MCAM is a novel metastasis marker and regulates spreading, apoptosis and invasion of ovarian cancer cells
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Melanoma cell adhesion molecule (MCAM) is a cell adhesion molecule that is abnormally expressed in a variety of tumours and is closely associated with tumour metastasis. The role of MCAM in ovarian cancer development has not been fully studied. In this study, through immunohistochemical staining of ovarian cancer tissue samples and RNA interference to silence MCAM in ovarian cancer cells, we examined the impact of MCAM on the biological functions of ovarian cancer cells and attempted to reveal the role of MCAM in ovarian cancer development. Our results showed that MCAM expression was particularly high in metastatic ovarian cancers compared with other pathological types of ovarian epithelial tissues. After MCAM silencing in the MCAM high-expression ovarian cancer cell line SKOV-3, the cell apoptosis was increased, whereas the cell spreading and invasion were significantly reduced, which may be related with dysregulation of small RhoGTPase (RhoA and Cdc42).These results suggest that MCAM expression in ovarian cancer is highly correlated with the metastatic potential of the cancer. MCAM is likely to participate in the regulation of the Rho signalling pathway to protect ovarian cancer cells from apoptosis and promote their malignant invasion and metastasis. Therefore, MCAM can be used not only as a molecular marker to determine the prognosis of ovarian cancer but also as a therapeutic target in metastatic ovarian cancer.
KeywordsMCAM Ovarian cancer Spreading Invasion Apoptosis
Ovarian cancer has become one of the primary tumours that pose a serious threat to women's lives and health globally. Although the incidence rate is not high, the death rate is the highest among all gynaecological tumours . The major cause of death in advanced ovarian cancer is metastasis, which is a complex process that involves changes in many molecules, including adhesion molecules, proteolytic enzymes, chemokines and so on. Among them, the adhesion molecules between cell–cell and cell matrix have drawn much attention in cancer metastasis research .
It is generally believed that the lack of function of cell adhesion molecules will facilitate tumour cell dissemination. For example, in epithelial ovarian cancer, opioid-binding cell adhesion molecule is often inactivated by allelic deletion or by methylation . In addition, the down-regulation of CD9 indicates a poor prognosis because this change can cause a reduction in the expression of certain integrins, thus leading to the metastasis of ovarian cancer . Interestingly, the elevated expression of certain other adhesion molecules, such as p-cadherin, can promote ovarian cancer metastasis . Therefore, the role of adhesion molecules in the metastasis of ovarian cancer is complex and requires further study.
A member of the immunoglobulin superfamily, melanoma cell adhesion molecule (MCAM; also known as CD146 or MUC18) was first identified in melanoma . MCAM is a membrane calcium-independent glycoprotein adhesion molecule, the extracellular domain of which contains the typical V-V-C2-C2-C2 Ig-like domain and the intracellular structure of which contains several protein kinase recognition motifs, suggesting that MCAM may participate in cell signalling pathways inside and outside the cell . MCAM was initially considered to be the characteristic antigen that distinguishes malignant melanoma from benign or borderline melanoma. Follow-up studies found that MCAM is abnormally expressed in a variety of tumour tissues, including melanoma , prostate cancer , breast cancer  and non-small cell lung cancer  and that this abnormal expression is closely associated with tumour progression and metastasis. In 2006, Aldovini et al. reported that epithelial ovarian cancer patients with high expression of MCAM in tumour tissues had a significantly higher relapse rate than MCAM expression-negative patients and that the survival period of the former group was significantly shorter .
In this study, we have found that borderline ovarian tumours and malignant epithelial ovarian cancer have higher MCAM-positive rates compared with normal ovarian epithelium and benign ovarian tumours. The MCAM expression rate is particularly high in metastatic ovarian cancer lesions. We further used RNA interference to silence MCAM gene expression in the ovarian cancer cell line SKOV-3, and our results showed that, after MCAM knockdown, the cancer cell apoptosis was increased, and the capacities of cell spreading on the extracellular matrix and invasion through matrigel were significantly reduced. The down-regulation of MCAM expression was also correlated with decreased Rho GTPases (Cdc42 and RhoA) activation. Our study has demonstrated that MCAM affects ovarian cancer cell apoptosis and invasion, indicating that, in addition to being used as a molecular marker to determine the prognosis of ovarian cancer, MCAM may also be used as a new target for clinical treatment.
Materials and methods
Cell culture and chemical reagents
Ovarian cancer cell lines SKOV-3 (purchased from the Cell Bank of the Chinese Academy of Science, Shanghai, China.), OVCA429 and RMUG-S (gifts from prof. Bin Ye, Harvard Medical School, Boston, MA ) were cultured at 37 °C in a humidified 5 % CO2 atmosphere in RPMI-1640 medium with 10 % fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) and Opti-MEM–1 Medium (Gibco, Invitrogen, Carlsbad, CA) were used for siRNA transfection. The siRNAs were synthesised by Shanghai GenePharma Co. Rabbit polyclonal antibodies used in this study were directed against MCAM (ProteinTech Group, Inc (Chicago, IL). Rabbit monoclonal antibodies used in this study were directed against RhoA and Cdc42 (Cell Signalling Technology, Inc., Danvers, MA). Mouse monoclonal antibodies used in this study were directed against Rac1 (Merck Millipore, Danvers, MA), tubulin (Sigma, St. Louis, MO) and Ki67 (Abcam, Hong Kong). IRDye 680/800 conjugated second antibodies were from LI-COR, Inc. (Lincoln, NE). Collagen I was purified in our laboratory , Collagen IV, fibronectin and laminin 1 were purchased from Merck Millipore (Danvers, MA). Cell Counting Kit8 (CCK8) was a product of Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). In Situ Cell Death Detection Kit was from Roche Applied Science (Mannheim, Germany)and FITC Annexin V Apoptosis Detection Kit was from BD Biosciences (San Jose, CA).
Clinical samples and immunohistochemical staining
Human ovary tissue microarrays (OV1005a and OV808) contained 45 cases of primary malignant epithelial ovarian cancer, 40 metastatic ovarian cancer, 7 borderline cystadenoma, 16 benign cystadenoma, 17 cancer adjacent normal ovary tissues and 3 normal ovary tissues were purchased from US Biomax Inc (Alenabio, Xi’an, China). Immunohistochemistry was performed for MCAM according to standard procedures as described . All of the sections were observed and photographed with a microscope (Axio Imager.A1, Carl Zeiss MicroImaging GmbH, Germany). After nuclear counterstaining with hematoxylin, the cytoplasmic and cytomembrane of epithelial cells immunostaining intensity was categorised semiquantitatively into four groups: negative (score 0): no staining at all, weakly positive (score 1): faint/barely perceptible staining in the majority of the epithelial cells, moderately positive (score 2): a moderate staining in the majority of the tumour cells, and strongly positive (score 3): a strong staining of the majority of the tumour cells. The final score was designated as negative or positive as follows: score of 0–1, negative, score of 2–3, positive. These scores were determined independently by two senior pathologists.
Quantitative real-time PCR
Total RNA extracted using Trizol reagent (TaKaRa, Japan), and reversely transcribed through PrimeScript RT-PCR kit (TaKaRa, Japan) according to the protocol. Real-time PCR analyses were performed with SYBR Premix Ex Taq (TaKaRa, Japan) on a 7300 Real-time PCR system (Applied Biosystems, Inc. USA) at the recommended thermal cycling settings: one initial cycle at 95 °C for 10 s followed by 40 cycles of 5 s at 95 °C and 31 s at 60 °C. Primer sequences used for MCAM detection were as follows, sense: 5′-GGGTACCCCATTCCTCAAGT-3′ and antisense: 5′-CCTGGACTCCTTCATGTGGT-3′ . The expression level were normalised to the internal reference gene 18s rRNA (sense, 5′-GTAACCCGTTGAACCCCATT-3′; antisense, 5′-CCATCCAATCGGTAGTAGCG-3′) .
Western blotting and GTPase pull-down assays
Cells were lysed in lysis buffer(50 mM Tris–HCl, 150 mM NaCl, 1 % Triton-X 100, 1 Mm each MgCl2, MnCl2 and CaCl2, 1 mM PMSF and 10 mM sodium fluoride), then mixed with Laemmli buffer. Proteins were separated by SDS-PAGE under reducing condition, followed by immunoblotting with specific primary antibodies (anti-MCAM and anti-tubulin) and species-specific secondary antibodies. Bound secondary antibodies were revealed by Odyssey imaging system (LI-COR Biosciences, Lincoln, NE). GTPase pull-down assays were performed according to standard procedures as described .
Small interfering RNAs duplexes for MCAM were as follows: MCAM-si1 sense, 5′-GACUUGGACACCAUGAUAUTT-3′, anti-sense, 5′-AUAUCAUGGUGUCCAAGUCTT-3′; MCAM-si2 sense, 5′-GGUGUUGAAUCUGUCUUGUTT-3′, anti-sense, 5′-ACAAGACAGAUUCAACACCTT-3′. Transfection steps were following the manufacture’s protocols.
Cell proliferation assay and apoptosis assay
Cell proliferation Assay was tested with the CCK8 Assay. And cell death was detected by Direct TUNEL labeling assay or flow cytometric analysis of FITC Annexin V staining. All processes were according to the manufacture’s protocols.
Cell invasion assay
Seventy microlitres of 1:6 diluted Matrigel (2–3 mg/ml protein) was added into the centre of each chamber (Merck Millipore, Danvers, MA) laid in the 24 wells plate (Corning, NY). After coating in incubator for 20–30 min, 1 × 105 cells in 150 μl of defined medium were plated into upper chamber, with 600 μl of medium to the lower chamber. After culturing for approximately 48 h, the cells were fixed with 0.5 ml of 1 % glutaraldehyde in 1× PBS. Then washed each well three times with 1× PBS, and stained with 0.6 ml of 0.5 % crystal violet solution. After removing cells on the upper chamber using a cotton swab, counted the number of cells at five fields per membrane with the microscope (Axio Imager.A1).
Cell adhesion and spreading assay
Assays were performed as described previously by Zhang et al. . The area of spreading cells’ surface was measured by an image software, Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD). And in each group, at least 50 adherent cells were calculated.
The results were presented as the means and SDs. The data was subjected to Student’s t-test (two tailed; p < 0.05 was considered significant) and χ2 test was used to analyse the distribution of MCAM-positive cases in relation to clinical and pathology category variables.
MCAM expression varies among different pathological types of ovarian epithelial tissues
Patient’s clinical and pathological characteristics and their association with MCAM expression
MCAM expression (n = 111)
Normal and benign
0.001 (versus normal)
0.020 (versus normal)
<0.001 (versus normal)
0.002 (versus carcinoma)
0.548 (versus grading 1)
0.514 (versus grading 1)
Knock-down MCAM in ovarian cancer cell lines
Silencing of MCAM induced apoptosis of ovarian cancer cell
MCAM silencing inhibited in vitro invasion of ovarian cancer cells
Silencing of MCAM decreased the ability of ovarian cancer cells to spread on extracellular matrix proteins
MCAM participates in the regulation of the Rho GTPase signalling pathway
Invasion and metastasis are complex pathological processes that involve not only interactions among tumour cells and interactions between tumour cells and host cells but also the complex regulation of many molecules. Changes in the expression of cell adhesion molecule (CAMs) have been confirmed in a variety of highly invasive tumours .
It has been thought that, during the tumour metastasis, cell adhesion ability decreases, contributing to the cells dissociation from the primary site. A typical example is the reduction in the E-cadherin expression level in a variety of tumours. It was found that E-cadherin expression levels were significantly lower in ascitic and metastatic ovarian cancer cells than in the primary lesion sites of ovarian cancer, and the lower the E-cadherin expression level is, the more invasive the ovarian cancer cells are . However, not all tumour metastases are related to the down-regulation of cell adhesion molecules. It is becoming increasingly clear that many cells deviated from the solid tumour in the form of tight or loose groups . Therefore, it is hypothesised that the metastatic tumour cells that lack E-cadherin may be connected by other adhesion molecules to form a colony. In contrast to E-cadherin, another type of adhesion molecule, the immunoglobulin superfamily (including NCAM, MCAM, ALCAM and L1CAM, among others), is often highly expressed in metastatic tumour tissues .
This study focused on MCAM, a cell–cell adhesion molecule. This molecule can mediate heterotypic and homeotypic cell–cell adhesion through interaction with unknown ligands . It was reported that, in mature normal tissues, MCAM is expressed mainly in endothelial cells and smooth muscle cells , and a certain amount is also expressed in certain activated lymphocytes and bone marrow cells . Previous research showed that abnormal expression of MCAM occurs in a variety of tumours and is related to tumour development. For example, the overexpression of MCAM in melanoma cells can promote the growth and metastasis of xenograft tumours in nude mice . In contrast, MCAM expression in breast cancer is reduced . However, CD146 down-modulation is associated with the reversal of several biological characteristics leading to a less aggressive phenotype of breast cancer cells . The fact that MCAM plays different roles in different tumours reflects the complexity of cancer molecular biology.
Our research has shown that normal ovarian surface epithelial cells do not express MCAM and that the MCAM-positive tumour ratio is very low in benign ovarian tumours. However, the MCAM-positive tumour rate significantly increased in borderline ovarian tumours and malignant epithelial ovarian tumours, suggesting that MCAM expression is correlated with tumour malignancy. It is noteworthy that the MCAM-positive rate is especially high in metastatic ovarian cancer lesions, indicating that MCAM expression may be involved in the metastasis of ovarian cancer.
Furthermore, we found that when MCAM was silenced, the growth of the ovarian cancer cell lines SKOV-3 and OVCA-429 were significantly inhibited. Becker et al. reported that reducing MCAM or beta3 integrin expression in melanoma cells by RNA interference can inhibit cell growth . The specific molecular mechanisms by which MCAM affects tumour growth are not yet fully understood. We examined the cell proliferation and apoptosis and found that the apoptosis of ovarian cancer cell lines SKOV-3 and OVCA-429 were increased by silencing of MCAM. We have also found that MCAM interference in ovarian cancer cells led to a significant reduction in their in vitro invasion through Matrigel and spreading on extracellular matrix. Earlier studies have shown that the overexpression of MCAM in melanoma increased the expression of matrix metalloproteinase-2 (MMP-2), thereby contributing to the degradation of the extracellular matrix by tumour cells and promoting metastasis. Conversely, MCAM antibody blockage can down-regulate MMP-2 expression . It has been shown that the expression of MMPs was regulated by small Rho GTPases (Cdc42, Rac1 and RhoA), which are involved in many normal and pathological cellular processes, including cancer invasion and metastasis [31, 32]. Small Rho GTPases were also demonstrated to be important regulators of apoptosis in both normal and tumour cells . In this study, we found that the activities of Rho GTPases (Cdc42 and RhoA) were decreased by silencing of MCAM. Taken together, MCAM might regulate the Rho signalling pathway to promote ovarian cancer cell malignant invasion and metastasis and protect them from apoptosis.
In conclusion, we have demonstrated that MCAM have multiple effects on epithelial ovarian cancer cell properties, including invasion, apoptosis and spreading on extracellular matrix, which may be related to the dysregulation of small Rho GTPase (RhoA and Cdc42). In general, the inhibition of MCAM leads to a change in interaction among tumour cells and between tumour cells and the extracellular matrix, leading to the alterations in cancer invasion, metastasis and apoptosis. The findings above suggest that MCAM plays an important role in protecting epithelial ovarian cancer cell from apoptosis and promoting their metastasis, indicating that MCAM can be used as a potential target for the clinical treatment of epithelial ovarian cancer. More in-depth study will be required to clarify the value of MCAM in clinical applications.
The work was supported by the National Natural Science Foundation of China (81071738), the Innovation Program of Shanghai Municipal Education Commission (12YZ043) and the Shanghai Jiaotong Medical/ Engineering Foundation (YG2010MS76).
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