Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer

  • Authors:
    • Mitsuhiro Nakamura
    • Xiuzhi Zhang
    • Yasunari Mizumoto
    • Yoshiko Maida
    • Yukiko Bono
    • Masahiro Takakura
    • Satoru Kyo
  • View Affiliations

  • Published online on: December 23, 2013     https://doi.org/10.3892/ijo.2013.2230
  • Pages: 669-677
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Abstract

A small subset of cells with CD133 expression is thought to have increased chemoresistance and tumorigenicity, features of cancer stem cells (CSCs); the molecular mechanisms by which these properties arise remain unclear. We characterized CD133+ endometrial cancer cells based on microarray analyses of Ishikawa cells. Of the genes upregulated in CD133+ cells compared with CD133- cells, we noted several key factors involved in the aggressive behavior of cells, including ABCG2 and matrix metalloproteinase (MMP). Flow cytometric analyses identified a side-cell population (SP) with CSC features in Ishikawa cells, and they were found to be more enriched in CD133+ cells than CD133- cells. In particular, CD133+/SP cells exhibited higher proliferative and colony‑forming activity than CD133+/non-SP cells. Matrigel invasion assay revealed that CD133+ cells have enhanced invasive capacity with elevated MT1-MMP expression. siRNA‑based knockdown of MT1-MMP largely abolished the invasive capacity of CD133+ cells, but not CD133- cells due to low levels of constitutive MT1-MMP1 expression. These findings demonstrate that increased chemoresistance and tumorigenic potential of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression. These results will be of assistance in the establishment of molecular target therapy to CSCs in endometrial cancer.

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).

Table I.

PCR primer and condition details.

Table I.

PCR primer and condition details.

GenePrimerAnnealing temperatureCycle no.
ABCG2F: GTTCTCTTCTTCCTGACGACCA
R: CCACACTCTGACCTGCTGCTA
6030
MT1-MMPF: TCGGCCAAAGCAGCAGCTTC
R: CTTCATGGTGTCTGCATCAGC
5935
GAPDHF: CTCAGACACCATGGGGAAGGTGA
R: ATGATCTTGAGGCTGTTGTCATA
6028

[i] F, forward; R, reverse.

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).

Table II.

The genes of higher expression in CD133+ Ishikawa cells than CD133− cells.

Table II.

The genes of higher expression in CD133+ Ishikawa cells than CD133− cells.

ARRDC2ZNF334GJB6TREX1MMP14(MT1-MMP)STARD8Q86WM5_HUMANLHX1SLC26A3IGX1_HUMAN
ZNF137PLD2NMD3A_HUMANQ96DP9_HUMANTCL1ACDAHBXAPFAM49ADIAPH2DDHD1
OR9G4CXCL13NKX2-4MARCOGBG1_HUMANSENP7TMEM40ZNF584PCDH12C13orf23
Q7Z2R7_HUMANRRADGSH1DPTC4orf6Q8N134_HUMANGIMA5_HUMANCAPN3ABCG2TRPV6
LAF4OPN1SWC21orf63CBLN1NUDT10Q86VH3_HUMANC1QTNF3IER3GPSM1FSIP1
GLSATOH1C20orf144KR108_HUMANQ8NH32_HUMANOR6Y1CYP3A5NTF3PCYT1BFRMD3
Q86W74_HUMANVSIG4ZC3HAV1DPP10TLL2C21orf121NOSTRINPITRM1RBM10P2RY1
FAM53BSLC14A1Q9NXW5_HUMANHPRHMX2LRRK1C20orf85Q9P1C0_HUMANADM2VGLL1
GALPSG10Q9H357_HUMANSCRG1_HUMANQ8NGE6_HUMANCRSP2C2orf13EFHBTNFRSF8MSH4
VISL1_HUMANSLC27A4ENK13_HUMANQ8IY46_HUMANZNF346SEPT4_HUMANQ9H8T6_HUMANPPY2BMP6ACOX3
PPP1R14DXDHRALGPS2DEPDC1ALKBHSH2D3CSIA7B_HUMANALS2CR8Q7Z470_HUMANQ8NAH5_HUMAN
PKN2ZNF195PAQR3_HUMANRAPGEF1GPATC4TMSL6C10orf81KIAA0258WBSCR23Q9NRZ3_HUMAN
Q9P135_HUMANZNF541Q70YC7_HUMANKLF8SCELQ8N2X2_HUMANLMOD1Q9NSJ0_HUMANRAP1GDS1NF2
KBTB6_HUMANHOXC13IGJDLX3PRSS23CE290_HUMANLPLZFXHIP14_HUMANKRT23
GPR19Q8NHA3_HUMANOR8D1ASCC3L1PHACTR3FSTL3SP3ALDH1A1SPARCFHL2
NT5EEDNRAHAMPSLC22A16TMPRSS3DBTC9orf60ASB6CLDN9HSPB3
CTSGIGHG3GPR87_HUMANCPA1PRKYTUBB4QDSCR1L2ELNTMCC3SLC19A2
SNX9CEACAM5TEAD1ACCN5PDZK10CD24CD3GABCG8DNAJA4PTK2B
PCDHB10LOH11CR2AFBXO15DPYSNKPD1ABCG4TGM4ZIM3RALGPS2CSNK1G3
FIGNTNFRSF14BPESC1Q86XT6_HUMANUNC5A_HUMANPIP5KL1TIGD7RASGRP2Q8N9Q3_HUMANFSD1
PLAG1CASP8DMRT1Q8NGP1_HUMANFXYD6BIRC8THEGQ5VSL2_HUMANLEPREL1MMP10
CRABP1IGFBP5PTHLHQ8ND77_HUMANRTN1TFAP4KRTAP2-4FSHPRH1Q8N3F2_HUMANLRRK2
TMED6CAV1Q8NAM0_HUMANABCC9SYT3MLSTD1ZNF214TYB4_HUMANQ9P1G6_HUMANITM2A
CABYRKYNUABI3BPZFYVE9ITGB3APXLRGS7ALOX15BCHRAC1RGS16
Q9Y4N5_HUMANTDGF1HALRGS3LYZL1LAS1LSPG7LARSZNF93GRINL1A
Q8NEE2_HUMANKIF21BGPR120IFR28_HUMANPERQ1GPR119GPR26SALL1ATXN7L1ADHFE1
Q8N896_HUMANWDR9_HUMANSUHW4BEX1ZFP67OR5W2SLC16A5CLDN1C19orf18
XP_376267.2C9orf102HCP5TFF3OSGEPL1C10orf59FUBP3_HUMANIL17DQ9NWZ4_HUMAN
CD37RPS6KB1TRAM2PTPREZNF311DDAH1Q96JG6_HUMANMYCNOSINHBB
Q9P143_HUMANZDHHC8UPK1BHS3ST3A1CXCL1ACOXLTHAP2_HUMANQ14560_HUMANIL20RA
SEMA4FCOPAOXCT2ABCC8Q96FU4_HUMANSPATA13Q9NSH8_HUMANQ8NEF7_HUMANGPRC5C
ESR2SLMAPASB4F10Q8N4W5_HUMANSERPINB8C9orf90GKTMEPAI
OLFM1DIO1RASGEF1CHDDUSP16ATP2B4NID2CA9CCL15
FPRL1O14634_HUMANCM35H_HUMANCPEB2FUT10THSD1CHRNA2CBLN3_HUMANCTAG2
TBA3_HUMANARHGAP25Q86V40_HUMANAPEX2ZNF12ACTL7BCLDN16NRGNRHBDF1
PSMF1NCF2SYT5ELAC1ACOX2EBF2HGFCALR3Q9NPS2_HUMAN
CKLFS100A12PLXNA4SIRT6_HUMANPPP1R1AGK2ALDH1A3LENEPZNF588
TRIM58HPCL4_HUMANPBX1TEX15FSCN1SAMD1Q8N9M7_HUMANQ14946_HUMANCCL24
UTRNTRIM6RAB14_HUMANC20orf54RPP30FGFR1FGFBP1ZNF367ARPM2_HUMAN
SLC6A9Q9H8Q9_HUMANFZD4Q9HAU7_HUMANSERPINI2PIK3CGMARK4Q6UXT6_HUMANNXN
OR51E1ZNF623ALPK2DSG1PTGESNRAPQ9BT82_HUMANCTAGE6ZNF44
Q30181_HUMANC20orf96GABRG2Q8N3H6_HUMANCST5KRT17RERGKCNQ3FBXW8_HUMAN
CCDC11Q5VZR3_HUMANSPACA3CA6HIST1H1BADPRHL1IVNS1ABPGPR126SCGB1A1
PCDH15AKR1B10ANKRD2TAGLNAHRRSLC22A17TNFRSF19LTPM1NKX3-1
CELCMKOR1Q86SX0_HUMANWWOXNALP2TLX3CSN1S1Q5TAX4_HUMANPROM1
OR7E5PSOCS6C1SRCN3C10orf63RTDR1CRLF1EIF2C3Q9NSC1_HUMAN

Table III.

The genes of lower expression in CD133+ Ishikawa cells than CD133− cells.

Table III.

The genes of lower expression in CD133+ Ishikawa cells than CD133− cells.

DMPKRBP4PRSS1ZNF261
Q6ZQS7_HUMANOR56B4USH1CSEMG1
GRIN2CC20orf19Q86XE0_HUMANB3GALT3
GPR54MALLHFPL2ARSA
KLF1ZNF322ARHEBL1KIAA1683
CCNA1ANKRD24TRIM48Q5QPC4_HUMAN
MLL3DNASE1MBD3L2O75372_HUMAN
Q8NI68_HUMANZNF228TESK2CLCN4
IGF2RL41_HUMANLPHN1DCHS1
IGF1UTF1TNIP1POLL
BIRC4TLE2CSF3RPAX5
ITGALHIST1H2ABMYO18AACRC
C20orf23Q8N9V4_HUMANGRAPTPK1
RFPL1Q96NA9_HUMANKCNC4MSN
Q6PJR0_HUMANZNF431POMCRFPL2
RIMS2C6orf148LRRIQ1Q8N9H1_HUMAN
FEVQ9NRE5_HUMANNXPH4DCP1B
C21orf81FGD1PEX6HAGHL
RHDRHOI_HUMANBCL2L13RCOR2
ATP1A2POLR1ATMEFF1Q96MC9_HUMAN
ALPPL2TRIM43VPREB3MSMB
Q9HCN2_HUMANQ9NYD4_HUMANCOX1_HUMANOR11H4
RASD1_HUMANZNF423Q8IW70_HUMANSOS2
NTRI_HUMANOCRLC14orf49CNTNAP4

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).

Figure 3.

Increased invasive ability of CD133+ cells is dependent on elevated MT1-MMP expression in endometrial cancer cells. (A) RT-PCR and western blot analyses confirmed the elevated expression of MT1-MMP in CD133+ cells compared to CD133− cells in Ishikawa or MFE280 cells. (B) CD133+ and CD133− Ishikawa or MFE280 cells were suspended in the upper wells of Matrigel chambers at 50,000 cells/chamber; the cells that had migrated through the membrane to the lower surface after a 22-h incubation were microscopically counted to obtain the invasion ratio. RT-PCR assays confirmed the elevated expression of MT1-MMP mRNA in both cells. Each point represents the mean ± SD of three independent experiments performed in triplicate. Columns, mean; bars, mean ± SD. *P<0.05; **P<0.01. (C) Ishikawa or MFE280 cells were transfected with siRNA against MT1-MMP (si-MT1-MMP) or negative control (si-Control) and the invasion ratio was evaluated in a similar manner. RT-PCR assays (PCR) and western blot analysis showed effective inhibition of MT1-MMP mRNA and protein expression by siRNA. Relative expression of MT1-MMP in cells transfected with negative control siRNA was measured by densitometric analysis using NIH image and described as 100%. Gelatin zymography to monitor the extent of latent proMMP-2 and active MMP-2 found the effective inhibition of enzymatic activity of MT1-MMP by siRNA. Each point represents the mean ± SD of three independent experiments performed in triplicate. Columns, mean; bars, mean ± SD. *P<0.05. (D) Sorted CD133+ and CD133− cells in Ishikawa or MFE280 cells were transfected with siRNA against MT1-MMP (si-MT1-MMP) or negative control siRNA (si-Control), and the invasion ratio was evaluated in a similar manner. Inhibition of MT1-MMP by siRNA was confirmed by quantitative RT-PCR (qPCR) and western blot analysis. Relative expression of MT1-MMP in cells transfected with negative control siRNA was described as 100%. Each point represents the mean ± SD of three independent experiments performed in triplicate. Columns, mean; bars, mean ± SD. *P<0.05; **P<0.01.

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 (3032). 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).

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2014-March
Volume 44 Issue 3

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Online ISSN:1791-2423

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Spandidos Publications style
Nakamura M, Zhang X, Mizumoto Y, Maida Y, Bono Y, Takakura M and Kyo S: Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer. Int J Oncol 44: 669-677, 2014
APA
Nakamura, M., Zhang, X., Mizumoto, Y., Maida, Y., Bono, Y., Takakura, M., & Kyo, S. (2014). Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer. International Journal of Oncology, 44, 669-677. https://doi.org/10.3892/ijo.2013.2230
MLA
Nakamura, M., Zhang, X., Mizumoto, Y., Maida, Y., Bono, Y., Takakura, M., Kyo, S."Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer". International Journal of Oncology 44.3 (2014): 669-677.
Chicago
Nakamura, M., Zhang, X., Mizumoto, Y., Maida, Y., Bono, Y., Takakura, M., Kyo, S."Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer". International Journal of Oncology 44, no. 3 (2014): 669-677. https://doi.org/10.3892/ijo.2013.2230