Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.     

microRNA in the control of stem-like phenotype of cancer cells

Current therapies against metastatic tumors are still ineffective. Cancer stem cells — a small subset of cells inside the tumor that possesses a self-renewal capacity — might be responsible for the recurrence of the tumor after anti-cancer therapies. Their immortality and unique drug resistance impede their eradication during therapy. The ‘stemness’ of these cells is controlled by microRNAs. These molecules possess the ability to downregulate gene expression by binding to the target mRNA. It turns out that microRNAs control the expression of approximately 60% of the genes in human cells. MicroRNA aberrant expression can lead to cancer development and progression. Therefore, recent research has focused on unraveling the role of microRNA in maintaining a stem-like phenotype in malignant tumors and cancer stem cells. This review summarizes our current knowledge about microRNAs that control the self-renewal capacity of cancer stem cells and indicates the importance of profound research aimed at developing efficient miRNA-targeted therapies.     

Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods

The cancer stem cell (CSC) hypothesis has provided insights into the initiation and recurrence of brain tumor. Specific identification and targeted elimination of these CSCs within the tumor mass represents a promising therapeutic strategy for refractory brain tumors. In this study, we attempted to identify CSCs in the rat C6 glioma cell line by three different identification methods. It is interesting to note that single-cell clonal analysis showed most C6 cells are cancer stem-like cells with characteristics of self-renewal, multilineage differentiation potentials in vitro, and tumorigenic capacity in vivo. It is surprising to note that CD133 failed to identify the total cancer stem-like cell population in the C6 line, since both CD133 (+) and CD133 (-) C6 cells have cancer stem-like cell fractions. Moreover, Hoechst 33342 staining, which is used in flow cytometry to isolate the side population (SP), was found to be harmful to C6 cells. Therefore, CD133 (-) and non-SP C6 cells may also harbor cancer stem-like cells. These results imply the limitation of using current identification methods in C6 line and underscore the importance of defining the genetic and molecular basis of CSCs.     

The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype

Glioblastomas are highly lethal cancers that contain cellular hierarchies with self-renewing cancer stem cells that can propagate tumors in secondary transplant assays. The potential significance of cancer stem cells in cancer biology has been demonstrated by studies showing contributions to therapeutic resistance, angiogenesis, and tumor dispersal. We recently reported that physiologic oxygen levels differentially induce hypoxia inducible factor-2α (HIF2α) levels in cancer stem cells. HIF1α functioned in proliferation and survival of all cancer cells but also was activated in normal neural progenitors suggesting a potentially restricted therapeutic index while HIF2α was essential in only in cancer stem cells and was not expressed by normal neural progenitors demonstrating HIF2α is a cancer stem cell specific target. We now extend these studies to examine the role of hypoxia in regulating tumor cell plasticity. We find that hypoxia promotes the self-renewal capability of the stem and non-stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation as well as upregulation of important stem cell factors, such as OCT4, NANOG, and c-MYC. The importance of HIF2α was further supported as forced expression of non-degradable HIF2α induced a cancer stem cell marker and augmented the tumorigenic potential of the non-stem population. This novel finding may indicate a specific role of HIF2α in promoting glioma tumorigenesis. The unexpected plasticity of the non-stem glioma population and the stem-like phenotype emphasizes the importance of developing therapeutic strategies targeting the microenvironmental influence on the tumor in addition to cancer stem cells.     

Induction of cancer cell stemness by chemotherapy

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.     

Isolation of colorectal cancer stem-like cells

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.     

Epithelial to Mesenchymal Transition Is Mechanistically Linked with Stem Cell Signatures in Prostate Cancer Cells

Background

Current management of patients diagnosed with prostate cancer (PCa) is very effective; however, tumor recurrence with Castrate Resistant Prostate Cancer (CRPC) and subsequent metastasis lead to poor survival outcome, suggesting that there is a dire need for novel mechanistic understanding of tumor recurrence, which would be critical for designing novel therapies. The recurrence and the metastasis of PCa are tightly linked with the biology of prostate cancer stem cells or cancer-initiating cells that is reminiscent of the acquisition of Epithelial to Mesenchymal Transition (EMT) phenotype. Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells.

Methodology/Principal Findings

In this study, we found that PCa cells with EMT phenotype displayed stem-like cell features characterized by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1, consistent with enhanced clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice, which was associated with decreased expression of miR-200 and/or let-7 family. Reversal of EMT by re-expression of miR-200 inhibited prostasphere-forming ability of EMT-type cells and reduced the expression of Notch1 and Lin28B. Down-regulation of Lin28B increased let-7 expression, which was consistent with repressed self-renewal capability.

Conclusions/Significance

These results suggest that miR-200 played a pivotal role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in PCa. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to Mesenchymal-Epithelial Transition (MET) phenotype using novel agents would be useful for the prevention of tumor recurrence especially by eliminating those cells that are the “Root Cause” of tumor development and recurrence.     

Brain Tumor Stem Cells

Primary malignant brain cancer, one of the most deadly diseases, has a high rate of recurrence after treatment. Studies in the past several years have led to the hypothesis that the root of the recurrence may be brain tumor stem cells (BTSCs), stem-like subpopulation of cells that are responsible for propagating the tumor. Current treatments combining surgery and chemoradiotherapy could not eliminate BTSCs because these cells are highly infiltrative and possess several properties that can reduce the damages caused by radiation or anti-cancer drugs. BTSCs are similar to NSCs in molecular marker expression and multi-lineage differentiation potential. Genetic analyses of Drosophila CNS neoplasia, mouse glioma models, and human glioma tissues have revealed a link between increased NSC self-renewal and brain tumorigenesis. Furthermore, data from various rodent models of malignant brain tumors have provided compelling evidence that multipotent NSCs and lineage-restricted neural progenitor cells (NPCs) could be the cell origin of brain tumors. Thus, the first event of brain tumorigenesis might be the occurrence of oncogenic mutations in the stem cell self-renewal pathway in an NSC or NPC. These mutations convert the NSC or NPC to a BTSC, which then initiates and sustains the growth of the tumor. The self-renewal of BTSCs is controlled by several evolutionarily conserved signaling pathways and requires an intact vascular niche. Targeting these pathways and the vascular niche could be a principle in novel brain tumor therapies aimed to eliminate BTSCs.     

Stem cell: balancing aging and cancer

Stem cells are defined by their self-renewing capacity and the ability to differentiate into one or more cell types. Stem cells can be divided, depending on their origin, into embryonic or adult. Embryonic stem cells derive from early stage embryos and can give rise to cells from all three germ layers. Adult stem cells, first identified in hematopoietic tissue, reside in a variety of adult tissues. Under normal physiologic conditions, adult stem cells are capable of differentiating into the limited cell types that comprise the particular tissue or organ. Adult stem cells are responsible for tissue renewal and exhaustion of their replicative capacity may contribute to tissue aging. Loss of unlimited proliferative capacity in some of the adult stem cells and/or their progenitors may have involved the evolutionary trade-off: senescence prevents cancer but may promote aging. Embryonic stem cells exhibit unlimited self-renewal capacity due to the expression of telomerase. Although they possess some cancer cell characteristics, embryonic stem cells exhibit a remarkable resistance to genomic instability and malignant transformation. Understanding the tumor suppressive mechanisms employed by embryonic stem cells may contribute to the development of novel cancer treatments and safe cell-based therapies for age-related diseases.     

Isolation and characterization of stem-like cells from a human ovarian cancer cell line

Increasing evidence supports the existence of a subpopulation of cancer cells capable of self-renewal and differentiation into diverse cell lineages. These cancer stem-like or cancer-initiating cells (CICs) also demonstrate resistance to chemo- and radiotherapy and may function as a primary source of cancer recurrence. We report here on the isolation and in vitro propagation of multicellular ovarian cancer spheroids from a well-established ovarian cancer cell line (OVCAR-3). The spheroid-derived cells (SDCs) display self-renewal potential, the ability to produce differentiated progeny, and increased expression of genes previously associated with CICs. SDCs also demonstrate higher invasiveness, migration potential, and enhanced resistance to standard anticancer agents relative to parental OVCAR-3 cells. Furthermore, SDCs display up-regulation of genes associated with epithelial-to-mesenchymal transition (EMT), anticancer drug resistance and/or decreased susceptibility to apoptosis, as well as, down-regulation of genes typically associated with the epithelial cell phenotype and pro-apoptotic genes. Pathway and biological process enrichment analyses indicate significant differences between the SDCs and precursor OVCAR-3 cells in TGF-beta-dependent induction of EMT, regulation of lipid metabolism, NOTCH and Hedgehog signaling. Collectively, our results indicate that these SDCs will be a useful model for the study of ovarian CICs and for the development of novel CIC-targeted therapies.     

上皮间充质转化促进肿瘤细胞干性获得的研究进展

上皮间充质转化是上皮细胞丢失细胞极性和细胞黏附,而获得间充质细胞迁移和侵袭特性的生物学过程.肿瘤干细胞是存在于肿瘤中具有自我更新和异质性分化能力的一小群细胞,在肿瘤的发生发展过程中起重要的作用.上皮间充质转化(EMT)与肿瘤的转移密切相关,而近几年的研究表明,EMT也可以促进肿瘤细胞获得干细胞的特性,因此使肿瘤治疗更困难,本文对EMT促肿瘤干细胞形成机制及其对临床治疗意义的研究进展作一综述.     

Determination of somatic and cancer stem cell self-renewing symmetric division rate using sphere assays

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cell's resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence.     

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.     

Identification of Cancer Stem-Like Side Population Cells in Purified Primary Cultured Human Laryngeal Squamous Cell Carcinoma Epithelia

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.     

The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.     

Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53^−/− astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53^−/− astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.     

Metabolic regulation of cancer cell side population by glucose through activation of the Akt pathway

Side population (SP) cells within tumors are a small fraction of cancer cells with stem-like properties that can be identified by flow cytometry analysis based on their high ability to export certain compounds such as Hoechst 33342 and chemotherapeutic agents. The existence of stem-like SP cells in tumors is considered as a key factor contributing to drug resistance, and presents a major challenge in cancer treatment. Although it has been recognized for some time that tumor tissue niches may significantly affect cancer stem cells (CSCs), the role of key nutrients such as glucose in the microenvironment in affecting stem-like cancer cells and their metabolism largely remains elusive. Here we report that SP cells isolated from human cancer cells exhibit higher glycolytic activity compared to non-SP cells. Glucose in the culture environment exerts a profound effect on SP cells as evidenced by its ability to induce a significant increase in the percentage of SP cells in the overall cancer cell population, and glucose starvation causes a rapid depletion of SP cells. Mechanistically, glucose upregulates the SP fraction through ATP-mediated suppression of AMPK and activation of the Akt pathway, leading to elevated expression of the ATP-dependent efflux pump ABCG2. Importantly, inhibition of glycolysis by 3-BrOP significantly reduces SP cells in vitro and impairs their ability to form tumors in vivo. Our data suggest that glucose is an essential regulator of SP cells mediated by the Akt pathway, and targeting glycolysis may eliminate the drug-resistant SP cells with potentially significant benefits in cancer treatment.     

Isolation of Cancer Stem Like Cells from Human Adenosquamous Carcinoma of the Lung Supports a Monoclonal Origin from a Multipotential Tissue Stem Cell

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.     

Therapeutic implications of cancer stem cells

Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.