DNA Replication Licensing Protein MCM10 Promotes Tumor Progression and Is a Novel Prognostic Biomarker and Potential Therapeutic Target in Breast Cancer

Abstract

Breast cancer is one of the most common malignancies in women worldwide. In breast cancer, the cell proliferation rate is known to influence the cancer malignancy. Recent studies have shown that DNA replication initiation/licensing factors are involved in cancer cell proliferation as well as cancer cell migration and invasion. Licensing factors have also been reported as important prognostic markers in lung, prostrate, and bladder cancers. Here, we studied the role of MCM10, a novel licensing factor, in breast cancer progression. From the public database, NCBI, we investigated six independent breast cancer patient cohorts, totaling 1283 patients. We observed a significant association between high MCM10 mRNA expression with tumor grading and patients' survival time. Most importantly, using breast cancer cohorts with available treatment information, we also demonstrated that a high level of MCM10 is associated with a better response to conventional treatment. Similarly, in in vitro studies, the expression level of MCM10 in breast cancer cell lines is significantly higher compared to paired normal breast epithelium cells. Knockdown of MCM10 expression in the cancer cell line showed significantly decreased tumorigenic properties such as cell proliferation, migration and anchorage independence. The MCF7 breast cancer cell line, after MCM10 expression knockdown, showed significantly decreased tumorigenic properties such as cell proliferation, migration, and anchorage independent growth. Mechanistically, MCM10 expression is observed to be regulated by an Estrogen Receptor (ER) signaling pathway, where its expression is suppressed by the inhibition of the ER or serum withdrawal. Our results suggest that MCM10 plays an important role in breast cancer progression and is a potential prognostic/predictive biomarker and therapeutic target for breast cancer patients.

Keywords: MCM10; breast cancer; knockdown; overexpression; proliferation; survival.

Figure 1

The association between MCM10 expressions, tumor grade and estrogen receptor status. Box plots showing the mean, 95% confidence interval and range of MCM10 mRNA expression in breast cancer datasets in tumors with different histologic grade in (A) GSE1456 (n = 147); (B) GSE3494 (n = 234); (C) GSE 7390 (n = 196) and (D) GSE11121 (n = 200); the association between MCM10 expressions and estrogen receptor status. Box plots showing the mean, 95% confidence interval and range of MCM10 mRNA expression in breast cancer datasets in tumors with different estrogen receptor status in (E) GSE2034 (n = 286); (F) GSE3494 (n = 232) and (G) GSE7390 (n = 198).

Figure 1

The association between MCM10 expressions, tumor grade and estrogen receptor status. Box plots showing the mean, 95% confidence interval and range of MCM10 mRNA expression in breast cancer datasets in tumors with different histologic grade in (A) GSE1456 (n = 147); (B) GSE3494 (n = 234); (C) GSE 7390 (n = 196) and (D) GSE11121 (n = 200); the association between MCM10 expressions and estrogen receptor status. Box plots showing the mean, 95% confidence interval and range of MCM10 mRNA expression in breast cancer datasets in tumors with different estrogen receptor status in (E) GSE2034 (n = 286); (F) GSE3494 (n = 232) and (G) GSE7390 (n = 198).

Figure 2

The association between MCM10 expressions and patient survival. Kaplan-Meier plots showing the proportion of patient survival for those with low or high MCM10 expression levels in breast cancer datasets (A) GSE1456 (n = 159); (B) GSE2034 (n = 286); (C) GSE3494 (n = 236); (D) GSE7390 (n = 198); (E) GSE11121 (n = 200); and (F) GSE12276 (n = 204).

Figure 3

MCM10 expression in patient tumor biopsy. (A) MCM10 expression quantified by qPCR in paired normal and tumor biopsy samples (n = 16, *** p = 0.006); (B,C) immunohistochemistry staining of patients’ biopsy, High MCM10 DAB staining is observed in patients’ biopsies; (C) compared to normal breast tissue (B). Magnified images showing nuclear staining of MCM10, in red arrows.

Figure 4

Relative expression of MCM10 in different cells in vitro. (A,B) relative protein quantification by Western blots and qPCR for the normal breast epithelial cell line MCF10A and for the breast cancer cell lines MCF 7 and MDA 231. High expression of MCM10 was found in MDA 231 cells, which are the most aggressive cells. (n = 3, * p < 0.05) (C,D) expression of MCM10 under stress induced by Fetal bovine serum (FBS) starvation in MDA231 cells. Starved MDA 231 cells showed an increase in MCM10 protein level after six hours in FBS. Interestingly, addition of proliferation inhibitor Aphidicolin further blocked expression of MCM10 observed by Western blots as well as qPCR. (n = 3, ** p < 0.01) (E,F) Tamoxifen treatment to MCF 7 cells reduced MCM10 expression in a time dependent manner observed by qPCR and Western blots. (n = 3, * p < 0.05).

Figure 5

Cancer hallmarks of stable MCM10 knockdown (KD) in MCF 7 cells. (A,B) relative expression of MCM10 in stable MCM10-KD MCF 7 cell lines assessed by qPCR and Western blots (n = 3, *** p < 0.001); (C) apoptosis analysis by Annexin V/Propidium Iodide flow cytometry showed no difference between stable MCM10-control MCF7 cell lines and stable MCM10-KD MCF 7 cell lines (n = 3, * p < 0.05); (D) following MCM10 KD, the MCM10-KD MCF7 cells showed a decrease in cell proliferation monitored for seven days using phase contrast images in Incucyte ZOOM analysis software (n = 6); (E) MTT assay using stable cells showed a similar observation indicating decreased proliferation in MCM10-KD MCF 7 cells (n = 3, * p < 0.05); (F) analysis cell cycle markers by western blot and QPCR showed a decrease in cycling D1 expression indicating impaired cell proliferation in in MCM10-KD MCF 7 cells (n = 3, * p < 0.05); (G) wound healing migration assay performed by using Incucyte ZOOM showed a decrease in cell migration in MCM10-KD MCF 7 cell lines. Wound heal quantified by relative Gap size using Incucyte ZOOM software (n = 6, * p < 0.05); (H) Transwell cell migration assay was used to confirm our observation and showed a similar decrease in the number of migrating cells in MCM10-KD MCF 7 cells (n = 3, ** p < 0.01); (I) colony formation assay performed using soft agar showed a decrease in the number of colonies in MCM10-KD MCF 7 cells compared to the control cells. (n = 3, * p < 0.05).

Figure 6

Effect of MCM10 knockdown in tumor progression in vivo. (A) tumor growth monitored by tumor volume measurement and showed a significant difference at the 12th week after grafting MCM10-KD MCF7 cell (n = 12, *** p < 0.001); (B) representative tumors at 14 weeks after grafting; (C) relative quantification of MCM10 in mice tumors (n = 3, * p < 0.05).

Figure 7

The association between MCM10 expression and response to neoadjuvant chemotherapy. Box plots showing the mean, 95% confidence interval and range of MCM10 mRNA expression in breast cancer datasets in patients with various response to chemotherapy in (A) GSE 22226 (n = 124); (B) GSE22358 (n = 122) and (C) GSE42822 (n = 91). Kaplan-Meier plots showing proportion of patients survived for those with low or high level expression of MCM10 in GSE22226; (D) the whole patient cohort; (E) patients who did not achieve pCR and (F) patients who achieved pCR.