SV40 T/t-common polypeptide enhances the sensitivity of HER2-overexpressing human cancer cells to anticancer drugs cisplatin and doxorubicin

Cancer stem cells (CSCs) are involved in tumorigenesis and progression of prostate cancer (PCa). Conventional anticancer therapeutics failed to eradicate CSCs, which may eventually lead to the disease relapse and metastasis. Therefore, targeting prostate CSCs may be an ideal strategy to cure PCa. Genistein is a major isoflavone constituent of soybeans and soy products, which has been shown to exhibit potent anticancer effect on many cancers. We have previously reported that genistein can inhibit PCa cell invasion by reversing epithelial to mesenchymal transition, suggesting that genistein may be effective against metastatic PCa. In addition, we have recently demonstrated that PCa tumorsphere cells (TCs) possess CSC properties. Here, we found that tumorsphere formation and colony formation of Pca cells were noticeably suppressed in the presence of genistein. Pretreatment of PCa TCs with genistein also suppressed tumorigenicity in vivo. Additionally, genistein treatment inhibited tumor growth of PCa TCs. Further studies showed that genistein treatment not only led to the down-regulation of PCa CSC markers CD44 in vitro and in vivo, but also inhibited Hedgehog-Gli1 pathway, which may contribute to the anti-CSC effect of genistein in PCa TCs. Therefore, our findings demonstrated that genistein may be a dietary phytochemical with potential to target prostate CSCs.     

Tumorigenic potential of circulating prostate tumor cells

Circulating tumor cells (CTCs) have received intense scientific scrutiny because they travel in the bloodstream and are therefore well situated to mediate hematogenous metastasis. However, the potential of CTCs to actually form new tumors has not been tested. Popular methods of isolating CTCs are biased towards larger, more differentiated, non-viable cells, creating a barrier to testing their tumor forming potential. Without relying on cell size or the expression of differentiation markers, our objective was to isolate viable prostate CTCs from mice and humans and assay their ability to initiate new tumors. Therefore, blood was collected from transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and from human patients with metastatic castration-resistant prostate cancer (PCa). Gradient density centrifugation or red cell lysis was used to remove erythrocytes, and then leukocytes were depleted by magnetic separation using CD45 immunoaffinity beads. CTCs fractions from TRAMP mice and PCa patients were verified by immunocytochemical staining for cytokeratin 8 and EpCAM, and inoculated into immunodeficient mice. TRAMP tumor growth was monitored by palpation. Human tumor growth formation was monitored up to 8 months by ultrasensitive PSA assays performed on mouse serum. We found viable tumor cells present in the bloodstream that were successfully isolated from mice without relying on cell surface markers. Two out of nine immunodeficient mice inoculated with TRAMP CTCs developed massive liver metastases. CTCs were identified in blood from PCa patients but did not form tumors. In conclusion, viable CTCs can be isolated without relying on epithelial surface markers or size fractionation. TRAMP CTCs were tumorigenic, so CTCs isolated in this way contain viable tumor-initiating cells. Only two of nine hosts grew TRAMP tumors and none of the human CTCs formed tumors, which suggests that most CTCs have relatively low tumor-forming potential. Future studies should identify and target the highly tumorigenic cells.     

Tumor-initiating stem cells in liver cancer

It is widely accepted that cancer is a disease of stem cells. Definite evidence suggests that tumors harbor a small population of cancer stem cells (CSC) that both give rise to the bulk of the tumor and are tumorigenic in experimental models. Mounting evidence suggests that these cells are responsible for regrowth of a tumor following unsuccessful treatment and for the establishment of metastases. The concept of CSC has been demonstrated in several human cancers including leukemia, breast, prostate, lung, pancreas, colon and brain tumors. Recently, several studies have demonstrated that liver cancer, like other tumors, are derived from a small population of liver cancer stem/progenitor cells. Although still controversial, Liver CSC will likely become the most crucial target in the treatment of liver cancer, and a thorough understanding of its origin, molecular profile and particularly of how the Liver CSC differs from the normal stem cells, might allow it to be targeted selectively and eliminated, thus improving therapeutic outcome. In this review we will summarize the recent evidence for Liver CSC, the relationship between normal and liver stem cells, and the possibility that transformation of different cell types in the liver may generate different types of liver cancers.     

Systematic dissection of phenotypic,functional, and tumorigenic heterogeneity of human prostate cancer cells

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA^−/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR⁺PSA⁺, AR⁺PSA⁻, AR⁻PSA⁻, and AR⁻PSA⁺ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA^−/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA⁺ versus PSA^−/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA^−/lo PCa cells possess distinct epigenetic profiles. As the PSA^−/lo PCa cell population is heterogeneous, in the second part, we employ two PSA⁻ (Du145 and PC3) and two PSA⁺ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.     

Prostate cancer stem cells and their potential roles in metastasis

Most solid tumors have now been reported to contain stem cell-like cells called cancer stem cells (CSCs). These cells are endowed with high tumorigenic capacity and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. Since these self-renewing cancer cells may be the sole population to develop a primary tumor, it is predicted that CSCs may also represent the lethal seeds of metastasis, as supported by a flurry of recent studies on the relationship between CSCs and metastasis. Herein, we summarize current knowledge of stem/progenitor cells and CSCs, especially in the context of normal human prostate and prostate cancer. We further update the recently gained knowledge on the involvement of CSCs in metastasis. We also discuss the fundamental influence of tumor microenvironment on the manifestation of CSCs and metastasis. Finally, we discuss the clinical implication of CSC-based therapy.     

BRCA1 and EZH2 cooperate in regulation of prostate cancer stem cell phenotype

Prostate cancer (PCa) is the second most common malignancy and the sixth leading cause of cancer-related death among men worldwide. Prostate carcinogenesis is driven by the accumulation of genetic and epigenetic aberrations, which regulate cancer cell transition between a stem- and nonstem-cell state and accelerate tumor evolution. Elevated expression of enhancer of zeste homolog 2 (EZH2) histone methyltransferase, a core member of the polycomb repressive complex 2 (PRC2), results in cancer progression through histone methylation-driven tumor cells dedifferentiation. Previous studies demonstrated that tumor suppressor breast cancer 1 (BRCA1) is a negative regulator of PRC2-dependent H3K27 methylation. Our recent studies revealed that inhibition of EZH2-mediated histone methylation radiosensitizes prostate cancer stem cells (CSCs) population. However, the link between BRCA1 and EZH2 in regulation of prostate CSCs remains elusive. Present study demonstrated that BRCA1 and EZH2 are coregulated in patients’ tumors and PCa cell lines, and cooperate in regulation of CSC phenotype and properties. Knockdown of BRCA1 expression significantly increases the number and the size of tumor spheres. Inhibition of BRCA1 and EZH2 expression leads to an increase of aldehyde dehydrogenase (ALDH)-positive cell population that is, at least partially, attributed to the upregulation of ALDH1A3 protein. Treatment with a global histone methylation inhibitor 3-Deazaneplanocin A abrogates this regulation, downregulates BRCA1 and EZH2 expression and has an inhibitory effect on the tumorigenic properties of radioresistant PCa cells in vivo. We found that EZH2/BRCA1 signaling mechanisms play an important role in the maintenance of prostate CSC properties and may be a promising target for tumor treatment.     

Revisiting the concept of cancer stem cells in prostate cancer

The cancer stem cell (CSC) model proposes that cells within a tumor are organized in a hierarchical lineage relationship and display different tumorigenic potential, suggesting that effective therapeutics should target rare CSCs that sustain tumor malignancy. Here we review the current status of studies to identify CSCs in human prostate cancer as well as mouse models, with an emphasis on discussing different functional assays and their advantages and limitations. We also describe current controversies regarding the identification of prostate epithelial stem cells and cell types of origin for prostate cancer, and present potential resolutions of these issues. Although definitive evidence for the existence of CSCs in prostate cancer is still lacking, future directions pursuing the identification of tumor-initiating stem cells in the mouse may provide important advances in evaluating the CSC model for prostate cancer.     

Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo

Cancer may arise from a cancer stem/progenitor cell that shares characteristics with its normal counterpart. We report the reconstitution of the original human prostate cancer specimen from epithelial cell lines (termed HPET for human prostate epithelial/hTERT) derived from this sample. These tumors can be described in terms of Gleason score, a classification not applied to any of the transgenic mouse models currently developed to mimic human disease. Immunohistochemical and Western blot analyses indicate that they do not express androgen receptor or p63, similar to that reported for prostate stem cells. These cell lines also express embryonic stem markers (Oct4, Nanog, and Sox2) as well as early progenitor cell markers (CD44 and Nestin) in vitro. Clonally derived HPET cells reconstitute the original human tumor in vivo and differentiate into the three prostate epithelial cell lineages, indicating that they arise from a common stem/progenitor cell. Serial transplantation experiments reconstitute the tumors, suggesting that a fraction of parental or clonally derived HPET cells have self-renewal potential. Thus, this model may enhance our understanding of human tumor development and provide a mechanism for studying cancer stem/progenitor cells in differentiation, tumorigenesis, preclinical testing, and the development of drug resistance.     

The mammary progenitor marker CD61/beta3 integrin identifies cancer stem cells in mouse models of mammary tumorigenesis

The cells of origin and mechanisms that underpin tumor heterogeneity in breast cancer are poorly understood. Here, we have examined three mouse models of mammary tumorigenesis (MMTV-wnt-1, MMTV-neu, and p53(+/-)) for changes in their epithelial cell hierarchy during the preneoplastic and neoplastic stages of tumor progression. In preneoplastic tissue, only MMTV-wnt-1 mice showed a perturbation in their epithelial subpopulations. In addition to an expanded mammary stem cell pool, repopulating cells capable of yielding extensive mammary outgrowths in vivo were revealed in the committed luminal progenitor population. These findings indicate that wnt-1 activation induces the appearance of aberrant progenitor cells, and suggest that both mammary stem and progenitor cells can serve as the cellular targets of wnt-1-induced tumorigenesis. In tumors arising in MMTV-wnt-1 tumors, the luminal epithelial progenitor marker CD61/beta3 integrin identified a cancer stem cell (CSC) population that was highly enriched for tumorigenic capability relative to the CD61(-) subset. CD61 expression also defined a CSC subset in 50% of p53(+/-)-derived tumors. No CSCs, however, could be identified in the more homogeneous MMTV-neu/erbB2 model, suggesting an alternative model of tumorigenesis. Overall, our findings show the utility of the progenitor marker CD61 in the identification of CSCs that sustain specific mammary tumors.     

Annexin A1 is involved in the acquisition and maintenance of a stem cell-like/aggressive phenotype in prostate cancer cells with acquired resistance to zoledronic acid

In this study, we have characterized the role of annexin A1 (ANXA1) in the acquisition and maintenance of stem-like/aggressive features in prostate cancer (PCa) cells comparing zoledronic acid (ZA)-resistant DU145R80 with their parental DU145 cells. ANXA1 is over-expressed in DU145R80 cells and its down-regulation abolishes their resistance to ZA. Moreover, ANXA1 induces DU145 and DU145R80 invasiveness acting through formyl peptide receptors (FPRs). Also, ANXA1 knockdown is able to inhibit epithelial to mesenchymal transition (EMT) and to reduce focal adhesion kinase (FAK) and metalloproteases (MMP)-2/9 expression in PCa cells. DU145R80 show a cancer stem cell (CSC)-like signature with a high expression of CSC markers including CD44, CD133, NANOG, Snail, Oct4 and ALDH7A1 and CSC-related genes as STAT3. Interestingly, ANXA1 knockdown induces these cells to revert from a putative prostate CSC to a more differentiated phenotype resembling DU145 PCa cell signature. Similar results are obtained concerning some drug resistance-related genes such as ATP Binding Cassette G2 (ABCG2) and Lung Resistant Protein (LRP). Our study provides new insights on the role of ANXA1 protein in PCa onset and progression.     

4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance,proliferation and metastasis

Cancer death is a leading cause of global mortality. An estimated 14.1 million new cancer cases and 8.2 million cancer deaths occurred worldwide in 2012 alone. Cancer stem cells (CSCs) within tumors are essential for tumor metastasis and reoccurrence, the key factors of cancer lethality. Here we report that 4EGI-1, an inhibitor of the interaction between translation initiation factors eIF4E1 and eIF4G1 effectively inhibits breast CSCs through selectively reducing translation persistent in breast CSCs. Translation initiation factor eIF4E1 is significantly enhanced in breast CSCs in comparison to non-CSC breast cancer cells. 4EGI-1 presents increased cytotoxicity to breast CSCs compared to non-CSC breast cancer cells. 4EGI-1 promotes breast CSC differentiation and represses breast CSC induced tube-like structure formation of human umbilical vein endothelial cells (HUVECs). 4EGI-1 isomers suppress breast CSC tumorangiogenesis and tumor growth in vivo. In addition, 4EGI-1 decreases proliferation in and induces apoptosis into breast CSC tumor cells. Furthermore, 4EGI-1 selectively inhibits translation of mRNAs encoding NANOG, OCT4, CXCR4, c-MYC and VEGF in breast CSC tumors. Our study demonstrated that 4EGI-1 targets breast CSCs through selective inhibition of translation critical for breast CSCs, suggesting that selective translation initiation interference might be an avenue targeting CSCs within tumors.     

Role of stem cell derived exosomes in tumor biology

Exosomes are nano‐scale messengers loaded with bio‐molecular cargo of RNA, DNA, and Proteins. As a master regulator of cellular signaling, stem cell (both normal, and cancer stem cells) secreted exosome orchestrate various autocrine and paracrine functions which alter tumor micro‐environment, growth and progression. Exosomes secreted by one of the two important stem cell phenotypes in cancers a) Mesenchymal stem cells, and b) Cancer stem cells not only promote cancerous growth but also impart therapy resistance in cancer cells. In tumors, normal or mesenchymal stem cell (MSCs) derived exosomes (MSC‐exo) modulate tumor hallmarks by delivering unique miRNA species to neighboring cells and help in tumor progression. Apart from regulating tumor cell fate, MSC‐exo are also capable of inducing physiological processes, for example, angiogenesis, metastasis and so forth. Similarly, cancer stem cells (CSCs) derived exosomes (CSC‐exo) contain stemness‐specific proteins, self‐renewal promoting regulatory miRNAs, and survival factors. CSC‐exo specific cargo maintains tumor heterogeneity and alters tumor progression. In this review we critically discuss the importance of stem cell specific exosomes in tumor cell signaling pathways with their role in tumor biology.     

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.     

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem‐like cells

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem‐like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum‐AD and PLum‐AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53‐independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self‐renewal ability of the highly androgen‐resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.     

Cancer Stem Cells and Response to Therapy

The cancer stem cell (CSC) model states that cancers are organized in cellular hierarchies, which explains the functional heterogeneity often seen in tumors. Like normal tissue stem cells, CSCs are capable of self-renewal,either by symmetric or asymmetric cell division, and have the exclusive ability to reproduce malignant tumors indefinitely. Current systemic cancer therapies frequently fail to eliminate advanced tumors, which may be dueto their inability to effectively target CSC populations. It has been shown that embryonic pathways such as Wnt, Hedgehog, and Notch control self-renewal and cell fate decisions of stem cells and progenitor cells. These are evolutionary conserved pathways, involved in CSC maintenance. Targeting these pathways may be effective in eradicating CSCs and preventing chemotherapy or radiotherapy resistance.     

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.     

CXCR4 mediates the proliferation of glioblastoma progenitor cells

Increasing evidence points to a fundamental role for cancer stem cells (CSC) in the initiation and propagation of many tumors. As such, in the context of glioblastoma multiforme (GBM), the development of treatment strategies specifically targeted towards CSC-like populations may hold significant therapeutic promise. To this end, we now report that the cell surface chemokine receptor, CXCR4, a known mediator of cancer cell proliferation and invasion, is overexpressed in primary glioblastoma progenitor cells versus corresponding differentiated tumor cells. Furthermore, administration of CXCL12, the only known ligand for CXCR4, stimulates a specific and significant proliferative response in progenitors but not differentiated tumor cells. Taken together, these results implicate an important role for the CXCR4 signaling mechanism in glioma CSC biology and point to the therapeutic potential of targeting this pathway in patients with GBM.