Hangwen Li

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.

PC3 human prostate carcinoma cell holoclones contain self-renewing tumor-initiating cells

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.

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.

Drug-tolerant cancer cells show reduced tumor-initiating capacity: depletion of CD44 cells and evidence for epigenetic mechanisms.

Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44+ cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration, whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many “stemness” genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44+ tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms.

Sodium/Glucose Co-transporter 1 Expression Increases in Human Diseased Prostate

Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that takes up glucose into cells independent of the extracellular concentration of glucose. This transporter plays a critical role in maintaining glucose homeostasis at both physiological and pathological levels. The expression level of SGLT1 in normal and diseased human prostatic tissue has not been determined. We produced two rabbit polyclonal antibodies against human SGLT1, one each for immunohistochemical and Western blot analyses, and characterized the expression of SGLT1 in human prostate tissues: normal prostate (n=3), benign prostatic hyperplasia (BPH) (n=53), prostatic intraepithelial neoplasia (PIN) (n=9), and prostate cancer (PCa) (n=44). In normal prostate tissue, SGLT1 was weakly expressed exclusively in the epithelium. The transporter was significantly increased in the basal cells and stromal cells of BPH, increased in the epithelial cells of PIN, and frequently overexpressed in stromal cells and universally overexpressed in the tumor cells of PCa. The pattern of expression was shown as membranous/ cytoplasmic staining in low-grade cancer cells and nuclear envelope staining in high-grade cancer cells. The SGLT1-positive stromal cells of BPH and PCa tissues were negative for tenascin, a marker of reactive stromal cells. We concluded that SGLT1 is up-regulated in BPH and PCa, and SGLT1 may serve as a potential therapeutic target for treating these prostate disorders.

Prostate Cancer Stem/Progenitor Cells

The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. Here we provide evidence that both cultured prostate cancer cells and xenograft prostate tumors contain stem-like cells that can initiate serially transplantable tumors. We also present a hypothetical model of tumorigenic hierarchy of cancer cells in prostate tumors. Further studies on these important tumorigenic cells will help to understand prostate tumor biology and to develop novel diagnostic and prognostic markers and therapeutic agents.

Abstract C65: Drug-tolerant cancer cells show reduced tumor-initiating capacity: Depletion of CD44+ cells and evidence for epigenetic mechanisms

Abstract Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even nontumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44+ cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many ‘stemness’ genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44+ tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms. Citation Format: Xin Chen, Izabela Fokt, Stanislaw Skora, Waldemar Priebe, Yongyi Bi, Dean G. Tang, Hong Yan, Qiuping Zhang, Jichao Qin, Hangwen Li, Can Liu, Tammy Davis, Luis D. Coletta, Jim Klostergaard. Drug-tolerant cancer cells show reduced tumor-initiating capacity: Depletion of CD44+ cells and evidence for epigenetic mechanisms [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C65.

Abstract 1503: Cancer testis antigen SPANXB2 and prostate cancer progression: Human prostate stroma promotes SPANXB2 expression

A critical problem in prostate cancer is our inability to reliably distinguish indolent from aggressive disease. Recent evidence has implicated a class of genes, termed Cancer Testis Antigens (CTA) that uniquely expressed on malignant tissue, in cancer progression. CTA SPANXB2 was reported to be associated with aggressive and metastasis of melanoma and other cancers. The role of SPANXB2 in prostate cancer is still uncertain. In preliminary studies, we observed that the CTA SPANXB2 is up-regulated in mRNA level in metastatic prostate cancer xenograft models (PC3 cells injected in mouse dorsal prostate with Matrigel). Compared with other injection sites, mouse dorsal prostate enriched prostate stroma. Therefore, we further tested whether prostate stroma is the key factor to influence SPANXB2 expression and enhance the metastasis potential of prostate cancer cells. We validated this through an in vitro epithelial-stroma interaction models: GFP labeled PC3 cells were placed on the top of human prostate stromal cells (HPS-19I); two days later PC3-GFP cells were isolated through flow cytometry with high purity (> 99%). By real time PCR, the expression level of SPANXB2 in these purified PC3-GFP cells was found to be upregulated up to 10 folds. We also tested DU145 and LNCaP in this same model, and similar results were obtained. We further examined the migration and invasion ability of these cells and found that stromal exposed cells gained an enhanced ability to migrate and invade. Interestingly, we also observed that the stem cells marker, CD24 was highly up -regulated up to 6 folds in PC3 cells after co-culture with HPS-19I. In addition, we treated PC3 cells with TGF-β1 and TGF-β2, and noted that SPANXB2 and CD24 were significantly increased in PC3 cells after 24 hours treatment with TGF-β2. Our results reveal that i) the interaction between human prostate stromal cells and prostate cancer cells promote CTA-SPANXB2 and CD24 expression in prostate cancer cells, ii) these interactions further enhance the migration and invasion ability of prostate cancer cells and iii) TGF-β2 may regulate SPANXB expression relevant cells. These data provide rationale for studying SPANXB2 in stromal-epithelial interactions and may be potentially used to pinpoint druggable targets that drive metastasis and that distinguish indolent from aggressive prostate cancer. Keywords: Cancer testis antigen, SPANXB2, Prostate cancer, Metastasis, Stoma Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1503. doi:1538-7445.AM2012-1503

Abstract 476: Defining two populations of prostate cancer cells with distinct molecular, biological, and tumor-propagating properties

Prostate cancer (PCa) is a heterogeneous malignancy containing different types of tumor cells. The cellular basis underlying PCa cell heterogeneity and functional importance of different PCa cell populations in maintaining tumor homeostasis and mediating castration-resistant progression remain poorly understood. By whole-genome microarray analysis, time-lapse videomicroscopy, serial tumor transplantations, and other functional assays, here we report the distinct molecular, cell biological, and tumor-propagating properties of PCa cells that express high (i.e., PSA + ) and low (PSA −/lo ) levels of PSA. PSA −/lo PCa cells are relatively quiescent and resistant to multiple stresses, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity in intact male mice. They preferentially express stem cell-associated genes and epigenetic profiles and can generate PSA + cells by either asymmetric or symmetric cell division. Of great clinic significance, PSA −/lo PCa cells can initiate robust tumor development in fully castrated hosts and survive experimental androgen-deprivation therapy. In contrast, PSA + PCa cells, despite being highly tumorigenic in androgen-proficient hosts, possess more limited tumor-propagating capacity, mainly undergo symmetric division, and are sensitive to castration. Our data together suggest that the two populations of PCa cells appear to play differential roles in tumor maintenance and PSA −/lo cells may represent an important source of castration-resistant PCa cells. Our findings have important implications in understanding PCa cell heterogeneity, tumor response to the mainstay antiandrogen therapies, and emergence of castration-resistant PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 476. doi:10.1158/1538-7445.AM2011-476