Subcellular localization of MCM2 correlates with the prognosis of ovarian clear cell carcinoma

Abstract

Highly malignant tumors overexpress the minichromosome maintenance 2 (MCM2) protein in the nucleus, which is associated with advanced tumor grade, advanced stage, and poor prognosis. In this study, we showed that MCM2 is highly expressed in clinical samples of ovarian clear cell carcinoma. Although MCM2 expression was mainly localized to the nuclei as in other cancers, a few cases exhibited cytoplasmic localization of MCM2. Surprisingly, tumor samples with cytoplasmic MCM2 demonstrated excellent prognosis, showing 100% survival during the observation period of more than 200 months. However, cases with nuclear expression of MCM2 exhibited approximately 78% 5-year-survival rate. In a previous study, we showed that Friend leukemia virus (FLV) envelope protein gp70 bound to MCM2, impaired its nuclear translocation, and enhanced DNA damage-induced apoptosis in FLV-infected hematopoietic cells with high levels of MCM2. As expected, clear cell carcinoma cells with cytoplasmic expression of MCM2 exhibited significantly higher apoptotic cell ratio than that of cells with nuclear MCM2 expression. In vitro experiments using ovarian cancer cells with cytoplasmic expression of MCM2 demonstrated that transfection of MCM2-ΔN enhanced DNA damage-induced apoptosis. Therefore, cytoplasmic localization of MCM2 significantly correlated with increased apoptosis in clear cell carcinoma cells, resulting in improved prognosis.

Keywords: MCM2; cancer therapy target; clinicopathologic study; ovarian cancer; pathology.

Figure 1

Histological features by hematoxylin and eosin (H&E) staining (A, C, E, G) and the immunohistochemical expression patterns of minichromosome maintenance 2 (MCM2; (B, D, F, H) in serous carcinoma, endometrioid carcinoma, and clear cell carcinoma using the monoclonal antibody BM28. Magnification of all images shown is ×400, and scale bar indicates 50 μm. Although MCM2 was primarily localized to the nuclei of cancer cells (B, D, F), a subset of clear cell carcinoma cases exhibited cytoplasmic MCM2 expression in addition to nuclear staining (H). Clear cell carcinoma with nuclear expression of MCM2 is designated as CCC-N, whereas clear cell carcinoma showing cytoplasmic/nuclear localization of MCM2 is indicated as CCC-C.

Figure 2. Overall survival curves for clear cell carcinoma with nuclear expression of MCM2 (CCC-N) and clear cell carcinoma with cytoplasmic/nuclear expression of MCM2 CCC-C cases

As indicated, CCC-C cases exhibited a 100% 5-year survival rate, whereas CCC-N cases had a 5-year survival rate of 78%. Kaplan-Meier estimation of survival curves revealed significant differences in overall survival between CCC-N and CCC-C cases (P < 0.05).

Figure 3. Immunohistochemical staining for CC3

(A) and the frequency of CC3-positive cells in clear cell carcinoma with nuclear expression of MCM2 (CCC-N; n = 55) and clear cell carcinoma with cytoplasmic/nuclear expression of MCM2 (CCC-C; n = 22) cases (B). CC3 was frequently positive in the nuclei of CCC-C cases. The frequency of CC3-positive cells in CCC-C was significantly higher than that in CCC-N (B, P < 0.01). P values were calculated using Student's t-test

Figure 4. Transfection of 3×FLAG-MCM2-ΔN that lacks the NLS domain of minichromosome maintenance 2 (MCM2) resulted in the cytoplasmic expression of MCM2 in OVTOKO and OVISE cells

(A) Schematic diagram of full-length MCM2 (MCM2-FL) and MCM2 deletion mutants [MCM2-ΔN (aa 156–904)]. (B) Western blot analysis to confirm that both 3×FLAG-MCM2-FL and 3×FLAG-MCM2-ΔN were expressed as the appropriate-size protein. (C, D) Subcellular localization of transduced MCM-FL and MCM-ΔN in OBTOKO and OVISE cells was demonstrated by fluorescent immunostaining using an anti-FLAG antibody. MCM-FL was localized to the nucleus, while MCM2-ΔN was expressed in both cytoplasm and nucleus of OVTOKO (C) and OVISE (D) cells.

Figure 4. Transfection of 3×FLAG-MCM2-ΔN that lacks the NLS domain of minichromosome maintenance 2 (MCM2) resulted in the cytoplasmic expression of MCM2 in OVTOKO and OVISE cells

(A) Schematic diagram of full-length MCM2 (MCM2-FL) and MCM2 deletion mutants [MCM2-ΔN (aa 156–904)]. (B) Western blot analysis to confirm that both 3×FLAG-MCM2-FL and 3×FLAG-MCM2-ΔN were expressed as the appropriate-size protein. (C, D) Subcellular localization of transduced MCM-FL and MCM-ΔN in OBTOKO and OVISE cells was demonstrated by fluorescent immunostaining using an anti-FLAG antibody. MCM-FL was localized to the nucleus, while MCM2-ΔN was expressed in both cytoplasm and nucleus of OVTOKO (C) and OVISE (D) cells.

Figure 5. The apoptotic cell ratio induced by cisplatin treatment in full-length minichromosome maintenance 2 (MCM2-FL)- and NLS domain-deficient MCM2 (MCM2-ΔN)-transduced cells was determined by FACS analysis after annexin V staining

P values were calculated using Student's t-test. The annexin V-positive cell ratio was assessed in control and MCM2-FL- and MCM2-ΔN-transduced OVTOKO and OVISE cells with or without cisplatin treatment. The transfection of MCM2-FL and MCM2-ΔN resulted in a significant increase of the apoptotic cell ratio in OVTOKO cells compared with control cells. In contrast, MCM2-FL and MCM2-ΔN transfection did not cause a significant increase of apoptotic cells in OVISE cells. After treatment with cisplatin, cells with MCM2-ΔN transduction exhibited significantly stronger induction of apoptosis than MCM-FL-transduced OVTOKO and OVISE cells did. Each experiment was performed three times.