Background
Epithelial ovarian cancer (EOC) is the fifth leading cause of female cancers in USA with a high fatality rate (about 65 %) [
1]. The high lethality of the cancer is because the early stage of the disease is mostly asymptomatic and therefore remains undiagnosed until the cancer has already disseminated throughout the peritoneal cavity [
2]. The early stage disease can be treated successfully, however, effective therapy for the advanced-stage disease is lacking because of the strong chemo-resistance of recurrent ovarian cancer [
2]. The major challenges for combating ovarian cancer are: (a) the ovarian cancer is histologically and molecularly heterogeneous with at least four major subtypes [
3,
4], (b) there is a lack of reliable specific diagnostic markers for an effective early diagnosis of each subtype, though molecular signatures of the major subtypes are available [
5], and (c) very little is known of how ovarian tumor emerges and how it progresses to malignancy ([
6] for a review).
In general, tumorigenesis is a complex process involving changes of several biological characteristics [
7], including the aberrant expression of cell adhesion molecules [
8]. Tumor progression is induced by a complex cross-talk between tumor cells and stromal cells in the surrounding tissues [
8]. These interactions are, at least in part, mediated by cell adhesion molecules (CAMs), which govern the social behaviors of cells by affecting the adhesion status of cells and cross-talk and modulating intracellular signal transduction pathways [
8]. Thus the altered expression of CAMs can change motility and invasiveness, affect survival and growth of tumor cells, and alter angiogenesis [
8]. As such, CAMs may promote or suppress the metastatic potential of tumor cells [
9]. Aberrant expression of various CAMs, such as mucins [
10], integrins [
11], CD44 [
12], L1CAM [
13], E-cadherin [
14], claudin-3 [
15], EpCAM [
16], and METCAM/MUC18 [
17,
18], has been associated with the malignant progression of ovarian cancer.
We have been focusing our studies on the possible role of METCAM/MUC18 in the progression of several epithelial tumors [
19]. Human METCAM/MUC18 (or MCAM, Mel-CAM, S-endo1, or CD146), an integral membrane cell adhesion molecule (CAM) in the Ig-like gene superfamily, has an N-terminal extra-cellular domain of 558 amino acids, a transmembrane domain, and a short intra-cellular cytoplasmic domain (64 amino acids) at the C-terminus [
19,
20]. The extra-cellular domain of the protein comprises a signal peptide sequence and five immunoglobulin-like domains and one X domain [
19,
20]. The cytoplasmic domain contains five consensus sequences potentially to be phosphorylated by PKA, PKC, and CK2 [
19,
20]. Thus human METCAM/MUC18 is capable of performing typical functions of CAMs, such as governing the social behaviors by affecting the adhesion status of cells and modulating cell signaling. Therefore, an altered expression of METCAM/MUC18 may affect motility and invasiveness of many tumor cells in vitro and tumorigenesis and metastasis in vivo [
19].
Human METCAM/MUC18 is only expressed in several normal tissues, such as hair follicular cells, smooth muscle cells, endothelial cells, cerebellum, normal mammary epithelial cells, basal cells of the lung, activated T cells, and intermediate trophoblasts [
19,
21]. Human METCAM/MUC18 is also expressed in several epithelial tumors, such as melanoma, prostate cancer, osteosarcoma, breast carcinoma, and intermediate trophoblast tumors [
19,
21]. Over-expression of METCAM/MUC18 promotes the tumorigenesis of prostate cancer [
22] and breast carcinoma [
23,
24], but it has a minimal effect on the tumorigenesis of melanoma [
25]. Over-expression of METCAM/MUC18 also initiates the metastasis of prostate cancer [
26] and promotes the metastasis of melanoma [
25] and breast carcinoma [
27].
On the contrary, the possibility that the over-expression of METCAM/MUC18 might play a tumor suppressor role was first suggested by Shih et al. [
28], who found that METCAM/MUC18 expression suppressed tumorigenesis of a breast cancer cell line MCF-7 in SCID mice. However, this notion was contradicted by recently published evidence, which supported the positive role of METCAM/MUC18 in the progression of breast cancer cells [
23,
24,
27], similar to its role in the progression of melanoma and prostate cancer cells.
The role of METCAM/MUC18 in the progression of ovarian cancer has not been well studied, except that the METCAM/MUC18 expression has been recently reported to correlate with the progression of ovarian cancer [
17,
18], and perhaps affects the behaviors of ovarian cancer cells [
29]. To directly test the role of METCAM/MUC18 in the progression of epithelial ovarian cancer, we first chose to use SK-OV-3 cells for testing the effect of over-expression of METCAM/MUC18 on in vitro motility and invasiveness, in vivo tumor formation in nude mice after subcutaneous (
SC) injection, and in vivo progression in nude mice after intraperitoneal (
IP) injection. We found that the over-expression of METCAM/MUC18 inhibited in vitro motility and invasiveness and suppressed in vivo tumorigenesis and the malignant progression of the human ovarian cancer cell line SK-OV-3. We conclude that METCAM/MUC18 is a novel tumor and metastasis suppressor for the progression of human ovarian cancer cells.
Discussion
In this study, we initiated the investigation by determining expression levels of METCAM/MUC18 in several ovarian cancer cell lines. We found that METCAM/MUC18 was expressed at a level of 31–50 % in two out of three cell lines established from primary adenocarcinomas (HEY and CAOV3), but poorly expressed (1–11 %) in two cell lines established from malignant ascites (SKOV3 and NIHOVCAR3). It appeared that METCAM/MUC18 was expressed poorer in malignant cell lines than in primary adenocarcinomas, suggesting that METCAM/MUC18 may play a negative role in the progression of ovarian cancer. To further support this hypothesis, we provided in vitro evidence to show that a high expression level of METCAM/MUC18 inhibited the migration and invasion of SKOV3 cancer cells. We also provided in vivo evidence in animal tests to show that METCAM/MUC18 expression inhibited the tumorigenicity at the subcutaneous sites as well as the tumorigenicity and ascites formation in the intra-peritoneal cavity of an athymic nude mouse model. Since the METCAM/MUC18 expressed in the tumors and ascites cells were similar to that in the injected clones/cells, the protein was not modified to manifest these processes. Taken together, we conclude that METCAM/MUC18 serves as a tumor suppressor as well as a metastasis suppressor for the human ovarian cancer cells SK-OV-3. METCAM/MUC18 may suppress tumorigenesis and malignant progression of ovarian cancer cells in nude mice by decreasing their abilities in proliferation, aerobic glycolysis, and angiogenesis, and by decreasing their abilities in EMT, but not altering the apoptosis/anti-apoptosis and survival pathways.
This conclusion contradicts the results of a positive correlation of clinical prognosis with the increased expression of METCAM/MUC18 in malignant ovarian cancer specimens [
17,
18,
29]. This suggests that the positive correlation in this case is fortuitous and that we should not assume a positive role of METCAM/MUC18 in the progression of ovarian cancer without the support of tests in an animal model. Our results also contradict the previously established notion that METCAM/MUC18 serves as a tumor promoter in both prostate cancer cells [
22] and breast cancer cells [
23,
24], and as a metastasis promoter in human melanoma cells [
25], prostate cancer [
26], and breast cancer [
27]. The conclusion, nevertheless, appears to be consistent with the first notion suggested by one group that METCAM/MUC18 is a tumor suppressor in human breast cancer cell line MCF-7 [
28]; albeit the notion was later proven to contradict to the evidence from two different groups [
23,
24,
27]. Regardless, the role of METCAM/MUC18 as a tumor suppressor was not only conclusively demonstrated in a human ovarian cancer cell line, SK-OV-3 (as shown here), but also in another human ovarian cancer cell line BG-1 [Wu, unpublished results], as well as in a mouse melanoma cell line, K1735-9 [
34] and one NPC cell line, NPC-TW01 ([
35,
36], & Wu, unpublished results). METCAM/MUC18 has also been demonstrated as a metastasis suppressor in the two human ovarian cancer cell lines, SK-OV-3 (as also shown here) and BG-1 [Wu, unpublished results], and one mouse melanoma cell line, K1735-9 [
34]. Thus sufficient evidence is provided to support the novel suppressor role of METCAM/MCU18 in the progression of these human cancers.
E-cadherin, a cell adhesion molecule, has been demonstrated as a tumor suppressor role in many tumors derived from epithelium; however, E-cadherin has not been found to play a tumor or metastasis promoter role in any tumor [
8]. Thus the most intriguing, unique biological function of METCAM/MUC18 in tumorigenesis and metastasis is that it seems to play a dual role in the progression of some tumor cell lines. It can be a tumor/metastasis promoter in prostate cancer cell lines [
22,
26], breast cancer cell lines [
23,
24,
27], and most melanoma cell lines [
19,
25,
34]. It can also be a tumor/metastasis suppressor in the progression of other tumor cell lines in animal studies, such as, two ovarian cancer cell lines (in this report and Wu, unpublished results), one mouse melanoma subline ([
34] and Wu, unpublished results), nasopharyngeal carcinoma ([
35,
36] and Wu, unpublished results), and perhaps hemangioma [
37]. It is not clear why METCAM/MUC18 plays a dual role in tumorigenicity and metastasis. One point is clear, which is that METCAM/MUC18 plays an opposite role in different cancer types or in different clones/sublines of the same cancer type [
38]. Thus it is logical to propose that the effect of METCAM/MUC18 on the progression of epithelial cancers is modulated by different intrinsic factors in different tumor cells/types. The dual role of METCAM/MUC18 is very likely due to the presence of different interacting partners intrinsic to each cancer cell type and different clone, or perhaps due to different heterophilic ligands, which unfortunately have not been identified [
19,
34,
38]. Interactions of METCAM/MUC18 with different sets of intrinsic partners may result in the promotion or suppression of tumorigenicity and metastasis via increasing or decreasing aerobic glycolysis, proliferation, angiogenesis, other growth-promoting pathways, as well as altering tumor cell motility, invasiveness, and vascular metastasis, as suggested in this report. In the future, the identification of these partners and/or ligands is essential to understand further detailed mechanisms.
Interestingly, many molecules have recently been shown to play a dual role in the progression of cancer. The most well-known examples are TGF-β, which is context dependent and acts as a tumor suppressor in the early stage of tumorigenesis, but as a progression promoter in the late stage [
7], and VEGF, which also plays a dual role in tumor progression [
39].
One point worth noting is that the tumors induced by the METCAM clone 2D were confined to small regions, as shown in the results of H&E and IHC, whereas the tumors induced by the control (vector) clone 3D developed serious tumors, suggesting that tumors from the 2D clone appeared to be dormant; thus METCAM/MUC18 may function similarly to other tumor suppressors in other tumor cells [
40].
Another point also worth noting is that tumorigenicity of the control (vector) clone 3D in the dorsal site appeared to be significantly better than that in the ventral site (P value = 0.016), whereas tumorigenicity of the 2D clone in the ventral site was significantly better than that in the dorsal site (P value = 0.024). We don’t know why different SC sites have different effects on tumorigenicity. This also requires further investigation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GJW conceived of the idea and study, participated in its design and coordination, carried out in vivo animal studies, performed the statistical analysis, and revised the manuscript many times suitable for publication. GFZ carried out western blots analyses, migration and invasion studies, and colony formation study, participated in in vivo animal studies, performed the statistical analysis, and drafted the manuscript. Both authors read and approved the final manuscript.