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- Materials and Methods
A monoclonal antibody (mAb) MY.1E12 was applied to detect MUC1 with sialylated glycans in a total of 55 formalin-fixed, paraffin-embedded surgical specimens of ovarian clear cell adenocarcinomas. A reverse correlation between the binding levels of this mAb and patient survival was demonstrated. To examine the role of MUC1 in ovarian clear cell carcinomas, two cDNA encoding MUC1 were transfected into ES-2 ovarian clear cell carcinoma cells. By comparing these cells, the role of MUC1 in tumorigenicity, chemosensitivity and survival under anoikis conditions were assessed. The results indicate that MUC1 expressed on ovarian clear cell carcinoma cells is causally involved in the malignant behavior. (Cancer Sci 2007; 98: 1586–1591)
Ovarian epithelial cancer is a highly lethal neoplasm due to its metastatic capacity to the upper abdomen and its resistance to chemotherapy in its advanced stages.(1,2) Advanced ovarian cancer is difficult to treat because it is impossible to completely resect diffuse tumors and to remove residual tumor cells by chemotherapy.(1,2) Ovarian cancer has various histological types with different clinical features and prognosis,(3) which are associated with various types of mucins and carbohydrate antigens.(4) Clear cell adenocarcinoma of the ovary, whose incidence is increasing uniquely in Japan, exhibits several characteristics.(4) Clear cell adenocarcinoma accounts for 15–20% of all cases of epithelial ovarian cancer.(4,5) Up to 60% of patients with clear cell adenocarcinoma have stage I disease.(6) Although patients with stage I ovarian cancer show a favorable prognosis, patients with clear cell adenocarcinoma are known to have a worse prognosis than those with other histological types of epithelial ovarian carcinomas.(7) However, whether subpopulations of patients with clear cell carcinoma show better or worse prognosis is not known.
MUC1 is a mucin expressed by almost all epithelial cells and many carcinomas, and its expression and distribution are known to correlate with the progression of a variety of malignant diseases, as explored extensively in our and other laboratories.(8–10) MUC1 is a transmembrane glycoprotein with a large extracellular domain that extends 200–500 nm above the cell membrane. The protein backbone of the extracellular domain consists mostly of 30–90 repeats of 20 amino acids that are highly similar.(8,11,12) Proline residues hold the protein backbone straight, whereas serine and threonine residues, which are O-glycosylated, make the structure very rigid.(13) O-Glycans of MUC1 are extended and modified in diverse ways depending on the cell type during their maturation and transport through the intercellular compartment.(14) Despite many reports on the correlation of unique glycosylation status of MUC1 with disease progression, the role of MUC1 glycoforms in ovarian cancer biology is not well understood. Dong and colleagues reported that low expression of MUC1 in the apical membrane was associated with an early stage and a good outcome of invasive ovarian tumors, though their study included very few cases of clear cell tumors.(15) In our previous studies using 17 ovarian carcinoma cell lines,(4) cell lines from ovarian clear cell adenocarcinoma expressed MUC1 but no other mucins as far as tested at the mRNA and protein levels. In contrast, cell lines derived from other histological types expressed other mucins too. Therefore, we chose ovarian clear cell adenocarcinoma cells to address the biological and clinical significance of MUC1. In the present study, we used mAb MY.1E12,(16) which was previously generated in our laboratory, to detect MUC1 with sialylated O-glycans(17) in formalin-fixed, paraffin-embedded surgical specimens of clear cell adenocarcinoma. A correlation between the level of expression of sialylated MUC1 and survival was demonstrated in ovarian clear cell adenocarcinoma.
In the present report, the significance of MUC1 expression with respect to chemosensitivity and sensitivity to apoptosis induced by loss of attachment status was also investigated by the use of established ovarian clear cell carcinoma cell lines. Chemosensitivity should represent another important aspect of diversity in the malignant behavior of ovarian clear cell carcinoma, and MUC1 overexpression on tumor cells was shown to confer resistance to anticancer drugs. Moreover, MUC1 overexpression was also shown to attenuate apoptosis induced under anoikis condition.
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- Materials and Methods
Ovarian malignancies have a worse prognosis than other gynecologic malignancies and early diagnosis is difficult due to the anatomical location and lack of symptoms at an early stage. Einhorn and colleagues pointed out that the clinical stage and histological type are important determinants of prognosis and that these factors correlate with tumor spread.(3) Dong and colleagues reported that a high expression of MUC1 in the apical membrane was associated with non-mucinous ovarian tumors, whereas a low expression of MUC1 in the same lesion was associated with an early stage and a good outcome of invasive ovarian tumors.(15) In our previous study at the mRNA, ovarian clear cell adenocarcinoma cell lines expressed MUC1 but no other mucins tested, whereas mucinous adenocarcinomas expressed MUC1 and other mucins. Interestingly, all cell lines derived from clear cell adenocarcinoma expressed MUC1 mRNA, and the glycoprotein product detected on the cell surface contained sialoglycans at a variety of levels, as differential expression was revealed by the binding of mAb MY.1E12.(4) Thus, MUC1, particularly that with sialoglycans, might influence clinical features and biological behaviors of clear cell carcinomas. Clear cell carcinomas have uniquely been increasing in Japan, and the mechanism of progression and acquired chemoresistance is not well understood. In the present study, we found that the level of MUC1 with sialoglycans recognized by mAb MY.1E12 was inversely correlated with patient prognosis. However, sialylated MUC1 expression is associated with the clinical stage of ovarian cancer, and it therefore may not be considered as an independent prognostic factor in ovarian clear cell carcinomas. In ovarian carcinoma, the degree of histological differentiation is generally classified as grade 1 through to grade 3, determined according to the increase in the proportion of solid growth within the adenocarcinoma. But no classification system has been established for the differentiation of clear cell adenocarcinoma, and all of their tumors are classified as grade 3. According to our immunohistochemical results, the level of sialylated MUC1 expression in ovarian clear cell adenocarcinoma may be available as an index of malignancy, particularly in stage I disease. Further investigation including more samples is needed.
MUC1 on tumor cells has been shown to suppress homotypic cellular aggregation,(23) to prevent cell–matrix adhesions, and to promote invasion in Matrigel.(24,25) Moreover, MUC1 was reported to inhibit cytotoxic lymphocyte–target cell interactions in vitro.(26,27) Furthermore, MUC1 was reported to induce apoptosis of lymphocytes.(28,29) Glycoforms of MUC1 associated with these functions were not previously known. Therefore, ovarian clear cell carcinoma patients with high expression of sialylated MUC1 may be at an advanced stage and have a poor prognosis. To examine the role of MUC1 in the pathobiology of ovarian clear cell adenocarcinoma, we transfected full-length cDNA encoding MUC1 with 22 and 42 tandem repeats into ES-2 cells, which are derived from ovarian clear cell adenocarcinoma and express very low levels of MUC1.(4) Although Wesseling and colleagues(24) reported that transfection of MUC1 into a melanoma cell line induced loss of adhesion to the culture dish, we did not observe such a change after MUC1 transfection into the ES-2 cell line. Moreover, morphological differences were not observed between mock- and MUC1-transfectant cells. This discrepancy was probably dependent on the cellular origin that greatly influences the glycosylation of MUC1. A role of MUC1 in regulating tumor cell growth was previously reported in several cancer types,(30–32) yet the results are contradictory among different reports. Our present results show the growth advantage of MUC1-transfectant cells only in vivo. Recently, MUC1 was shown to be associated with all four EGF receptors, and that increased MUC1 expression potentiated EGF signaling through activation of the mitogenic MAP kinase pathways.(33) Another study showed that MUC1 constitutively associates with the EGF receptor, and that EGF receptor-mediated phosphorylation of MUC1 induces its binding to c-Src tyrosine kinase and β-catenin.(34) However, it is still controversial whether these characteristics are associated with any of the MUC1 glycoforms or MUC1 on different cells with unique glycosylation. Effects of MUC1 on growth signaling cannot explain our result that MUC1-transfectant cells grow faster in vivo, but not in vitro, than mock transfectant cells. In vivo growth of tumor cells does not necessarily correspond with in vitro growth because of hypoxia, attack by natural killer cells and macrophages, and transient loss of anchorage. It is known that MUC1 secreted or expressed on plasma membranes of tumor cells interferes with natural killer cell-mediated lysis.(35) As stated later, MUC1 renders resistance to apoptosis induced by anoikis. Therefore, it is likely that MUC1 overexpression in ovarian carcinoma cells provides an advantage to in vivo tumorigenicity and growth without directly influencing growth signaling. These possibilities should be further examined in the near future.
In advanced ovarian cancer, cancer cells are shed from the primary tumor into the peritoneal cavity, followed by attachment of cells to the peritoneal mesothelium that lines the bowel and abdominal wall. It is likely that specific adhesion molecules mediate this interaction between ovarian cancer cells and the peritoneal mesothelium. Among the surface membrane molecules that are capable of mediating cell adhesion, CD44H is a major receptor for hyaluronan that was reported to be partly responsible for promoting the adhesion of ovarian cancer cells to the peritoneum.(36,37) When released from a primary tumor into the peritoneal cavity, cancer cells lose their anchorage and undergo apoptosis. Frisch and Francis first reported that apoptosis was induced by disruption of the interactions between epithelial cells and the extracellular matrix, and termed this phenomenon ‘anoikis’.(22) To understand the mechanism of peritoneal dissemination, especially the events during the spread of cancer cells through the peritoneal cavity, the survival of floating cancer cells is critical. Our present in vitro model was considered to be suitable for studying such situations, and our results demonstrated that the expression of sialylated MUC1 led to resistance to apoptosis. Alteration of the environment (i.e. from anchorage to non-adherent conditions) may induce apoptosis, whereas a high level of sialylated MUC1 may support tumor cell survival under such conditions.
Ovarian clear cell adenocarcinoma is one of the more intriguing epithelial ovarian cancers due to its distinct clinical features. Many gynecologists feel that clear cell adenocarcinoma of the ovary is less responsive to chemotherapy than the more common ovarian serous adenocarcinoma.(5,38–41) The high recurrence rate of early disease may be due to chemoresistance. In support of this, cultured clear cell adenocarcinoma cell lines were found to exhibit resistance to CDDP.(42) However, clear cell adenocarcinoma is regarded clinically as a high-grade differentiated tumor and all patients with clear cell adenocarcinoma are recommended to have adjuvant chemotherapy. Investigation of the mechanism of chemoresistance in a subpopulation limited to ovarian clear cell adenocarcinoma may provide new strategies for chemotherapy. We demonstrated that MUC1 modified the cells to be more resistant against anticancer drugs than cells without MUC1. Although CDDP and TX have different mechanisms of cytotoxity,(43,44) MUC1 expression induced resistance against both of them.
In conclusion, increased expression of MUC1 shown by the binding of mAb MY.1E12, specific for MUC1, with sialoglycans was associated with advanced stage of ovarian clear cell adenocarcinoma, and MUC1 containing sialoglycans expressed on clear cell adenocarcinoma cells influenced chemoresistance and apoptosis.