Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer
- Authors:
- Published online on: December 23, 2013 https://doi.org/10.3892/ijo.2013.2230
- Pages: 669-677
Abstract
Introduction
Endometrial cancer is the third most common gynecologic cancer in Japan, and its morbidity and mortality have dramatically increased in the past 30 years (1). The majority of patients with early stage endometrial cancer are cured through surgery, while the patients with advanced stage or recurrent lesions are treated by chemotherapy. Paclitaxel and/or cisplatin-based chemothetrapies have been applied to these patients, but with limited efficacy, for which new molecular target therapies are urgently needed. Recent studies have demonstrated the potential of molecular target therapy against cancer stem cells (CSCs) (2).
The cells with CSC-like properties has been demonstrated as tumor-initiating cells (TICs) in a variety of solid tumors including breast cancer (3), brain tumors (4,5), prostate cancer (6,7), lung cancer (8), pancreatic cancer (9), colorectal cancers (10,11) and melanoma (12). CD133, a 5-transmembrane glycoprotein with a molecular weight of 117 kDa, has been widely used to isolate TICs and is now considered to be a potential marker of TICs in a variety of tumor types.
A previous study (13), together with our previous report (14), showed that CD133 is a potential marker of CSCs in endometrial cancer cells. Sorted CD133+ cells had elevated levels of expression of self-renewal genes, such as Nanog and BMI, compared to CD133− cells (14). CD133+ cells were able to generate both CD133+ and CD133− cells, exhibiting self-renewal capacity, while CD133− cells could not. Furthermore, CD133+ cells showed increased proliferative potential in vitro and tumorigenicity in vivo, and showed apparent resistance to cytotoxicity from chemotherapeutic agents. Immunohistochemical analysis of endometrial cancer specimens revealed that overall survival was worse for tumors with high CD133 expression than low CD133 expression (14). These studies have raised several questions: why are CD133+ cells aggressive, leading to the worse prognosis? What are the signaling pathways or molecules causing the effect? Such information may support the establishment of molecular target therapy to CSCs in endometrial cancer.
To answer these questions, we have sought to characterize CD133+ endometrial cancer cells using microarray analyses to identify genes involved in their CSC-like features. Our study clearly demonstrates that an increased chemoresistance and tumorigenic potentials of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression.
Materials and methods
Cell culture
The human endometrial cancer cell lines, Ishikawa and MFE280, were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 μg/ml), and penicillin (100 IU/ml) in the presence of 5% CO2.
Flow cytometry and cell sorting
Cells were incubated in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA with phycoerythrin (PE)-conjugated CD133/2 (clone 293C3) antibodies (Miltenyi Biotec, Auburn, CA, USA). Mouse IgG2b-PE (Miltenyi Biotec) was used as the isotype control antibody. To identify and isolate SP cells, cells were stained with Hoechst 33342 (Sigma-Aldrich), either alone or in combination with 100 μM verapamil (Sigma-Aldrich). For flow cytometry and cell sorting, samples were analyzed using the JSAN desktop cell sorter and AppSan software (Bay Bioscience Co. Ltd., Kobe, Japan).
Cell proliferation assay
Cell proliferation was determined using the WST-1 reagent (Roche Diagnostics, Tokyo, Japan). Briefly, 2×103 cells were seeded in 96-well plates and incubated in normal medium conditions at 37°C. On designated days, WST-1 reagent (10 μl) was added to each well, and the cells further incubated for 2 h at 37°C. Absorbance was measured using a microplate reader at test and reference wavelengths of 450 and 655 nm, respectively.
Soft agar colony formation assay
Diluted single cells (5×104) were seeded onto 60-mm dishes containing 0.33% soft agar in DMEM supplemented with 10% heat-inactivated FBS on top of 0.5% base agar in DMEM supplemented with 10% heat-inactivated FBS. Colonies with diameters larger than 0.25 mm after 14 days incubation were counted.
Chemosensitivity assay
Cells (1×104) were seeded in 24-well plates, incubated for 24 h, then treated with designated concentrations of paclitaxel (provided by Bristol Pharmaceuticals, Tokyo, Japan). After incubation for 48 h, the cells were counted using a hemacytometer with trypan blue staining.
RNA analysis
Total RNA was isolated from the cells using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Complementary DNA was synthesized from 2 μg of RNA using the Omniscript RT kit (Qiagen) with random primers and amplified together with Taq polymerase (Nippon Gene, Tokyo, Japan) for the amplification of ABCG2, MT1-MMP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers and PCR conditions are listed in Table I. Real-time PCR (quantitative PCR; qPCR) was performed using a LightCycler and a SYBR-Green system (Applied Biosystems, Foster City, CA, USA). Microarray analyses were performed using 3D-Gene Human Oligo chip 24k (Toray, Tokyo, Japan).
Western blot analysis
Whole cell extracts were prepared using 1X lysis buffer (Cell Signaling, Danvers, MA, USA) and concentrations were determined using the Bradford protein assay (Life Science, Hercules, CA, USA). SDS-PAGE and western blot analysis were performed as described previously, using 50–100 μg of protein (15,16). MT1-MMP antibody (clone 114-6G6, Fuji Chemical Industries Ltd, Takaoka, Japan) was used at 1:25, ABCG2 (ab3380, Abcam) and GAPDH (Abcam) at 1:10,000.
Knockdown study of MT1-MMP
Cells were seeded and transfected with 30 nM of negative control small interfering RNA (siRNA) or human MT1-MMP siRNA oligonucleotides (Applied Biosystems) using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s protocol.
Invasion assay
The invasive ability of CD133+ or CD133− cells was assayed in vitro using a Biocoat Matrigel Invasion Chamber (Becton-Dickinson Biosciences, Bedford, MA, USA), as described previously (17). Cells were suspended in the upper wells of Matrigel chambers, in DMEM containing 0.1% BSA. After a 22-h incubation, cells on the upper surface of the membrane were removed by wiping with cotton swabs, and cells that had migrated through the membrane to the lower surface were fixed with methanol and stained with Mayer’s Hematoxylin and eosin. The cells on the lower surface of the membrane were counted microscopically to obtain the invasion index. Chemotaxis assays were performed in the same manner, except that the filters were not coated with Matrigel, and the number of cells on the lower surface of the membrane was considered the migration index. The invasive ability of cells was described as the ratio of the invasion index to the migration index.
Gelatin zymography
The supernatants of cells were subjected to gelatin zymography with Gelatin zymography kit (Primary Cell, Sapporo, Japan) according to the manufacturer’s directions.
Statistical analysis
Statistical analysis was carried out using the statistical package StatView version 5.0 (Abacus Concepts, Berkeley, CA, USA). We used the Student’s t-test for in vitro experiments. A p-value of <0.05 was considered to indicate statistical significance.
Results
SP cells are enriched in CD133-expressing Ishikawa cells
In our previous study (14), we examined the frequency of CD133+ cells in 6 endometrial cancer cell lines, among which Ishikawa and MFE280 cells exhibited significant levels of CD133 expression detectable by FACS analysis. Furthermore, only Ishikawa cells had distinct side population fraction, another hallmark of CSC. Thus, in the present study, we mainly used Ishikawa cells for the subsequent analyses. To characterize CD133+ cells in endometrial cancer, we first performed cDNA microarray analyses with Ishikawa cells. A total of 440 genes were found to be overexpressed in CD133+ cells at least 2-fold compared with CD133− cells: genes of ABC transporters, cytokines, growth factors and invasion molecules were included in the total (Table II). In contrast, a total of 96 genes were downregulated in CD133+ cells at least 2-fold compared with CD133− cells (Table III). Among these genes, we paid special attention to the multi-drug resistance gene ABCG2, because of the previous findings that CD133+ endometrial cancer cells are more resistant to chemotherapeutic agents such as paclitaxel and cisplatin than CD133− cells (14). Increased expression of ABCG2 in CD133+ Ishikawa cells was confirmed by RT-PCR and western blot analyses (Fig. 1A).
Based on the microarray analyses, we were interested in the relationship between CD133+ cells and SP cells, since the latter specialized populations are known to highly express ABCG2. Flow cytometry was used to examine the SP fraction in CD133+ or CD133− Ishikawa cells. The ratio of the SP cells in CD133+ cells was calculated as 0.69% (±0.14%) based on the control with verapamil, whereas it was 0.49% (±0.17%) in CD133− cells. These findings indicate that there are more SP cells in CD133+ cells (Fig. 1B).
A recent report indicated that SP cells in endometrial cancer are potential CSCs (18). Therefore, we confirmed such potential of Ishikawa-SP cells by chemosensitivity or colony-formation assay. Ishikawa-SP or non-SP cells were treated with paclitaxel at 2 or 10 nM for 48 h and the cells counted. As shown in Fig. 1C, SP cells are more resistant than non-SP cells to paclitaxel at 10 nM (Fig. 1C). A colony-formation assay was performed, in which Ishikawa-SP or non-SP cells were seeded onto soft agar and colonies larger than 0.25 mm in diameter after incubation for 14 days were counted. The SP cells showed significantly greater colony-forming ability than non-SP cells (Fig. 1D), consistent with a previous report (18).
CD133-expressing SP cells have increased proliferative and anchorage-independent growth
We next examined the tumorigenic potential of SP cells with or without CD133 expression. CD133+/SP, CD133+/non-SP, CD133−/SP and CD133−/non-SP Ishikawa cells were sorted, purified and cultured in normal growth medium for 8 days, and cell growth compared by WST assay. As shown in Fig. 2A, in normal growth medium, CD133+/SP Ishikawa cells grew significantly faster than any other group.
To evaluate anchorage-independent growth of these cells, we assessed their colony-forming ability in soft agar. CD133+/SP cells formed more colonies than CD133+/non-SP, CD133−/SP or CD133−/non-SP cells (Fig. 2B). These results suggest that CD133+/SP Ishikawa cells have the highest potential as CSCs.
CD133-expressing cells have increased invasive ability via elevated levels of MT-MMT expression
We previously reported CD133 to be a prognostic factor in endometrial cancer (14). The precise mechanisms, however, remained unclear, but we speculated that increased invasive ability of CD133+ cells might be the key. Among the genes highly expressed in CD133+ cells, we took particular notice of matrix metalloproteinase genes (MMPs) involved in cellular invasion. We investigated the expression of a total of 26 MMPs in both CD133+ and CD133− Ishikawa cells and found that only MMP14 (MT1-MMP) showed higher expression (>2-fold) in CD133+ cells than CD133− cells. We confirmed by RT-PCR and western blot analysis that MT1-MMP was preferentially expressed in CD133+ cells (Fig. 3A).
MT1-MMP plays a critical role in tumor invasion and metastasis. We evaluated the invasion ability of CD133+ or CD133− endometrial cancer cells using an in vitro invasion assay. Significant differences in invasive ability were observed between CD133+ and CD133− Ishikawa cells (50.5 vs 21.4%) (Fig. 3B), which were confirmed in another cell type (MFE280 cells): CD133+ MFE280 cells showed higher invasive ability than CD133− cells (73.6 vs 46.1%). These results suggest that CD133+ endometrial cancer cells have increased invasive activity compared with CD133− cells.
To confirm whether elevated MT-MMP1 expression is essential for increased invasive ability of CD133+ endometrial cancer cells, we performed siRNA knockdown experiments of MT1-MMP. RT-PCR and western blot analysis showed successful knockdown of MT1-MMP by siRNA (Fig. 3C). Gelatin zymography also showed significant inhibition of enzymatic activity of MT1-MMP by siRNA. In vitro invasion assay revealed that the knockdown of MT1-MMP led to decreased invasive ability in both Ishikawa and MFE280 cells, from 147.6 to 52.0% and from 38.0 to 19.1%, respectively (Fig. 3C), confirming that MT1-MMP influences their invasive capacity. We also examined the invasive ability of sorted CD133+ and CD133− endometrial cancer cells, with or without knockdown of MT1-MMP. In CD133+ Ishikawa and MFE280 cells, invasive ability was significantly decreased by successful knockdown of MT1-MMT from 159.7 to 63.6%, and 10.6 to 6.3%, respectively: knockdown efficacies in mRNA expression were 60 and 45%, respectively, compared with mock transfected cells (Fig. 3D). Knockdown of CD133− Ishikawa and MFE280 cells was not sufficient, and invasive ability was not affected in these cells (47.9 vs 51.6%, 3.7 vs 2.3%, respectively). This was because constitutive levels of mRNA MT1-MMP expression were much lower in CD133− Ishikawa and MFE280 cells than in CD133+ Ishikawa and MFE280 cells. These findings indicate that increased expression of MT1-MMP in CD133+ endometrial cancer cells contributes to their invasive ability.
Discussion
Several lines of evidence have identified CSC populations using various CSC markers in many malignant tumors (19). The characteristic of CSCs are high potential for tumorigenicity, tumor invasion and metastasis (20,21), and chemoresistance (22). Previously, we demonstrated that CD133 is not only a CSC marker but also an independent prognostic factor in endometrial cancer. In this study, we focused on the invasive ability of CD133+ cells, in order to dissect the mechanisms of the aggressive behavior of endometrial CSCs.
The SP phenotype is mediated by expression of ABCG2 protein, a superfamily of ATP-binding cassette (ABC) transporters, which is associated with multi-drug resistance (23,24). SP cells are known to be resistant to chemotherapeutic agents and have been identified as CSCs in malignant solid tumors including hepatocellular carcinoma (25), lung cancer (26), ovarian cancer (27), breast cancer (28) and pancreatic cancer (29). Our study demonstrated that Ishikawa cells contained SP cells (0.69%). Ishikawa-SP cells are more resistant to paclitaxel than non-SP cells. Furthermore, Ishikawa-SP cells exhibited increased colony-forming ability in soft agar, compared with non-SP cells, which shows that they have potential as CSCs. This is consistent with a recent study (18). We speculate that the enriched SP fraction in CD133+ cells contributes to their increased chemoresistance.
Our data indicate that in Ishikawa cells, both CD133+ and SP cells were capable of exhibiting the CSC phenotype. What does this mean? Are there multiple types of distinct CSCs or multiple markers of CSCs in this tumor type? Do CD133+ cells significantly overlap with SP cells? The ratio of the SP cells was 0.69% in CD133+ cells, compared with 0.47% in CD133− cells: therefore, overlapping population was not large. Nevertheless, CD133+ and SP cells showed CSC-like characteristics in vitro. Although we have not done in vivo analysis, Kato et al recently observed a CSC-like tumorigenic phenotype of SP cells (18). Thus, both CD133 and SP may be independently considered as CSC markers according to the current experimental criteria. We further investigated the characteristics of SP or non-SP cells in CD133+ and CD133− cells. SP/CD133+ Ishikawa cells had the greatest advantage of proliferation and tumorigenicity in vitro compared with SP/CD133−, non-SP/CD133+ and non-SP/CD133− cells. The frequency of CD133+ cells in Ishikawa cells was approximately 10.0%, while the ratio of SP fraction in CD133+ Ishikawa cells was 0.69%. Based on these results, about 0.069% SP/CD133+ cells were contained in Ishikawa cells, which exhibit the highest CSC activity. Taken together, we speculated that multiple types of CSCs with distinct markers may be present, at least satisfying the minimum experimental conditions for the definition of CSCs, but the small subset with concurrent expression of markers appears to have the highest CSC activity.
Accumulating evidence has revealed that CSCs have great invasive ability (30–32). We confirmed that CD133+ endometrial cancer cells exhibited higher expression of MT1-MMP, through which they appeared to show increased invasive ability. Annabi et al reported that MT1-MMP and MMP9 contributed to the invasive phenotype in CD133+ brain cancer stem cells (30), and Kohga et al showed that MMP2, which is activated by MT1-MMP, is required for invasive ability in CD133+ hepatocellular carcinoma cells (31), which is basically consistent with our results.
Invasion of cancer cells, including lymph node and distant metastasis, is believed to be associated with epithelialmesenchymal transition. Kabashima et al demonstrated that TGF-β-induced epithelial-mesenchymal transition (EMT)-and invasion-associated gene alterations such as reduction of E-cadherin and induction of Snail and MMP2 in a side population of pancreatic cancer (32). Circulating tumor cells in patients with advanced prostate and breast cancer expressed epithelial protein such as adhesion molecule, mesenchymal proteins including N-cadherin and vimentin, and the CSC marker CD133 (33). Our experimental model, in which CD133+ endometrial cancer cells exhibited increased invasive capacity via elevated MT1-MMP, might be suitable to study the role of EMT in metastasis. We are currently investigating whether CD133+ endometrial cancer cells are likely to show EMT phenotypes during the process of invasion.
The present microarray analyses revealed a total of 440 genes upregulated and 96 genes downregulated in CD133+ cells, compared to CD133− cells. There might be some genes other than MT1-MMP involved in aggressive behavior of CD133+ cells. For example, Q9HCN2, a gene encoding p53AIP, known to be a p53-regulated apoptosis-inducing protein (34), was downregulated in CD133+ cells. Thus, impaired apoptosis pathway might be associated with aggressive phenotypes of CD133+ cells. Further extensive analysis with microarray data will hopefully identify genes critical for determining phenotypes of CD133+ cancer stem cells.
In summary, we found the characteristic features of CD133+ endometrial cancer cells, enriched SP cells and elevated MT1-MMP expression, through which they achieve increased chemoresistance as well as invasive capacity. A subpopulation of SP cells with CD133 expression showed the greatest CSC-like activity. Further characterization of CD133+ cells is required to identify the more condensed population of CSCs and to provide a novel molecular target for this tumor type.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
References
|
Matsuda T, Marugame T, Kamo K, et al: Cancer incidence and incidence rates in Japan in 2006: Based on data from 15 population-based cancer registries in the monitoring of cancer incidence in Japan (MCIJ) project. Jpn J Clin Oncol. 42:139–147. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kyo S: Endometrial cancer stem cells: Are they a possible therapeutic target? Curr Obstet Gynecol Rep. 2:1–10. 2013. View Article : Google Scholar | |
|
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ and Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 100:3983–3988. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Singh SK, Hawkins C, Clarke ID, et al: Identification of human brain tumour-initiating cells. Nature. 432:396–401. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q and Hjelmeland AB: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 444:756–760. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Gu G, Yuan J, Wills M and Kasper S: Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo. Cancer Res. 67:4807–4815. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Miki J, Furusato B, Li H, et al: Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res. 67:3153–3161. 2007. View Article : Google Scholar | |
|
Eramo A, Lotti F, Sette G, et al: Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 15:504–514. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Hermann PC, Huber SL, Herrler T, et al: Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 1:313–323. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
O’Brien CA, Pollett A, Gallinger S and Dick JE: A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 445:106–110. 2007.PubMed/NCBI | |
|
Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M and Peschle C: Identification and expansion of human colon cancer - initiating cells. Nature. 445:111–115. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Monzani E, Facchetti F, Galmozzi E, et al: Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential. Eur J Cancer. 43:935–946. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Rutella S, Bonanno G, Procoli A, et al: Cells with characteristics of cancer stem/progenitor cells express the CD133 antigen in human endometrial tumors. Clin Cancer Res. 15:4299–4311. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Nakamura M, Kyo S, Zhang B, et al: Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer. Hum Pathol. 41:1516–1529. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Nakamura M, Bodily JM, Beglin M, Kyo S, Inoue M and Laimins LA: Hypoxia-specific stabilization of HIF-1alpha by human papillomaviruses. Virology. 387:442–448. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Mizumoto Y, Kyo S, Mori N, et al: Activation of ERK1/2 occurs independently of KRAS or BRAF status in endometrial cancer and is associated with favorable prognosis. Cancer Sci. 98:652–658. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Mizumoto Y, Kyo S, Ohno S, et al: Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements. Oncogene. 25:5673–5682. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Kato K, Takao T, Kuboyama A, et al: Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage. Am J Pathol. 176:381–392. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Visvader JE and Lindeman GJ: Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 8:755–768. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Maeda S, Shinchi H, Kurahara H, et al: CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer. Br J Cancer. 98:1389–1397. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Kojima M, Ishii G, Atsumi N, Fujii S, Saito N and Ochiai A: Immunohistochemical detection of CD133 expression in colorectal cancer: a clinicopathological study. Cancer Sci. 99:1578–1583. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Ma S, Lee TK, Zheng BJ, Chan KW and Guan XY: CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene. 27:1749–1758. 2008. | |
|
Kim M, Turnquist H, Jackson J, et al: The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res. 8:22–28. 2002.PubMed/NCBI | |
|
Doyle LA and Ross DD: Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene. 22:7340–7358. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Chiba T, Kita K, Zheng YW, et al: Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 44:240–251. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Ho MM, Ng AV, Lam S and Hung JY: Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 67:4827–4833. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al: Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci USA. 103:11154–11159. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Christgen M, Ballmaier M, Bruchhardt H, von Wasielewski R, Kreipe H and Lehmann U: Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line. Mol Cell Biochem. 306:201–212. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Wang YH, Li F, Luo B, et al: A side population of cells from a human pancreatic carcinoma cell line harbors cancer stem cell characteristics. Neoplasma. 56:371–378. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Annabi B, Rojas-Sutterlin S, Laflamme C, et al: Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype. Mol Cancer Res. 6:907–916. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Kohga K, Tatsumi T, Takehara T, et al: Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma. J Hepatol. 52:872–879. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Kabashima A, Higuchi H, Takaishi H, et al: Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer. 124:2771–2279. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Armstrong AJ, Marengo MS, Oltean S, et al: Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol Cancer Res. 9:997–1007. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Oda K, Arakawa H, Tanaka T, et al: p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell. 102:849–862. 2000. View Article : Google Scholar : PubMed/NCBI |
