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

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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. Their essential roles in proliferation make them Gene Expression Assays, respectively, in accordance with effective targets for drugs to kill cancer cells with high poten- the manufacturer's instructions (Applied Biosystems). All re- cy. Because cell proliferation is a highly conserved cellular verse transcription (RT) reactions, including no-template process, anti-MCM molecules may be effective against a controls and RT minus controls, were run in a 7500 Fast broad spectrum of different cancers regardless of their tissue Real Time PCR System (Applied Biosystems). Samples were or organ of origin. Although anti-MCM small molecules are normalized to RNU48 for miR expression or glyceraldehyde- most likely to prevent proliferation of cancer cells by block- 3-phosphate dehydrogenase (GAPDH) for gene expression ing replication licensing or DNA synthesis during S phase, a normalization (Applied Biosystems). Gene expression levels recent observation suggests that they may also kill cancer by were quantified using the 7500 Fast Real Time Sequence De- inducing cancer cell–specific apoptosis (33, 34). tection System software (Applied Biosystems). Comparative There are no reports on the effect of genistein, a natural, real-time PCR was done in triplicate, including no-template nontoxic dietary isoflavone, or trichostatin A (TSA), a potent controls. Relative expression was calculated using the com- anticancer drug, on the MCM gene family. In this study, parative Ct method. we show that both genistein and TSA possess an anti- Immunohistochemistry. Immunostaining was done on MCM effect by causing a significant decrease in MCM2 tissue microarray slides containing human normal adjacent

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Figure 1. The MCM gene family is overexpressed in human prostate cancer. A, relative mRNA expression levels in matched normal (N 1-6) and tumor (T 1-6) samples assessed by qRT-PCR. MCM members are significantly overexpressed in tumor samples compared with matched normal samples. *, statistically significant at P ≤ 0.05. B, 1 to 4, representative pictures of MCM2 immunohistochemical staining at distinct disease stages. 1, normal; 2, BPH; 3, primary cancer; 4, metastatic. In tumor samples, staining was uniformly confined to the nuclear compartment, with rare cytoplasmic staining. C, summarized quick score (quick score = percent cells stained × intensity of stain) results from immunohistochemistry of MCM2 in prostatic tissue arrays. prostate tissue samples (n = 15), malignant tumor (n = 72), System (Santa Cruz Biotechnology) as per the manufacturer's and metastatic carcinomas (n = 8). The slides were deparra- instructions. finized and antigen retrieval was carried out by microwaving Immunoblotting. Protein was isolated from 70% to 80% the slides in 10 mmol/L sodium citrate buffer. Slides were confluent plates of cultured cells using the M-PER Mammalian incubated overnight with anti-MCM2 antibody (Cell Signal- Protein Extraction Reagent (Pierce Biotechnology) following ing). Staining was done using the ImmunoCruz Staining the manufacturer's directions. Protein concentrations were

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Figure 2. Effect of genistein and TSA on MCM gene family. A and B, relative mRNA expression levels of MCM2 family members in PC3 and LNCaP cells as assessed by qRT-PCR after treatment with genistein (G) and TSA alone or in combination. mRNA levels of MCM members were significantly downregulated after treatment compared with vehicle control–treated cells. *, statistically significant at P ≤ 0.05. C and D, Western blot analysis showing downregulation of MCM members at the protein level after genistein, TSA, or cotreatment in PC3 and LNCaP cells. Control, vehicle control; 25G, 25 μmol/L genistein; 25G+T, 25 μmol/L genistein + 100 ng TSA; 50G, 50 μmol/L genistein; 50G+T, 50 μmol/L genistein + 100 ng TSA.

determined by the Bradford method. Equal amounts of duplexes specific for human MCM2 (Qiagen) or control protein were resolved on 10% or 15% SDS-polyacrylamide nonsilencing siRNA for 72 h. Initially, three different sets of gels and transferred to nitrocellulose membrane by voltage siRNA duplexes were tested to evaluate the target specificity gradient transfer. The resulting blots were blocked with 5% and knockdown efficiency. Two siRNA duplexes showing the nonfat dry milk and probed with antibodies specific for greatest knockdown effect were used for further experiments MCM2 (Cell Signaling), MCM3 (Cell Signaling), MCM4 (Ab- at 50 nmol/L concentration. cam), MCM5 (Abcam), MCM6 (Abcam), MCM7 (Cell Signaling), Cell cycle analysis. Cells were harvested, washed with cold CDT1 (Cell Signaling), CDK2 (Cell Signaling), CDC7 (Cell PBS, and resuspended in 4′,6-diamidino-2-phenylindole nu- Signaling), and GAPDH (Cell Signaling). Blots were then incu- clear stain (Beckman Coulter, Inc.). Fluorescence-activated bated with appropriate peroxidase-conjugated secondary cell sorting (FACS) analysis was done on stained cells imme- antibodies and visualized using enhanced chemiluminescence diately with a flow cytometer (Cell Lab Quanta SC, Beckman (Pierce Biotechnology). Coulter). MCM2 knockdown using small interfering RNA. Prostate Apoptosis assay. For measuring apoptosis, transfected cells cancer cells were plated 24 h before transfection. At 30% to were dual stained with the viability dye 7-aminoactinomycin D 50% confluence, cells were transfected using Lipofectamine (7-AAD) and Annexin V-FITC using Annexin V-FITC/7-AAD kit 2000 (Invitrogen) with small interfering RNA (siRNA) (Beckman Coulter) according to the manufacturer's protocol.

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Stained cells were immediately analyzed by flow cytometry confined to the basal proliferating layer of the prostate (Cell Lab Quanta SC). epithelium, whereas stromal cells showed no staining. The miR transfection. The day before transfection, cells were luminal differentiated cells in nonmalignant glands were plated in growth medium without antibiotics at a density rarely positive for MCM2 staining. Prostate cancer is charac- of approximately 40% to 50%. Transient transfection of terized by the loss of the basal cell layer (32). Therefore, miR-1296 at 50 nmol/L concentration was carried out with MCM2 staining was evident in the luminal epithelial cells Lipofectamine 2000 according to the manufacturer's proto- of malignant glands. Again, stromal cells were not positive col. Untransfected cells, mock, control miR, and a miR-1296 for MCM2 staining. Representative pictures of MCM2 immu- inhibitor were included as various controls. All miR transfec- nohistochemistry and quick score of immunostaining data tants were used after 72 h. are represented in Fig. 1B and C. Statistical analysis. Statistical analysis was done using Effect of genistein and TSA on the expression of MCM StatView version 5.0 for Windows as needed. The Student's gene family. Genistein and TSA alone or in combination sig- t test was used to compare the different groups. P values of nificantly downregulated the relative expression levels of all <0.05 were regarded as statistically significant and is repre- the MCM gene family members compared with the vehicle sented by an asterisk in the figures. control, although the degree of repression varied from one gene to another in both LNCaP and PC3 cells (Fig. 2A and B). To verify whether decreased transcription of these genes Results resulted in decreased levels of their respective proteins, we did Western blot analysis. Western blot analysis showed that MCM2-MCM7 expression profile. To examine the clinical the protein levels of all the MCM gene family were downre- relevance of the MCM gene family, their expression was gulated in genistein- or TSA-treated PC3 and LNCaP cells analyzed in matched carcinoma and normal prostate tissue (Fig. 2C and D; Supplementary Fig. S1). These results agree samples by Taqman quantitative real-time PCR analysis. with the quantitative RT-PCR (qRT-PCR) data and show that Almost all the carcinoma samples showed higher expression genistein and TSA downregulated transcription and transla- of all the MCM gene family members compared with their tion of the MCM gene family. matched normal samples. Expression of the MCM2 gene Knockdown of MCM2 by siRNA. To understand the role of was highest among all other members in prostate cancer MCM2 downregulation in prostate cancer, we undertook a samples (Fig. 1A). Thus, we selected this gene to further con- siRNA-mediated approach. Initially, three different sets of firm its overexpression in carcinomas by doing immuno- siRNA duplexes were tested to evaluate the target specificity histochemical staining on tissue microarrays containing and knockdown efficiency by quantitative real-time PCR human normal adjacent prostate tissue samples (n = 15), (Fig. 3A). Two siRNA duplexes (S-1 and S-2) showing the malignant tumor (n = 72), and metastatic carcinomas most efficient knockdown were used to further confirm the (n = 8). Immunohistochemistry on tissue microarrays re- efficiency at the protein level (Western blot analysis; Fig. 3B). vealed that MCM2 levels generally correlated with prostate Induction of cell cycle arrest and apoptosis. FACS anal- cancer progression (Fig. 1B). In fact, the mean value of ysis was done to test the effect of genistein and TSA on the MCM2 expression in metastatic lesions (257 ± 5) was 64.3- cell cycle, and the results were compared with that of the fold greater than in normal samples (4 ± 2.5) and 2.0-fold siRNA duplexes. As summarized in Fig. 4, genistein or TSA greater than in primary tumors (131 ± 11; Fig. 1C). Staining treatment resulted in a significant decrease in the percentage was uniformly confined to the nuclear compartment, with of cells in the S phase of the cell cycle in PC3 (24% to 4%; rare cytoplasmic staining. The cells exhibiting staining were Fig. 4A) and LNCaP (14% to 5%; Fig. 4B) cells compared with

Figure 3. Knockdown of MCM2 by siRNA. A, relative MCM2 mRNA levels assessed by qRT-PCR in PC3 and LNCaP cells transfected with 50 nmol/L siRNA duplexes (S-1, S-2, and S-3) against MCM2 and a nonsilencing siRNA duplex (NS). *, statistically significant at P ≤ 0.05. B, MCM2 protein levels were assessed by Western blot in PC3 and LNCaP cells transfected with 50 nmol/L siRNA duplexes (S-1 and S-2) and a nonsilencing siRNA duplex. All three siRNAs significantly downregulated MCM2 at the mRNA and protein level.

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Figure 4. FACS assay for cell cycle progression. A and B, effect of genistein and TSA alone or in combination on the cell cycle in PC3 and LNCaP cells. A significant decrease in the S phase of the cell cycle was observed in treated cells compared with vehicle-treated cells. C and D, cell cycle of PC3 and LNCaP cells transfected with siRNA duplexes (S-1 and S-2) showed a significant decrease in the S phase of the cell cycle. *, statistically significant at P ≤ 0.05.

vehicle-treated control. Knockdown by siRNA also showed a expression was analyzed in carcinoma (n = 23) and benign pros- decrease in the percentage of cells in S phase from 22% to 2% tate hyperplasia (BPH; n = 24) tissue samples. Almost all the car- (PC3 cells; Fig. 4B) or 14% to 6% (LNCaP cells; Fig. 4B) com- cinoma samples showed highly reduced miR-1296 expression pared with the nonsilencing siRNA control. There was no with respect to the BPH samples, and an overall lower relative change in the apoptotic population by genistein/TSA or average expression was observed in carcinoma compared with siRNA duplexes (results not shown). BPH samples (Fig. 6A). Transient transfection with miR-1296 at Effect of genistein and TSA on the expression of genes 50 nmol/L concentration in PC3 prostate cancer cells for 72 that are important for loading of the MCM complex onto hours resulted in a 500,000-fold induction in miR-1296 expres- chromatin to facilitate DNA replication. Central to the sion in comparison with the control miR (data not shown). DNA replication licensing system is the formation of pre- miR-1296 targets MCM2. To find out whether MCM2 is a RCs. The assembly of pre-RCs requires loading of the target of miR-1296, we used online search tools miRanda and MCM2-MCM7 complex onto chromatin, which depends on identified a target site with high complementarity to miR- CDT1, CDC7,andCDK2 genes. To check the effect of genis- 1296 in 3′-untranslated region (UTR) of MCM2. Overall, the tein/TSA on these genes, we did quantitative real-time PCR alignment score is 140, with a perfect match to the seed hep- and Western blot assays. In PC3 cells, genistein and TSA alone tamer (Fig. 6B, 1). MCM2 proved to be sensitive to miR-1296, or in combination significantly downregulated the expression and a significant decrease (51%) in MCM2 mRNA expression of CDK2, CDC7, and CDT1 genes at mRNA (Fig. 5A–C) and pro- levels was observed in miR-1296–transfected cells (Fig. 6B, 2). tein (Fig. 5D) levels compared with the vehicle control. These Western blot analysis confirmed that MCM2 protein levels results reveal that genistein and TSA have an inhibitory effect were also reduced in response to miR-1296 transfection on the DNA replication licensing machinery. (Fig. 6B, 3). Taken together, these data suggest that miR- miR-1296 expression is downregulated in prostate 1296 reduces MCM2 expression by inhibiting translation cancer. To examine the clinical relevance of miR-1296, its and/or causing mRNA instability/decay.

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We also transfected cells with a miR-1296 inhibitor (anti– erating cells versus quiescent somatic cells (16), thus allowing miR-1296; Applied Biosystems) designed specifically to bind for the identification of malignant cancer cells. In this study, and sequester mature miR-1296. The miR-1296 inhibitor we show that both genistein and TSA have an anti-MCM com- helped preserve MCM2 expression at both the mRNA and plex effect, causing the highest decrease in expression of the protein levels (Fig. 6B, 2 and 3). Taken together, these MCM2 in prostate cancer cells. We also used a RNAi approach results indicate that the MCM2 transcript is a target of to target the MCM2 gene to examine its functional aspects in miR-1296. prostate cancer cells and compare the results with that of gen- miR-1296 decreases the number of S-phase cells. FACS istein and TSA. We found that miR-1296 is downregulated in analysis was done on miR-1296–transfected PC3 cells to test prostate cancer and that MCM2 is one of its targets. its effect on the cell cycle. miR-1296 caused a significant de- Genistein is believed to be a potent anticancer agent and crease in the percentage of S-phase cells (from 15% to 2%) com- has been shown to have antitumor effects in animal models pared with control miR, untransfected, and mock-transfected (35). Various soy products containing genistein have been controls (Fig. 6C). We also did a quantitative real-time PCR found to inhibit the growth of transplanted human prostate assay to check the effect of genistein on miR-1296 expression. carcinoma, reduce the incidence of poorly differentiated A synergistic effect of genistein on miR-1296 was observed, as prostate adenocarcinoma in a transgenic mouse model, and there was an increase in miR-1296 expression of 3- to 5-fold inhibit 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine– with 25 or 50 μmol/L genistein (Fig. 6D). induced rat prostate cancer (36). Carcinogenesis or metas- tasis to the stomach, colon, bladder, and lung is also inhibited Discussion by genistein and related isoflavones (37–39). TSA, a histone deacetylase inhibitor and a potent anticancer drug, is cur- Cancer cells are highly proliferative; therefore, cell prolifer- rently undergoing clinical trials as a prostate cancer treat- ation markers are very effective and attractive as cancer ment but has high toxicity. Currently, there are no reports diagnostic markers (15). At present, MCM proteins are very on the effect of genistein, a natural, nontoxic dietary isofla- good proliferation markers and are highly expressed in prolif- vone, or TSA on the MCM gene family. We found expression

Figure 5. Effect of genistein and TSA on genes that are important for loading the MCM complex onto chromatin for DNA replication. A to C, relative mRNA expression levels of CDK2, CDC7, and CDT1 in genistein- or TSA-treated PC3 cells. All three genes were highly downregulated in PC3 cells after different treatments. *, statistically significant at P ≤ 0.05. D, Western blot analysis showing downregulation of CDT1, CDC7, and CDK2 at protein levels in genistein- or TSA-treated PC3 cells. GAPDH was used as the loading control.

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Figure 6. miR-1296 status in human prostate cancer cell lines. A, relative miR-1296 expression levels in BPH (n = 24) and prostate tumors samples (n = 23) as assessed by qRT-PCR. —, average value. B, miR-1296 targets MCM2. 1, seed sequence of the miR-1296 target sites located in the 3′-UTR of the MCM2 gene; 2, expression of MCM2 mRNA in PC3 cells transfected with miR-1296, miR-1296 inhibitor, or a control miR (Cont-miR); 3, expression of MCM2 protein in PC3 cells transfected with miR-1296, miR-1296 inhibitor, or a control miR. C, effect of miR-1296 on PC3 cell cycle distribution. D, effect of genistein on miR-1296 expression in PC3 cells. *Statistically significant at P ≤ 0.05.

of MCM genes to be higher in prostate cancer compared they enter the S phase with underlicensed G1 chromatin and with normal tissues. The MCM proteins are essential DNA are eliminated (40). replication factors that are highly expressed in malignant The loading of the MCM2-MCM7 complex is dependent on cancer and precancerous cells but downregulated in differ- CDT1 (licensing; refs. 5, 6), CDC7, and CDK2 that recruit ad- entiated somatic cells (16, 26, 27, 32). Treatment with gen- ditional factors to pre-RCs, resulting in the formation of pre- istein or TSA alone or in combination significantly ICs (7, 8). In addition, CDC7 and CDK2 activate the putative downregulated the expression of the MCM gene family in MCM2-MCM7 helicase, which, together with pre-IC forma- both androgen-dependent LNCaP or androgen-independent tion, results in recruitment of DNA polymerases and initia- PC3 cells. tion of DNA replication. Our results show that both Drugs targeting MCM proteins are likely to prevent the genistein and TSA alone or in combination significantly proliferation of cancer cells by blocking replication licensing downregulated the expression of CDK2, CDC7,andCDT1 or DNA synthesis during the S phase (40). Our results show genes at the mRNA and protein levels compared with the ve- that genistein, TSA, and siRNA decreased the S phase of the hicle control. These results show that genistein and TSA have cell cycle in both LNCaP and PC3 prostate cancer cells. It has inhibitory effects on the DNA replication licensing machinery been reported that when the negative regulator of replication in prostate cancer. licensing, geminin, is overexpressed in human primary cells, miRs are a class of naturally occurring small noncoding loading of the MCM complex to the chromatin is prevented RNAs that control gene expression by binding to sites in and the cells are blocked in the G1 phase of the cell cycle (33, the 3′-UTRs of target transcripts, resulting in translational 34). A “licensing checkpoint” may exist in normal human arrest and, in some instances, transcript degradation (41). cells, which blocks S-phase entry if replication licensing is in- Recent estimates suggest that one third of human mRNAs complete or compromised (33, 34). Cancer cells, on the other may be regulated by miRs (42). Studies have found that hand, are defective in this checkpoint. When treated with miR expression patterns are significantly different in normal anti-MCM drugs that compromise DNA replication licensing, and neoplastic tissues, suggesting that miRs may play a role

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Regulation of MCM Gene Family in Prostate Cancer

in tumorigenesis (43). In human cancers, some miRs may has anti-MCM effects in prostate cancer. We also found that have an oncogenic function due to their overexpression in MCM2 is a target of miR-1296. To our knowledge, this is the malignant tissue, whereas certain miRs may act as tumor first report showing the effect of genistein on the MCM li- suppressor genes because of decreased expression (43). Dif- censing gene family in prostate cancer. TSA is currently un- ferential expression of miRs has been reported in prostate dergoing clinical trials as a prostate cancer treatment but has cancer (44). We searched the online database to look for high toxicity. Genistein, a natural, nontoxic dietary isofla- miRs that regulate MCM2 and found that miR-1296 has a per- vone, may be an advantageous therapeutic agent for treating fect seed complimentary sequence in the 3′-UTR of the prostate cancer. The use of RNAi is currently being imple- MCM2 gene. The expression of miR-1296 was significantly mented as a gene-specific approach for molecular medicine. lower in prostate tumors than BPH tissues. Transfection of By the same principle, the specific downregulation of onco- miR-1296 in PC3 prostate cancer cells decreased the expres- genic genes by miR may contribute to the growing therapeu- sion of MCM2 mRNA and protein. To further validate that tic potential that small RNA-based drugs have in the miR-1296 inhibits MCM2 expression in prostate cancer, we treatment of cancer and other diseases. It is possible that transfected PC3 cells with the miR-1296 inhibitor, which is stimulation of silenced miRs by drug treatment may lead designed specifically to bind and sequester the mature to the downregulation of target oncogenes, thereby contrib- miR-1296 sequence. MCM2 mRNA expression in miR-1296 in- uting to novel therapeutic approaches in the treatment of hibitor–transfected cells was upregulated relative to control prostate cancer. miR–tansfected cells. MCM2 protein expression was also upregulated in miR-1296 inhibitor–transfected cells. These re- Disclosure of Potential Conflicts of Interest sults show that inhibition of miR-1296 upregulated MCM2 mRNA and protein expression in prostate cancer cells, con- No potential conflicts of interest were disclosed. firming that miR-1296 inhibits expression of the MCM2 gene. Acknowledgments We also observed a significant decrease in S-phase cells in response to miR-1296 compared with controls. Genistein We thank Dr. Roger Erickson for his support and assistance with the had a positive effect on miR-1296, as there was an increase preparation of the manuscript. of 3- to 5-fold in miR-1296 expression compared with vehicle control. Grant Support MCM proteins are sensitive markers for early cancer diag- NIH grants RO1CA 111470 and T32DK007790, Veterans Affairs Research nosis. Their essential role in cancer cell proliferation makes Enhancement Award Program, and Merit Review. them attractive therapeutic drug targets. Because cell prolif- The costs of publication of this article were defrayed in part by the payment eration is a highly conserved cellular process, anti-MCM of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. drugs may be effective against a broad spectrum of cancers, regardless of their organ or tissue of origin. Our study clearly Received 11/19/2009; revised 01/15/2010; accepted 01/20/2010; published showed that genistein, a natural, nontoxic dietary isoflavone, OnlineFirst 03/23/2010.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Cancer Correction Research Correction: Regulation of Minichromosome Maintenance Gene Family by MicroRNA-1296 and Genistein in Prostate Cancer

In the original version of this article (1), the incorrect GAPDH loading control for the Western blot in Fig. 5D was published. The error has now been corrected in the recent online HTML and PDF versions of the article. The authors regret this error.

Reference 1. Majid S, Dar AA, Saini S, Chen Y, Shahryari V, Liu J, et al. Regulation of minichromosome maintenance gene family by microRNA-1296 and genistein in prostate cancer. Cancer Res 2010;70:2809–18.

Published online July 2, 2018. doi: 10.1158/0008-5472.CAN-18-1117 Ó2018 American Association for Cancer Research.

3740 Cancer Res; 78(13) July 1, 2018 Published OnlineFirst March 23, 2010; DOI: 10.1158/0008-5472.CAN-09-4176

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

Shahana Majid, Altaf A. Dar, Sharanjot Saini, et al.

Cancer Res 2010;70:2809-2818. Published OnlineFirst March 23, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-4176

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