Mini-chromosome-maintenance-deficient 4 (MCM4) in breast cancer and its clinical significance

Abstract

MCM4 is a subunit of the MCM replication helicase complex (MCM2-7). The hexameric helicase complex plays an essential role in DNA replication and is also involved in regulating genome stability and DNA damage response. Previously, a murine hypomorphic Mcm4 mutant, Phe345Iso, was demonstrated to develop mammary adenocarcinomas in mouse. Another MCM complex subunit, MCM6, was found being up-regulated by estradiol treatment. Expression of MCM4 was found significantly elevated in cervical cancer cells, and the expression was associated with higher tumor staging in esophageal cancer. We hypothesized that MCM4 might be differentially expressed in breast cancers. To examine the expression of MCM4 protein, tissue microarrays containing 112 breast cancers of which 31 cases were paired with non-tumor counterparts were stained for protein expression using monoclonal anti-MCM4 antibody. MCM4 was found to be predominantly expressed in the nucleus. Semi-quantitative assessment showed nuclear score (reflected by percentage of nuclei stained) was significantly higher in tumors compared with non-tumors (p<0.001). Higher nuclear score was significantly associated with higher tumor grade and inversely associated with estrogen receptor (ESR) status. MCM4 mRNA expression was examined in 35 breast cancer cases with paired frozen tumor and non-tumor tissues. Significantly higher MCM4 expression was found in tumors. MCM4 was also expressed at a high level in 13 breast cancer cell lines. To investigate whether the observed aberrant expression pattern might be due to genetic and epigenetic alterations, gene amplification using quantitative PCR and promoter methylation using bisulfite sequencing methods were examined; however, both of these mechanisms were found not associated with the aberrant expression pattern of MCM4 in the breast tumors and/or cancer cell lines. Knockdown of MCM4 in ESR-alpha positive MCF7 cell line by transient transfection of MCM4 shRNA caused reduction of cell viability while the expression of other MCM family genes, such as MCM2 and 7, were substantially reduced. This however did not affect the expression of ESR-alpha, suggesting that ESR-alpha is unlikely to be a downstream target of MCM4. In contrast, estradiol treated MCF7 was found to have high levels of MCM4 expression. Our results suggest that MCM4 might play a role in breast carcinogenesis. Loss of MCM4 might affect the equilibrium and stability of other MCM family proteins. MCM4 might interact with ESR-alpha and estradiol in the development of the cancer. The current study opens a new avenue for studying the role of MCM4 in breast cancer. References Calaf GM et al. Mol Med, 2007; 13(5-6):255-256. Huang XP et al. Cancer Res Clin Oncol. 2005; 131, 677-682. Ishimi Y et al. J. Biochem. 2003; 270, 1089-1101. Shima N et al. Nature Genetics. 2007; 39, 1, 93-98.