Collene R. Jeter

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic 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 target, 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 stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors—including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary—have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic 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 target, 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.

Functional Evidence that the Self‐Renewal Gene NANOG Regulates Human Tumor Development

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell (ESC) self‐renewal gene NANOG is purportedly expressed by some epithelial cancer cells but a causal role in tumor development has remained unclear. Here, we provide compelling evidence that cultured cancer cells, as well as xenograft‐ and human primary prostate cancer cells express a functional variant of NANOG. NANOG mRNA in cancer cells is derived predominantly from a retrogene locus termed NANOGP8. NANOG protein is detectable in the nucleus of cancer cells and is expressed higher in patient prostate tumors than matched benign tissues. NANOGP8 mRNA and/or NANOG protein levels are enriched in putative cancer stem/progenitor cell populations. Importantly, extensive loss‐of‐function analysis reveals that RNA interference‐mediated NANOG knockdown inhibits tumor development, establishing a functional significance for NANOG expression in cancer cells. Nanog short hairpin RNA transduced cancer cells exhibit decreased long‐term clonal and clonogenic growth, reduced proliferation and, in some cases, altered differentiation. Thus, our results demonstrate that NANOG, a cell‐fate regulatory molecule known to be important for ESC self‐renewal, also plays a novel role in tumor development. Stem Cells 2009;27:993–1005

NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation.

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.

Prostate cancer stem/progenitor cells: Identification, characterization, and implications

Several solid tumors have now been shown to contain stem cell‐like cells called cancer stem cells (CSC). These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In this Perspective, we first provide our insight on how a CSC should be defined. We then summarize our current knowledge of stem/progenitor cells in the normal human prostate (NHP), an organ highly susceptible to hyperproliferative diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PCa). We further review the evidence that cultured PCa cells, xenograft prostate tumors, and patient tumors may contain stem/progenitor cells. Along with our discussion, we present several methodologies that can be potentially used to identify putative tumor‐reinitiating CSC. Finally, we present a hypothetical model for the hierarchical organization of human PCa cells and discuss the implications of this model in helping understand prostate carcinogenesis and design novel diagnostic, prognostic, and therapeutic approaches.

The PSA−/lo Prostate Cancer Cell Population Harbors Self-Renewing Long-Term Tumor-Propagating Cells that Resist Castration

Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA(+)) and low (PSA(-/lo)) levels of the differentiation marker PSA. PSA(-/lo) PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division to generate PSA(+) cells. Importantly, PSA(-/lo) PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and they harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH(+)CD44(+)α2β1(+) phenotype. In contrast, PSA(+) PCa cells possess more limited tumor-propagating capacity, undergo symmetric division, and are sensitive to castration. Altogether, our study suggests that PSA(-/lo) cells may represent a critical source of castration-resistant PCa cells.

Evidence of a Novel Cell Signaling Role for Extracellular Adenosine Triphosphates and Diphosphates in Arabidopsis

Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca2+]cyt). We have evaluated the ability of exogenously applied adenosine 5′-[γ-thio]triphosphate (ATPγS), adenosine 5′-[β-thio]diphosphate (ADPβS), and adenosine 5′-O-thiomonophosphate to alter [Ca2+]cyt in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPγS and ADPβS increase [Ca2+]cyt, and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca2+]cyt induced by nucleotide derivatives can be ablated by Ca2+-channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.

miRNA-128 Suppresses Prostate Cancer by Inhibiting BMI-1 to Inhibit Tumor-Initiating Cells

microRNA-128 (miR128) is reduced in prostate cancer relative to normal/benign prostate tissues, but causal roles are obscure. Here we show that exogenously introduced miR128 suppresses tumor regeneration in multiple prostate cancer xenograft models. Cancer stem-like cell (CSC)-associated properties were blocked, including holoclone and sphere formation as well as clonogenic survival. Using a miR128 sensor to distinguish cells on the basis of miR128 expression, we found that miR128-lo cells possessed higher clonal, clonogenic, and tumorigenic activities than miR128-hi cells. miR128 targets the stem cell regulatory factors BMI-1, NANOG, and TGFBR1, the expression of which we found to vary inversely with miR128 expression in prostate cancer stem/progenitor cell populations. In particular, we defined BMI-1 as a direct and functionally relevant target of miR128 in prostate cancer cells, where these genes were reciprocally expressed and exhibited opposing biological functions. Our results define a tumor suppressor function for miR128 in prostate cancer by limiting CSC properties mediated by BMI-1 and other central stem cell regulators, with potential implications for prostate cancer gene therapy.

Concise Review: NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self‐renewal and long‐term proliferative potential of stem‐like cancer cells, NANOG‐mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development. Stem Cells 2015;33:2381–2390

Critical and Distinct Roles of p16 and Telomerase in Regulating the Proliferative Life Span of Normal Human Prostate Epithelial Progenitor Cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, α2β1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67+ cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4–6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34+α6+ and Lrig-1+ bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.