Cancer stem cells (CSCs) or tumor progenitor cells are involved in tumor progression and metastasis1. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs2–5 and miRNA dysregulation 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 adhesion molecule CD44, either individually or in combination with other marker(s). Prostate cancer (PCa) stem/progenitor cells with enhanced clonogenic17 and tumor-initiating and metastatic18,19 capacities are also enriched in the CD44+ cell population, but whether miRNAs regulate the CD44+ PCa cells and PCa metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target20–24, was under-expressed in CD44+ PCa cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk PCa cells inhibited clonogenic expansion and tumor development. miR-34a re-expression in CD44+ PCa cells blocked whereas miR-34a antagomirs in CD44− PCa cells promoted tumor regeneration and metastasis. Systemically delivered miR-34a inhibited PCa metastasis and extended animal survival. Of significance, CD44 was identified and validated as a direct and functional target of miR-34a and CD44 knockdown phenocopied miR-34a over-expression in inhibiting PCa regeneration and metastasis. Our study reveals miR-34a as a critical negative regulator of CD44+ PCa cells and establishes a strong rationale for developing miR-34a as a novel therapeutic 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 exhibit oncogenic potential. We have recently reported that shRNA-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 quantitative RT-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 exhibited 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 prostate (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.

It has long been known that excessive mitotic activity due to H-Ras can block keratinocyte differentiation and cause skin cancer. It is not clear whether there are any innate surveillants that are able to ensure that keratinocytes undergo terminal differentiation, preventing the disease. IKKalpha induces keratinocyte terminal differentiation, and its downregulation promotes skin tumor development. However, its intrinsic function in skin cancer is unknown. Here, we found that mice with IKKalpha deletion in keratinocytes develop a thickened epidermis and spontaneous squamous cell-like carcinomas. Inactivation of epidermal growth factor receptor (EGFR) or reintroduction of IKKalpha inhibits excessive mitosis, induces terminal differentiation, and prevents skin cancer through repressing an EGFR-driven autocrine loop. Thus, IKKalpha serves as an innate surveillant.

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR−/lo). Xenograft modeling demonstrates that AR+ CRPC is enzalutamide-sensitive but AR−/lo CRPC is resistant. Genome editing-derived AR+ and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR+/hi and AR−/lo CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR−/lo PCa cells/clones.

MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44+ and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.

IKK (I B kinase) ␣ is essential for embryonic skin development in mice. Mice deficient in IKK␣ display markedly hyperplasic epidermis that lacks terminal differentiation, and they die because of this severely impaired skin. However, the function of IKK␣ in human skin diseases remains largely unknown. To shed light on the role of IKK␣ in human skin diseases, we examined IKK␣ expression and Ikk␣ mutations in human squamous cell carcinomas (SCCs). We found a marked reduction in IKK␣ expression in poorly differentiated human SCCs and identified Ikk␣ mutations in exon 15 of Ikk␣ in eight of nine human SCCs, implying that IKK␣ is involved in development of this human skin cancer. Furthermore, in a chemical carcinogen-induced skin carcinogenesis setting, mice overexpressing human IKK␣ in the epidermis under the control of a truncated loricrin promoter developed significantly fewer SCCs and metastases than did wild-type mice. The IKK␣ transgene altered the skin microenvironment conditions, leading to elevated terminal differentiation in the epidermis, reduced mitogenic activity in the epidermis, and decreased angiogenic activity in the skin stroma. Thus, overexpression of IKK␣ in the epidermis antagonized chemical carcinogen-induced mitogenic and angiogenic activities, repressing tumor progression and metastases.angiogenesis ͉ mitogenesis ͉ skin carcinogenesis ͉ differentiation ͉ tumor progression S quamous cell carcinomas (SCCs) can be very aggressive and metastatic. Previous studies have shown that Pten mutations are found frequently in human SCCs; however, these Pten mutations are not detected in all cases (1, 2). p53 mutations have also been identified in a large proportion of human SCCs (3); however, these p53 mutations can be latent in skin cells for years before the onset of this disease. Clearly, additional pivotal factors in the development of this human skin cancer remain to be identified.The I B kinase (IKK) complex consists of IKK␣, IKK␤, and IKK␥ (4-7), which phosphorylates I B␣ (S32͞S36) and I B␤ (S19͞S23) that are NF-B inhibitors. This phosphorylation event triggers the degradation of I B proteins via a 26S proteasomeubiquitination pathway, leading to NF-B translocation and activation. Previous findings have shown that IKK␣ is essential for embryonic skin development in mice (8-10). Mice lacking IKK␣ exhibit a strikingly hyperplastic epidermis, which lacks terminal differentiation, and they die of severely impaired skin soon after birth. The skin phenotypes of Ikk␣ Ϫ/Ϫ mice have not been observed in knockouts of any other NF-B family members. In contrast, mice overexpressing IKK␣ in the basal epidermis develop normally (11). The ectopic IKK␣ is able to induce terminal differentiation and to repress hyperplasia in the skin of Ikk␣ Ϫ/Ϫ mice, implying that IKK␣ plays a role in maintaining skin homeostasis (11).Although a role of IKK␣ in skin development in mice has been established, we still know little regarding the involvement of IKK␣ in human skin diseases. To shed light on the role of IKK␣ in human skin ...

MicroRNA-128 (miR-128) is reduced in prostate cancer (PCa) relative to normal/benign prostate tissues but causal roles are obscure. Here we show that exogenously introduced miR-128 suppresses tumor regeneration in multiple PCa xenograft models. Cancer stem-like cell (CSC) associated properties were blocked, including holoclone and sphere formation as well as clonogenic survival. Using a miR-128 sensor to distinguish cells on the basis of miR-128 expression, we found that miR-128-lo cells possessed higher clonal, clonogenic and tumorigenic activities than miR-128-hi cells. miR-128 targets the stem cell regulatory factors BMI-1, NANOG, and TGFBR1, the expression of which we found to vary inversely with miR-128 expression in PCa stem/progenitor cell populations. In particular, we defined BMI-1 as a direct and functionally relevant target of miR-128 in PCa cells, where these genes were reciprocally expressed and exhibited opposing biological functions. Our results define a tumor suppressor function for miR-128 in PCa by limiting CSC properties mediated by BMI-1 and other central stem cell regulators, with potential implications for PCa gene therapy.

We reported recently a marked reduction in IKB kinase A (IKKA) expression in a large proportion of human poorly differentiated squamous cell carcinomas (SCC) and the occurrence of Ikka mutations in human SCCs. In addition, overexpression of IKKA in the epidermis inhibited the development of skin carcinomas and metastases in mice. However, whether a reduction in IKKA expression promotes skin tumor development is currently unknown. Here, we assessed the susceptibility of Ikka hemizygotes to chemical carcinogen-induced skin carcinogenesis. Ikka +/À mice developed 2 times more papillomas and 11 times more carcinomas than did Ikka +/+ mice. The tumors were larger in Ikka +/À than in Ikka +/+ mice, but tumor latency was shorter in Ikka +/À than in Ikka +/+ mice. Some of the Ikka +/À papillomas and most Ikka +/À carcinomas lost the remaining Ikka wild-type allele. Somatic Ikka mutations were detected in carcinomas and papillomas. The chemical carcinogen-induced H-Ras mutations were detected in all the tumors. The phorbol ester tumor promoter induced higher mitogenic and angiogenic activities in Ikka +/À than in Ikka +/+ skin. These elevated activities were intrinsic to keratinocytes, suggesting that a reduction in IKKA expression provided a selective growth advantage, which cooperated with H-Ras mutations to promote papilloma formation. Furthermore, excessive extracellular signal-regulated kinase and IKK kinase activities were observed in carcinomas compared with those in papillomas. Thus, the combined mitogenic, angiogenic, and IKK activities might contribute to malignant conversion. Our findings provide evidence that a reduction in IKKA expression promotes the development of papillomas and carcinomas and that the integrity of the Ikka gene is required for suppressing skin carcinogenesis. [Cancer Res 2007;67(19):9158-68]