SURF4 maintains stem-like properties via BIRC3 in ovarian cancer cells
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Objective: As cancer stem cells (CSCs) are considered as the origin of tumor development, recurrence, and drug resistance, we aimed to explore the mechanism related to modulating stemness in CSCs, thus facilitating to search for new therapeutic strategy for ovarian cancer.Methods: In this study, ovarian cancer stem cells (OCSCs) induced from cell line 3AO and A2780 were enriched in serum-free medium (SFM).The effect of SURF4 on CSC-like properties was evaluated by sphere-forming assays, re-differentiation assays, quantitative real-time polymerase chain reaction, flow cytometry, Western blotting, cell viability assays and in vivo xenograft experiments.The downstream molecule participating in SURF4 maintaining stemness was screened by RNA-sequencing and identified by the experiments of gene function.Results: SURF4 was upregulated expressed in OCSCs.Knockdown of SURF4 reduced the expression of the related stem markers (SOX2 and c-MYC), inhibited self-renewal ability, and improved the sensitivity to chemotherapeutic drugs (paclitaxel and cisplatin) in OCSCs.SURF4 knockdown also inhibited tumorigenesis in nonobese diabetic/severe combined immunodeficiency mice.BIRC3 expression was controlled by SURF4, and BIRC3 showed the similar effect as SURF4 did, and BIRC3 overexpression partially recovered stem-like properties abolished by SURF4 knockdown.Conclusion: Our findings suggest that SURF4 possesses the ability to maintain stemness of OCSCs via BIRC3, and may serve as a potential target in stem cell-targeted therapy for ovarian cancer.Keywords:
Stem cell marker
The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.
Homeobox protein NANOG
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Glioblastoma multiforme (GBM) is the most lethal type of adult brain cancer and performs outrageous growth and resistance regardless of adjuvant chemotherapies, eventually contributing to tumor recurrence and poor outcomes. Considering the common heterogeneity of cancer cells, the imbalanced regulatory mechanism could be switched on/off and contribute to drug resistance. Moreover, the subpopulation of GBM cells was recently discovered to share similar phenotypes with neural stem cells. These cancer stem cells (CSCs) promote the potency of tumor initiation. As a result, targeting of glioma stem cells has become the dominant way of improving the therapeutic outcome against GBM and extending the life span of patients. Among the biomarkers of CSCs, CD-133 (prominin-1) has been known to effectively isolate CSCs from cancer population, including GBM; however, the underlying mechanism of how stemness genes manipulate CSC-associated phenotypes, such as tumor initiation and relapse, is still unclear.Tumorigenicity, drug resistance and embryonic stem cell markers were examined in primary CD133-positive (CD133(+)) GBM cells and CD133(+) subpopulation. Stemness signature of CD133(+) GBM cells was identified using microarray analysis. Stem cell potency, tumorigenicity and drug resistance were also tested in differential expression of SOX2 in GBM cells.In this study, high tumorigenic and drug resistance was noticed in primary CD-133(+) GBM cells; meanwhile, plenty of embryonic stem cell markers were also elevated in the CD-133+ subpopulation. Using microarray analysis, we identified SOX2 as the most enriched gene among the stemness signature in CD133(+) GBM cells. Overexpression of SOX2 consistently enhanced the stem cell potency in the GBM cell lines, whereas knockdown of SOX2 dramatically withdrew CD133 expression in CD133(+) GBM cells. Additionally, we silenced SOX2 expression using RNAi system, which abrogated the ability of tumor initiation as well as drug resistance of CD133(+) GBM cells, suggesting that SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells.SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells. Our results not only revealed the genetic plasticity contributing to drug resistance and stemness but also demonstrated the dominant role of SOX2 in maintenance of GBM CSCs, which may provide a novel therapeutic target to overcome the conundrum of poor survival of brain cancers.
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Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere-forming, Aldefluor, and side-population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness-related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/β-catenin activation were evaluated. We show that different cancer stem cell populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly-proliferating populations, overexpress SOX2, and KLF4 stemness-related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β-catenin signaling as evidenced by increased nuclear β-catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor-positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side-population phenotype is correlated with ABCG2 drug-efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. J. Cell. Physiol. 231: 876-886, 2016. © 2015 Wiley Periodicals, Inc.
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In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.
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Cancer stem cells (CSCs) or tumor initiating cells are rare cells that are able to establish a tumor or metastasis. Identification of those CSCs is, however, cumbersome even in established cell lines. Several cancer stem cell markers were reported to be expressed by ovarian cancer. Those cancer stem cells are gifted with lower vulnerability to irradiation and cytostatic drugs explaining the high incidence of recurrence after treatment. A variety of different cancer stem cell markers were described for epithelial tumors. Also, cancer cell lines were assessed for stem cell markers with no common denominator. The expression of CD24, CD44, CD117, CD133, ABCG2, ALDH was determined for cells from 22 patients. Ovarian cancer cells were collected from ascites. Part of the tumor cells were analyzed immediately and stained for the above mentioned cancer stem cell markers. The remainder of the cells was cultured for several weeks using standard stem cell culture conditions. We observed a large variety in expression of putative stem cell markers for primary tumors. After two weeks of culture spheres were seen in several cultures, indicative for cancer stem cells, though not all patients’ cells were able to form spheres. Our data show for the first time the heterogeneity in marker display in primary tumors. Also for the cultured cells stem cell markers were determined. None of the stem cell markers was expressed by all patients’ cells. No correlation with tumor type was demonstrated. The complexity of expression challenges the isolation of cancer stem cells
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Cancer stem cells (CSCs) are described as cells within a tumor that are able to indefinite self-renewal, form tumors when transplanted in vivo, differentiate into multiple lineages, and express genes such as OCT3/4, SOX2, KLF4, NANOG. Although these traits of CSCs are commonly accepted, there is still a lot of controversy regarding these cells. There are very few methods which allow to obtain these cells, like separation based on surface markers, presence of side population, sphere forming assays, and aldefluor assays. This paper seeks the confirmation of CSCs presence in cancer cell lines such as: breast, prostate, pancreatic, liver, brain, and cervical. Nowadays, researchers use two models of cell culture: established cancer cell lines (ECCLs) and primary cell culture. A major problem with these models is that tumors in organism evolve and cell cultures represent only small fragment of tumor development. Since CSCs were found, there exist high hopes of revealing new therapies targeting CSCs. However, the appearance of new populations with the ability to induce tumors should pour a bucket of water to create a cure for cancer.
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Cancer stem cells (CSCs) that closely correlated with tumor growth, metastasis, provide a plausible explanation for chemoresistance and cancer relapse. CSCs are usually isolated and enriched from carcinoma cells, which is inconvenient, low-efficient, and even unreliable. Here, we converted mouse induced pluripotent stem cells (miPSCs) into prostate cancer stem-like cells with carcinoma microenvironment following exposure to conditioned medium (CM) derived from RM9, a mouse prostate cancer cell line. These transformed cells, termed as miPS-RM9CM, displayed CSCs properties, including spheroids morphology and expression of both stemness genes and cancer stem cells surface markers, such as Oct3/4, Sox2, Nanog, Klf-4, c-Myc, CD44, and CD133. In addition, in vivo transplantation experiment was performed to confirm the tumorigenicity. Furthermore, we used the model to assess conventional chemotherapeutic agent, docetaxel. The results showed that miPS-RM9CM cells exhibited increased resistance to docetaxel, however, high susceptibility to the cancer cell stemness inhibitor I (BBI-608). Our current study demonstrates that CM from cultured RM9 cells play a crucial role in the determination of cell fate from miPSCs to cancer stem-like cells and provide a potentially valuable system for the study of CSCs.
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Abstract Lung cancer is the leading cause of death from cancer worldwide. NSCLC represents the most common subtype of lung cancer with the average five-year survival rate is only 16%. The majorities of these tumors are refractory to chemotherapeutics or acquire resistance to the therapy. In addition, most of the patients develop distant metastases even tough their primary tumors are surgically removed. Cancer stem cell hypothesis has emerged as a possible explanation for tumor growth, recurrence after treatment and metastasis in a variety of cancer. This hypothesis suggests that tumors are maintained by the subset of cancer stem cells (CSCs) that has the ability to self-renew and generate the diverse cells that comprise the tumor. While evidence supporting the existence of CSCs has been demonstrated in many solid cancers, it is largely underdeveloped in lung cancer. Here we have isolated cancer stem like cells from NSCLC cell lines either through isolating the side population (SP) cells based on their property to exclude Hoechst 33342 dye or enriched them by cultivating cancer cells in defined serum free medium containing N2-supplement, EGF and bFGF. Cells obtained from both these methods demonstrated higher clonogenic as well as sphere forming ability which is the typical characteristics associated with CSCs. We further characterized these cells for their ability to express progenitor/stem cell marker genes like Oct4, Sox2 and Nanog. Total RNA from SP cells showed higher expression of these genes as compared to main population. Similarly, the expression of Oct4, Sox2 and Nanog was found elevated after culturing H1650, H358, H322 and H292 cells in defined serum free medium for 10 days. Interestingly, CSCs enriched in this condition also showed the loss of E-cadherin expression and consequent gain of mesenchymal proteins like fibronectin and vimentin. Additionally, ABCG2, which is closely associated with the drug resistance and responsible for side population phenotype, was also upregulated in cancer stem like cells enriched from culturing in serum free defined media. In conclusion, results revealed that lung cancer stem like cells could be isolated and expanded from the established lung cancer cell lines by two different methodologies described here. These cells possess the characteristics of both stem cells and malignant tumors. Therefore, further studies towards targeting these cells may result in effective therapy against lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4309.
Homeobox protein NANOG
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HCCS
Liver Cancer
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