(6):1820–5]