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Regulation of Minichromosome Maintenance Gene Family by Microrna-1296 and Genistein in Prostate Cancer

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Regulation of Minichromosome Maintenance Gene Family by Microrna-1296 and Genistein in Prostate Cancer Published OnlineFirst March 23, 2010; DOI: 10.1158/0008-5472.CAN-09-4176 Prevention and Epidemiology Cancer Research Regulation of Minichromosome Maintenance Gene Family by MicroRNA-1296 and Genistein in Prostate Cancer Shahana Majid1, Altaf A. Dar2, Sharanjot Saini1, Yi Chen1, Varahram Shahryari1, Jan Liu1, Mohd Saif Zaman1, Hiroshi Hirata1, Soichiro Yamamura1, Koji Ueno1, Yuichiro Tanaka1, and Rajvir Dahiya1 Abstract The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer. Cancer Res; 70(7); 2809–18. ©2010 AACR. Introduction ORC1-6–dependent, CDC6-dependent, and CDT1-dependent manner (licensing; refs. 5, 6). At the G1-S transition, the activity DNA replication occurs in a precise fashion during eukary- of two kinases, CDC7 and cyclin E/A-CDK2, recruits additional otic cell division. This tight control is orchestrated by many factors to pre-RCs, resulting in the formation of pre–initiation regulatory molecules, including members of the minichromo- complexes (pre-IC; refs. 7, 8). In addition, CDC7 and CDK2 ac- some maintenance (MCM) gene family. Initially, MCM pro- tivate the putative MCM2-MCM7 helicase, which, together teins are recruited to sites of DNA replication and interact with pre-IC formation, results in recruitment of DNA poly- with each other, forming the MCM2-MCM7 complex during merases and initiation of DNA replication. The importance the G1 phase of the cell cycle (1). This complex has helicase of the replication licensing system is highlighted by the preva- activity and facilitates DNA replication (2). Therefore, the lence of genomic instability resulting from hyperactivation of MCM proteins are essential for proliferating cells and are fre- pre-RCs during the neoplastic process and impaired cell quently upregulated in a variety of dysplastic and cancer cells growth associated with failure to assemble pre-RCs (9). (3, 4). Central to the DNA replication licensing system is the The MCM2-MCM7 helicase is thought to exist as a hexam- formation of pre–replicative complexes (pre-RC) in late M er containing individual MCM polypeptides (MCM2/3/4/5/6/ and early G1 phases and their subsequent activation at the 7). MCM2-MCM7 subunits are evolutionarily conserved and G1-S boundary. Assembly of pre-RCs requires loading of DNA essential genes, with each member possessing conserved helicase and the MCM2-MCM7 complex onto chromatin in an Walker A, Walker B, and arginine finger motifs required for ATP binding and hydrolysis. The MCM complex is implicated Authors' Affiliations: 1Department of Urology, Veterans Affairs Medical both in the unwinding of origins of DNA replication during Center and University of California at San Francisco; 2California Pacific initiation and in replication fork progression (10). In addition Medical Center Research Institute, San Francisco, California to the hexamer, MCM subcomplexes (MCM7/4/6, MCM7/4/ Note: Supplementary data for this article are available at Cancer 6/2, and MCM3/5) have also been reported (9, 11). Within Research Online (http://cancerres.aacrjournals.org/). these subcomplexes, MCM7/4/6 is thought to possess core Corrected online May 10, 2018. ATPase and DNA helicase activity, whereas MCM5/3/2 pro- Corresponding Author: Rajvir Dahiya, Urology Research Center (112F), vides regulatory roles (12). Furthermore, pairwise ATPase Veterans Affairs Medical Center and University of California at in vitro San Francisco, 4150 Clement Street, San Francisco, CA 94121. studies have revealed that MCM3/7, MCM4/7, and Phone: 415-750-6964; Fax: 415-750-6639; E-mail: MCM2/6 pairs are able to catalyze ATP hydrolysis (13). [email protected]. As a hexamer, the ATPase activity of MCM2-MCM7 is not doi: 10.1158/0008-5472.CAN-09-4176 ©2010 American Association for Cancer Research. www.aacrjournals.org 2809 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 23, 2010; DOI: 10.1158/0008-5472.CAN-09-4176 Majid et al. required for loading of the complex onto chromatin, whereas gene expression in prostate cancer. We undertook a RNA ATP hydrolysis by the hexameric complex is essential for interference (RNAi) approach to target the MCM2 gene and initiation of DNA replication (14). studied its functional effects in PC3 and LNCaP prostate Because cancer cells are highly proliferative, whereas cancer cells, comparing the results with genistein and TSA. differentiated somatic cells are usually withdrawn from the We also showed that microRNA-1296 (miR-1296) regulates cell cycle, cell proliferation markers are effective in cancer MCM2 expression. diagnosis (15). MCM proteins are good proliferation markers and are expressed at high levels in proliferating cells but not Materials and Methods in quiescent somatic cells (16). The cellular levels of these proteins remain constant throughout the cell cycle of prolif- Tissue samples and cell culture. Tissue samples from rad- erating cells (17–20), allowing identification of cancer cells in ical prostatectomy were obtained from the Veterans Affairs all stages of the cell cycle. Most importantly, because replica- Medical Center (San Francisco, CA). Informed consent was tion licensing is essential for a quiescent cell to reenter the obtained from all patients. A board-certified pathologist pro- cell cycle, the MCM proteins are expressed as G0 cells enter cessed the specimens and samples were snap frozen in liquid the G1 phase even before they engage in active DNA synthesis nitrogen and stored at −80°C. Human prostate carcinoma cell (21). Therefore, the MCM proteins can be used to identify lines (LNCaP and PC3) were obtained from the American precancerous cells before malignant transformation is Type Culture Collection. The prostate cancer cell lines were completed, providing more sensitive markers for early cultured as monolayers in RPMI 1640 supplemented with cancer diagnosis. A recent study showed that both MCM2 10% fetal bovine serum (Hyclone), 50 μg/mL penicillin, and and Ki67 are expressed at high levels in the high growth frac- 50 μg/mL streptomycin (Invitrogen) and maintained in an tion of B-cell lymphomas (22). In contrast, only MCM2, but incubator with a humidified atmosphere of 95% air and 5% not Ki67, is expressed at high levels in the low growth CO2 at 37°C. Subconfluent cells (60–70% confluent) were fraction of B-cell lymphomas, indicating that DNA replica- treated with varying concentrations of genistein (0, 25, tion licensing occurs in premalignant cells before active and 50 μmol/L; Sigma) dissolved in DMSO for 96 h or TSA proliferation (22). In the last few years, several MCM (100 ng/mL; Sigma) for 24 h, and cells treated with vehicle proteins, including MCM5 (23), MCM2 (24–28), and MCM7 (DMSO) served as control. (29, 30), are effective molecular markers for proliferating ma- RNA extraction from clinical samples and cell lines. lignant cells in tumors and precancerous cells from prema- Total RNA was extracted using a combination of Trizol lignant lesions from the lung (26, 27), kidney (31), and reagent (Invitrogen) and RNeasy columns (Qiagen). Fresh prostate (32). Furthermore, increased expression of MCM2 prostate tissues, however, were homogenized in 1 mL Trizol in prostate (32) and lung cancer cells (26, 27) has been shown reagent. After the addition of 0.2 mL chloroform, samples to correspond with poorer patient survival rates, suggesting were centrifuged for 15 min at 14,000 rpm. The aqueous that the MCM proteins are sensitive and versatile diagnostic phase was moved to a new centrifuge tube and resuspended markers for early cancer detection and promising prognostic with one-half volume of 100% ethanol. Samples were then markers for monitoring responsiveness of various cancers to applied to an RNeasy Mini column. For DNA digestion, treatment regimens. Ambion DNA-Free kit was used according to the manufac- A critical step in anticancer drug development is the turer's protocol. RNA quality was assessed using a NanoDrop identification of drug targets that are cancer cell specific, ND-1000 (NanoDrop Technologies) spectrophotometer. essential for proliferation, and with activities suitable for Extracted RNA was stored at −80°C. Total RNA was extracted high-throughput screens. The MCM complex proteins are a from 80% confluent plates of cultured cells using a miRNA group of promising candidates that meet all these criteria. easy kit (Qiagen) according to the manufacturer's directions. The coupling of expression of the MCM proteins with prolif- Quantitative real-time PCR. Mature miRs and other eration offers the potential of identifying drugs with low tox- mRNAs were assayed using Taqman MicroRNA Assays and icity.
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