Introduction
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 [
1]. 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 [
2].
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 [
3]. 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 [
4]. Interestingly, the elevated expression of certain other adhesion molecules, such as
p-cadherin, can promote ovarian cancer metastasis [
5]. 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 [
6]. 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 [
7]. 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 [
8], prostate cancer [
9], breast cancer [
10] and non-small cell lung cancer [
11] 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 [
12].
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.
Discussion
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 [
20].
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 [
21]. 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 [
22]. 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 [
23].
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 [
24]. It was reported that, in mature normal tissues, MCAM is expressed mainly in endothelial cells and smooth muscle cells [
25], and a certain amount is also expressed in certain activated lymphocytes and bone marrow cells [
26]. 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 [
27]. In contrast, MCAM expression in breast cancer is reduced [
28]. However, CD146 down-modulation is associated with the reversal of several biological characteristics leading to a less aggressive phenotype of breast cancer cells [
10]. 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 [
29]. 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 [
30]. 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 [
33]. 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.