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
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.
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.
Abstract
Prostate cancer cells are heterogeneous in their tumorigenicity. For example, the side population cells isolated from LAPC9 xenografts are 100 to 1,000 times more tumorigenic than the ...corresponding non–side population cells. Highly purified CD44+ prostate cancer cells from several xenografts are also enriched in prostate cancer stem/progenitor cells. Because the CD44+ prostate cancer cell population is still heterogeneous, we wonder whether we could further enrich for tumorigenic prostate cancer cells in this population using other markers. Integrin α2β1 has been proposed to mark a population of normal human prostate stem cells. Therefore, we first asked whether the α2β1+/hi cells in prostate tumors might also represent prostate cancer stem cells. Highly purified (≥98%) α2β1+/hi cells from three human xenograft tumors, Du145, LAPC4, and LAPC9, show higher clonal and clonogenic potential than the α2β1−/lo cells in vitro. However, when injected into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse prostate or s.c., the α2β1+/hi prostate cancer cells are no more tumorigenic than the α2β1−/lo cells. Immunofluorescence studies reveal that CD44 and α2β1 identify an overlapping and inclusive population of prostate cancer cells in that ∼70% of α2β1+/hi cells are CD44+ and 20% to 30% of CD44+ cells are distributed in the α2β1−/lo cell population. Subsequently, we sorted out CD44+α2β1+/hi, CD44+α2β1−/lo, CD44−α2β1+/hi, and CD44−α2β1−/lo cells from LAPC9 tumors and carried out tumorigenicity experiments. The results revealed a hierarchy in tumorigenic potential in the order of CD44+α2β1+/hi ≈ CD44+α2β1−/lo > CD44−α2β1+/hi ≫ CD44−α2β1−/lo. These observations together suggest that prostate cancer cells are organized as a hierarchy. Cancer Res 2007;67(14):6796–805
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
). Xenograft modeling demonstrates that AR
CRPC is enzalutamide-sensitive but AR
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
and AR
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
PCa cells/clones.
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, alpha(2)beta(1) integrin, and beta-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.
Elucidating the cell of origin of cancer has great significance in stratifying patients into appropriate treatment groups and for developing novel targeted therapies. Early studies demonstrate that ...only stem‐like basal cells in the normal human prostate (NHP) can function as the cell of origin for prostate cancer (PCa). Here, we show that the organoids derived from bulk NHP luminal cells can also be tumorigenically transformed. We further show that the WIT medium, which is used to culture human mammary epithelial progenitor cells, when combined with the ROCK inhibitor, can readily propagate a population of progenitor‐like cells from the primary NHP luminal cell isolates. Such functionally defined luminal progenitors can be transformed by distinct sets of genetic perturbations (i.e., AR+AKT/ERG or c‐MYC+PTEN knockout) to form tumor glands. Genome‐wide RNA‐Seq analysis of freshly purified unperturbed human benign prostatic basal and luminal cells and culture‐expanded lineage‐specific stem/progenitor populations reveals that the luminal progenitors possess a distinct gene expression profile that is greatly enriched in advanced, castration‐resistant, and metastatic PCa, and it associates with poor patient survival. The ability of the simple two‐dimensional culture system reported herein to greatly enrich NHP progenitor‐like cells should facilitate biological and biochemical studies as well as high‐throughput screening in these cells and in progenitor‐like PCa cells. Stem Cells Translational Medicine 2017;6:748–760
Human cancers exhibit significant cellular heterogeneity featuring tumorigenic cancer stem cells (CSCs) in addition to more differentiated progeny with limited tumor-initiating capabilities. Recent ...studies suggest that microRNAs (miRNAs) regulate CSCs and tumor development. A previous library screening for differential miRNA expression in CD44+ (and other) prostate CSC vs. non-CSC populations identified miR-199a-3p to be among the most highly under-expressed miRNAs in CSCs. In this study, we characterized the biological functions of miR-199a-3p in CD44+ prostate cancer (PCa) cells and in tumor regeneration. Overexpression of miR-199a-3p in purified CD44+ or bulk PCa cells, including primary PCa, inhibited proliferation and clonal expansion without inducing apoptosis. miR-199a-3p overexpression also diminished tumor-initiating capacities of CD44+ PCa cells as well as tumor regeneration from bulk PCa cells. Importantly, inducible miR-199a-3p expression in pre-established prostate tumors in NOD/SCID mice inhibited tumor growth. Using target prediction program and luciferase assays, we show mechanistically that CD44 is a direct functional target of miR-199a-3p in PCa cells. Moreover, miR-199a-3p also directly or indirectly targeted several additional mitogenic molecules, including c-MYC, cyclin D1 (CCND1) and EGFR. Taken together, our results demonstrate how the aberrant loss of a miRNA-mediated mechanism can lead to the expansion and tumorigenic activity of prostate CSCs, further supporting the development and implementation of miRNA mimics for cancer treatment.
LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate ...cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR
and AR
xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.
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