Metformin selectively affects human glioblastoma tumor-initiating cell viability

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.     

Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

Fibroblast growth factor 10 (FGF10) is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multipotent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III, and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry, and Western blot analysis. Co-cultured stem cells began to express uroplakin II, III, and cytokeratin 8. Targeted FGF10 gene knockdown from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the Mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.     

Sustained Levels of FGF2 Maintain Undifferentiated Stem Cell Cultures with Biweekly Feeding

An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.     

A Synthetic Triterpenoid CDDO-Im Inhibits Tumorsphere Formation by Regulating Stem Cell Signaling Pathways in Triple-Negative Breast Cancer

Triple-negative breast cancer is associated with poor prognosis because of a high rate of tumor recurrence and metastasis. Previous studies demonstrated that the synthetic triterpenoid, CDDO-Imidazolide (CDDO-Im) induced cell cycle arrest and apoptosis in triple-negative breast cancer. Since a small subpopulation of cancer stem cells has been suggested to be responsible for drug resistance and metastasis of tumors, our present study determined whether the effects of CDDO-Im in triple-negative breast cancer are due to the inhibition of a cancer stem cell subpopulation. CDDO-Im treatment markedly induced cell cycle arrest at G2/M-phase and apoptosis in the triple-negative breast cancer cell lines, SUM159 and MDA-MB-231. Because SUM159 cells were more sensitive to CDDO-Im than MDA-MB-231 cells, the effects of CDDO-Im on the cancer stem cell subpopulation were further investigated in SUM159 cells. SUM159 cells formed tumorspheres in culture, and the cancer stem cell subpopulation, CD24−/EpCAM+ cells, was markedly enriched in SUM159 tumorspheres. The CD24−/EpCAM+ cells in SUM159 tumorspheres were significantly inhibited by CDDO-Im treatment. CDDO-Im also significantly decreased sphere forming efficiency and tumorsphere size in both primary and secondary sphere cultures. PCR array of stem cell signaling genes showed that expression levels of many key molecules in the stem cell signaling pathways, such as Notch, TGF-β/Smad, Hedgehog and Wnt, were significantly down-regulated by CDDO-Im in SUM159 tumorspheres. Protein levels of Notch receptors (c-Notch1, Notch1 and Notch3), TGF-β/Smad (pSmad2/3) and Hedgehog downstream effectors (GLI1) also were markedly reduced by CDDO-Im. In conclusion, the present study demonstrates that the synthetic triterpenoid, CDDO-Im, is a potent anti-cancer agent against triple-negative breast cancer cells by targeting the cancer stem cell subpopulation.     

Fibroblast Growth Factor Inhibits Expression of Neural Markers in Cultures of Zebrafish Early Embryo Cells

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures. Received March 10, 2000; accepted May 10, 2000.     

Wnt Pathway Activity in Breast Cancer Sub-Types and Stem-Like Cells

Introduction

Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.

Methods

We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.

Results

Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.

Conclusions

Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.     

FGF10 maintains stem cell compartment in developing mouse incisors

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.     

The cancer stem cell theory: is it correct?

The cancer stem cell hypothesis posits that tumor growth is driven by a rare subpopulation of cells, designated cancer stem cells (CSC). Studies supporting this theory are based in large part on xenotransplantation experiments wherein human cancer cells are grown in immunocompromised mice and only CSC, often constituting less than 1% of the malignancy, generate tumors. Herein, we show that all colonies derived from randomly chosen single cells in mouse lung and breast cancer cell lines form tumors following allografting histocompatible mice. Our study suggests that the majority of malignant cells rather than CSC can sustain tumors and that the cancer stem cell theory must be reevaluated.     

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions

Background

The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133⁺/α₂β₁ ^hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133^-/α₂β₁ ^low) counterparts, resulting in an informative cancer stem cell gene-expression signature.

Results

Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches.

Conclusion

We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.     

CDl33＋／ABCG2＋的人肺癌多耐药性肿瘤细胞亚群的分离及鉴定

肿瘤干细胞的多耐药性是导致肿瘤化疗失败的重要因素之一。因此,鉴定和明确耐药性肺癌干细胞亚群（cancer stem-cell subpopulations）的特征和机制是当前的热点。该研究从肺癌病人的肿瘤组织中分离出一个高表达CDl33和ABCG2蛋白的细胞亚群。发现该CDl33＋／ABCG2＋肺癌细胞高表达干细胞的生物标志,如：Nanog、Oct4、Sox2、Nestin、CD44、CDll7、CDl33和ABCG2等。不仅如此,这群细胞在体外具有高增殖性和高侵袈性,对于多种常用的化疗药物（如：顺铂和吉西他滨等）都具有耐受性。而且,少量的CDl33＋／ABCG2＋肺癌细胞即可以在免疫缺陷小鼠体内形成肿瘤。为了研究耐药性机制,我们检测了CDl33＋／ABCG2＋中ABCG2基因启动子区域的cpG岛（cpGislands）DNA甲基化修饰状态。实验结果表日月,该细胞中,ABCG2基因启动子区域的cpG岛处于去甲基化修饰状态。综上所述,作者成功地从肺癌病人肿瘤组织中分离、富集得到了CDl33＋／ABCG2＋细胞亚群,并利用该群细胞在体外建立了肿瘤耐药性研究模型。对于肺癌CDl33＋／ABCG2＋细胞的研究可为开发肺癌个体化治疗和新型化疗药物的设计和研发提供理论依据．     

Functional analyses of the cancer stem cell-like properties of human endometrial tumor initiating cells

Recent data suggest that rare stem cell populations with the capacity to self renew and drive tumor formation are a feature of solid tumors. Several investigators have identified putative stem cells from solid tumors and cancer cell lines following isolation of a side population (SP) defined by dye exclusion. We investigated this parameter in our efforts to identify an endometrial cancer (EnCa) stem cell population. Multiple EnCa cell lines were assessed and verapamil sensitive SP and non-SP cells were isolated from two human EnCa cell lines. The functional significance of the SP and non-SP derived from AN3CA was evaluated in vitro and in vivo. SP cells proliferated at a significantly slower rate than the non-SP fraction, and a larger proportion of the SP cells were in G1 phase of the cell cycle as compared to the non-SP fraction. The SP fraction was more resistant to the chemotherapeutic agent paclitaxel. The SP comprised ~0.02% of the initial AN3CA cell population and this proportion of SP cells was maintained within the larger heterogeneous population following repeated passages of purified SP cells. These findings suggest that SP cells derived from the AN3CA cell line have the stem cell properties of low proliferative activity, chemoresistance, and self-renewal. We also tested relative tumor formation activity of the SP and non-SP fractions. Only the SP fraction was tumorigenic. Additionally, we identified SP fractions in primary EnCa. Together these results are consistent with the hypothesis that EnCa contain a subpopulation of tumor initiating cells with stem like properties.     

Matrix metalloproteinase-10 promotes Kras-mediated bronchio-alveolar stem cell expansion and lung cancer formation

Matrix metalloproteinase 10 (MMP-10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (Mmp10(-/-)) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.     

Conditioned media from human pluripotent stem cell-derived cardiomyocytes inhibit the growth and migration of lung cancer cells

Conditioned media (CM) from various cell types contain significant levels of paracrine factors. Recently, therapeutic properties of CM derived from stem cells have been revealed. Based on the fact that heart cancer is extremely rarely, we hypothesized that the CM obtained from human pluripotent stem cell-derived cardiomyocytes might inhibit cancer cell growth and survival. To this end, lung cancer cell line A549 along with human foreskin fibroblasts (HFF) were treated with serial concentrations of cardiomyocyte CM (CCM) or fibroblast CM (FCM). We found that CCM markedly reduced the viability of lung cancer cells, while FCM did not compromise the viability of neither cancer cells nor HFF cells. Furthermore, we determined an optimized CCM concentration, 30 mg/mL, at which the growth, clonogenicity, and migration of A549 and Calu6 lung cancer cell lines were substantially impaired, whereas FCM did not influence these properties. Moreover, lung cancer cells exhibited cell cycle regulation upon treatment with CCM and the rate of apoptosis was markedly increased by cardiomyocyte CM in both lung cancer cell lines tested. Finally, in response to CCM treatment, A549 and Calu6 cells expressed lower levels of antiapoptotic and stemness genes, but higher levels of proapoptotic genes. In conclusion, this study provides cellular and molecular evidence for the antitumor ability of secretome obtained from stem cell-derived cardiomyocytes.     

The Tumor Growth Paradox and Immune System-Mediated Selection for Cancer Stem Cells

Cancer stem cells (CSCs) drive tumor progression, metastases, treatment resistance, and recurrence. Understanding CSC kinetics and interaction with their nonstem counterparts (called tumor cells, TCs) is still sparse, and theoretical models may help elucidate their role in cancer progression. Here, we develop a mathematical model of a heterogeneous population of CSCs and TCs to investigate the proposed “tumor growth paradox”—accelerated tumor growth with increased cell death as, for example, can result from the immune response or from cytotoxic treatments. We show that if TCs compete with CSCs for space and resources they can prevent CSC division and drive tumors into dormancy. Conversely, if this competition is reduced by death of TCs, the result is a liberation of CSCs and their renewed proliferation, which ultimately results in larger tumor growth. Here, we present an analytical proof for this tumor growth paradox. We show how numerical results from the model also further our understanding of how the fraction of cancer stem cells in a solid tumor evolves. Using the immune system as an example, we show that induction of cell death can lead to selection of cancer stem cells from a minor subpopulation to become the dominant and asymptotically the entire cell type in tumors.     

A Stable Chimeric Fibroblast Growth Factor (FGF) Can Successfully Replace Basic FGF in Human Pluripotent Stem Cell Culture

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.