Invasion Of Cancer Stem Cells Research Articles

Crosstalk between M2 macrophages and glioma stem cells.

Given its extremely poor prognosis, there is a pressing need for an improved understanding of the biology of glioblastoma multiforme (GBM), including the roles of tumor subpopulations that may contribute to their growth rate and therapy resistance. The most malignant phenotypes of GBM have been ascribed to the presence of subpopulations of cancer stem cells (CSCs), which are resistant to chemotherapeutic drugs and ionizing radiation and which promote invasiveness and metastasis. The mechanisms by which the CSC state is obtained and by which it promotes tumor maintenance are only beginning to emerge. We hypothesize that M2 polarized macrophages may affect CSC phenotypes via cell-cell communication. We investigated the interplay between glioma CSCs and macrophages via co-culture. The invasiveness of CSCs in the absence and presence of macrophages was assessed using collagen degradation and Transwell migration assays. The role of STAT3 as a CSC phenotypic mediator was assessed using siRNA-mediated gene silencing. We found that the levels of a M2 macrophage-specific secreted cytokine, TGF-β1, were elevated in the presence of CSCs, regardless of whether the cells were plated as contacting or non-contacting co-cultures. In addition, we found that the co-culture resulted in enhanced expression of M2 markers in macrophages that were previously polarized to the M1 phenotype. siRNA-mediated STAT3 silencing was found to reduce the chemo-responsiveness and migratory abilities of the CSCs. Combination treatment of STAT3 siRNA and DNA alkylating agents was found to further abrogate CSC functions. Our data indicate that the co-culture of CSCs and macrophages results in bi-directional signaling that alters the phenotypes of both cell types. These results provide an explanation for recently observed effects of macrophages on GBM tumor cell growth, motility and therapeutic resistance, and suggest potential therapeutic strategies to disrupt the CSC phenotype by impairing its communication with macrophages.

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumor formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signaling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signaling, and that drugs designed to target this site may be potent anti-cancer agents.

Increased cancer stem cell invasion is mediated by myosin IIB and nuclear translocation.

Despite many advances in the treatment of breast cancer, it remains one of the leading causes of death among women. One hurdle for effective therapy is the treatment of the highly invasive and tumorigenic subpopulation of tumors called cancer stem cells (CSCs). CSCs, when stimulated with EGF, migrate through a physiological 3D collagen matrix at a higher velocity than non-stem cancer cells (non-SCCs). This increased invasion is due, in part, by an enhanced nuclear translocation ability of CSCs. We observed no difference between CSC and non-SCC in cellular migration rates on a 2D surface. Furthermore, during transwell migration using large diameter transwell pores, both CSC and non-SCC populations migrated with similar efficiency. However, when challenged with more restrictive transwells, CSCs were dramatically more capable of transwell migration. These results implicate nuclear translocation as a major rate limiting factor for CSC dissemination. We further show that non-muscle myosin IIB is critical for this enhanced nuclear translocation and the ability for cancer stem cells to efficiently migrate through restrictive 3D environments. These studies suggest that cytoskeletal elements upregulated in CSCs, such as myosin IIB, may be valuable targets for intervention in cancer stem cell dispersal from tumors.

Abstract 3117: Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy

Abstract Colorectal cancer is one of the most common cancers and frequently metastasizes to the liver. TGF-β signaling has been shown to regulate cancer metastasis by promoting epithelial to mesenchymal transition and cancer stem cell (CSC) phenotypes. Smad2 is a key element downstream of TGF-β. In this study, we showed that hsa-hsa-miR-140-5p-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in colorectal cancer cell lines decreases Smad2 expression level, which leads to decreased level of cell invasion and proliferation, as well as increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy, an essential process for cancer cell survival and chemoresistance. We have systematically identified the targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished the tumor formation as well as metastasis in vivo by using subcutaneous xenografts and tail vein injection of CSCs in NOD/SCID mice. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues, quantified by qRT-PCR. The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in colorectal cancer progression and metastasis. As a result, it may have potential as a novel therapeutic molecule to treat colorectal cancer. Citation Format: Haiyan Zhai, Andrew Fesler, Jingfang Ju. Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2015-3117

Role of autophagy in the maintenance and function of cancer stem cells.

Recent advances in experimental technologies and cancer models have made possible to demonstrate that the tumor is a dynamic system comprising heterogeneous populations of cancer cells organized in a hierarchical fashion with cancer stem cells (CSCs) at the apex. CSCs are immature cells characterized by self-renewal property and long-term repopulation potential. CSCs have been causally linked to cancer initiation, propagation, spreading, recurrence and relapse as well as to resistance to anticancer therapy. A growing body of evidence suggests that the function and physiology of CSCs may be influenced by genetic/epigenetic factors and tumor environment. In this context, macroautophagy is a lysosomal degradative process (herein referred to as autophagy) critical for the adaptive response to stress and the preservation of cellular and tissue homeostasis in all eukaryotes that may have a crucial role of in the origin, maintenance and invasiveness of CSCs. The activation of the autophagic machinery is also considered as an adaptive response of CSCs to perturbation of tumor microenvironment, caused for instance by anticancer therapy. Nevertheless, compelling preclinical and clinical evidence on the cytoprotective role of autophagy for CSCs is still missing. Here, we summarize the results on the contribution of autophagy in CSCs and how it impacts tumorigenesis and tumor progression. We also discuss the therapeutical potential of the modulation of autophagy as a means to eradicate CSCs.

Increased fucosylation has a pivotal role in invasive and metastatic properties of head and neck cancer stem cells.

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with high mortality rates. Major challenges for OSCC management include development of resistance to therapy and early formation of distant metastases. Cancer stem cells (CSCs) have emerged as important players in both pathologic mechanisms. Increased fucosylation activity and increased expression of fucosylated polysaccharides, such as Sialyl Lewis X (SLex), are associated with invasion and metastasis. However, the role of fucosylation in CSCs has not been elucidated yet. We used the spheroid culture technique to obtain a CSC-enriched population and compared orospheres with adherent cells. We found that orospheres expressed markers of CSCs and metastasis at higher levels, were more invasive and tumorigenic, and were more resistant to cisplatin/radiation than adherent counterparts. We found fucosyltransferases FUT3 and FUT6 highly up-regulated, increased SLex expression and increased adhesion by shear flow assays in orospheres. Inhibition of fucosylation negatively affected orospheres formation and invasion of oral CSCs. These results confirm that orospheres are enriched in CSCs and that fucosylation is of paramount importance for CSC invasion. In addition, SLex may play a key role in CSC metastasis. Thus, inhibition of fucosylation may be used to block CSCs and metastatic spread.

Abstract 217: Salinomycin have antiproliferative and apoptotic effects on ovarian cancer stem-like cell

Abstract OBJECTIVE: Cancer stem cells (CSCs) represent a subpopulation of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance, drug resistance and metastasis of tumors. CSCs involved in drug resistance and relapse of cancers can significantly affect ovarian cancer therapy. The antibiotics salinomycin has recently been shown to be a potent compound to deplete chemoresistant cells in adenocarcinoma. The objective of the present study was to evaluate the effect of salinomycin on ovarian CSC whereby salinomycin mono-and combination treatment with paclitaxol regimend were analyzed. METHODS: The CD44+CD117+CSCs were isolated from the primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer by using immune magnetic-activated cell sorting system. To evaluate the effect of salinomycin on ovarian CSC, cells were treated in single and combined treatment. The expression was Nanog, Oct3/4, Sox2 and ABCG2 mRNA was determined by RT-PCR and protein expression was detected by Western blot analysis. The cell viability assay, gelatin zymography and apoptosis assay were applied to evaluate the effects of salinomycin compared with parental tumor cells and CSCs. RESULTS: The combination of salinomycin with paclitaxel (PTX) was enhanced change cell viability or activating apoptosis in CD44+CD117+ cells, whereas the salinomycin monotreatment did not cause significant changes. Salinomycin treatment reduced stemness gene expression and suppressed invasion of CSCs. CONCLUSIONS: Based on our findings, we concluded that anti-cancer effect of salinomycin with PTX reduced stemness and induced apoptosis in ovarian cancer and cancer stem cells. Citation Format: So-Jin Shin, Jin-Young Kim, Hyun-Gyo Lee, Eun-Ji Nam, Chi-Heum Cho. Salinomycin have antiproliferative and apoptotic effects on ovarian cancer stem-like cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2014-217

Oncogenic miR-20a and miR-106a enhance the invasiveness of human glioma stem cells by directly targeting TIMP-2

Emerging evidence has shown that cancer stem cells (CSCs) are the cellular determinants to promote cancer invasion and metastasis. However, the mechanism underlying CSC invasion remains unknown. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression, and their expressions are often dysregulated in cancers. In the present study, we demonstrated that two functionally related microRNAs, miR-20a and -106a (miR-20a/106a), were capable of enhancing the invasiveness of CD133(+) glioma stem cells (GSCs) isolated from both glioblastoma cell line U87 and primary human glioma specimens. We found that the level of miR-20a/106a in GSCs was significantly higher than that in the committed CD133(-) glioma cells, and correlated with the invasive capability of GSCs. By bioinformatic analysis, we identified tissue inhibitor of metalloproteinases-2 (TIMP-2) as one of the miR-20a/106a-targeted genes. TIMP-2 level correlated inversely with miR-20/106 expression. Directly targeting by miR-20a/106a on 3'-untranslation region (3'-UTR) of TIMP-2 mRNA was confirmed by 3'-UTR dual-luciferase reporter assay. Knockdown of miR-20a/106a in GSCs increased endogenous TIMP-2 protein abundance, thereby inhibiting GSC invasion. We also found that Nordy, a synthetic lipoxygenase inhibitor, inhibited GSC invasiveness by elevating the expression of TIMP-2 via downregulation of miR-20a/106a. Our results indicate that miR-20a/106a has a key role in GSC invasion and may serve as targets for treatment of glioblastoma.

MicroRNA-21 regulates the migration and invasion of a stem-like population in hepatocellular carcinoma

Due to invasion and intrahepatic metastasis, the prognosis for patients with hepatocellular carcinoma (HCC) is poor. However, the mechanisms underlying these processes of HCC remain unclear. Cancer stem cells may be involved in early systemic dissemination and metastasis formation and side population (SP) cells isolated from diverse cancer cells possess stem cell-like properties. However, the mechanisms involved in migration and invasion of cancer stem cells are not well understood. In this study, we identified and isolated populations of SP cells from HCC cell lines using flow cyto-metry. SP cells showed higher levels of migration and invasion capability. Higher expression of miR-21 was observed in SP cells. Silencing of miR-21 led to a reduction in the migration and invasion of these cells and overexpression of miR-21 can increase in cell migration and invasion. Overexpression of miR-21 did not cause degradation of PTEN or RECK or PDCD4 mRNA but drastically inhibited its protein expression. Consistent with these results, silencing miR-21 increased the levels of PTEN, RECK and PDCD4 protein, respectively. The role of silencing miR-21 was partially attenuated by silencing of PTEN or RECK or PDCD4 mRNA. The results of this study revealed the aberrant expression of miR-21 in SP cells and showed that miR-21 regulates the expression of multiple target proteins that are associated with tumor dissemination. MiR-21 is a pro-metastatic miRNA in SP cells and raises the possibility that therapy of HCC may be improved by pharmaceutical strategies directed towards miR-21.

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.

Expression of miR-125b in the new, highly invasive glioma stem cell and progenitor cell line SU3

MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells. However, the relationship between miRNA and glioma stem cells is still elusive. This study was designed to elucidate this potential relationship. We established a highly invasive glioma stem cell and progenitor (GSCP) cell line SU3. SU3 cell suspensions were injected into nude mice brains in situ, and the invasiveness of graft tumors was analyzed using hematoxylin and eosin staining as well as immunohistochemistry. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of miR-125b in SU3 and other cells. In vitro, SU3 cells expressed CD133 and nestin as well as differentiation markers glial fibrillary acidic protein (GFAP) and β-tubulin III, which were consistent with the characteristics of glioma stem cells. Scratch assays indicated that the migration ability of SU3 cells was stronger than that of U251 stem cells (U251s). In vivo, SU3 cells invaded into each part of the mouse brain from the caudate nucleus in a diffuse pattern and highly expressed invasive and proliferative cell markers matrix metalloprotease 2 (MMP2), MMP9, and Ki-67. Real-time PCR results revealed that the levels of miR-125b and MMP9 were significantly higher in SU3 and SU2, also a highly invasive GSCP cell line we established before, than in U251s. High expression of miR-125b both in newly established GSCPs, SU3, and long-term cultured GSCPs, SU2 suggests that miR-125b exhibits oncogene-like behavior. This behavior should be considered in further studies of miR-125b in cancer stem cells. Furthermore, MMP9, which plays a role in cancer stem cell invasion, may be a target gene of miR-125b.

Transgelin Promotes Migration and Invasion of Cancer Stem Cells

Recent studies have suggested the existence of a small subset of cancer cells called cancer stem cells (CSCs), which possess the ability to initiate malignancies, promote tumor formation, drive metastasis, and evade conventional chemotherapies. Elucidation of the specific signaling pathway and mechanism underlying the action of CSCs might improve the efficacy of cancer treatments. In this study, we analyzed differentially expressed proteins between tumerigenic and nontumorigenic cells isolated from the human hepatocellular carcinoma (HCC) cell line, Huh7, via proteomic analysis to identify proteins correlated with specific features of CSCs. The expression level of Transgelin was 25-fold higher in tumorigenic cells than nontumorigenic cells. Similar results were also observed in tumorigenic cells derived from colorectal adenocarcinoma and prostate carcinoma. More importantly, the elevated levels of Transgelin significantly increased the invasiveness of tumorigenic cells, whereas reduced levels decreased the invasive potential. Moreover, in tumors derived from Huh7-induced xenografts, Transgelin was also co-expressed with CXCR4, which is responsible for tumor invasion. Taken together, these results indicate that the metastatic potential of CSCs arises from highly expressed Transgelin.

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.