Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell-cycle G0–G1 arrest as well as downregulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In the current study, the potential ISO inhibition of bladder tumor formation has been explored in a xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anticancer activities have been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by directly targeting Sp1 mRNA 3′-untranslated region (UTR). Similar to ISO treatment, ectopic expression of miR-137 alone led to G0–G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by overexpression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced inhibition of Sp1/Cyclin D1 expression, induction of G0–G1 cell growth arrest, and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3′-UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0–G1 growth arrest, and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anticancer activity of ISO in the therapy of human bladder cancer. Mol Cancer Ther; 15(3); 512–22. ©2016 AACR.
Bladder carcinoma is highly prevalent and the second most common genitourinary malignant disease in the United States (1). Bladder carcinoma is threatening for human beings when it invades muscles. Among all the cancer types, bladder carcinoma ranks the fifth in total healthcare cost with an annual expenditure of approximately $4 billion in the United States alone (2). Although MVAC (methotrexate, vinblastine, adriamycin, and cisplatin) chemotherapy has been widely used for treatment of advanced bladder cancers, it is accompanied with major toxic side effects (3). Therefore, development of less toxic alternate chemotherapeutic therapies and/or dietary management strategies is of high significance for prevention and therapy of this disease (2, 3). Isorhapontigenin (ISO) is a new derivative of stilbene compound isolated from Chinese herb Gnetum cleistostachyum, and its chemical structure is shown in our previous publications (4, 5). Recently, our group has reported that ISO effectively suppresses bladder cancer cell growth in vitro (4, 5) and has found that ISO treatment induces G0–G1 cell growth arrest and inhibits anchorage-independent growth of human bladder cancer cells through downregulated Cyclin D1 gene transcription via inhibition of Sp1 transactivation in bladder cancer cells (4).
Sp1 is the first transcription factor to be isolated from mammalian cells and belongs to the specificity Protein/Kruppel-like Factor (SP/KLF) family (6), which are characterized by their COOH-terminal domains containing three C2H2-type zinc fingers that recognize GC-rich motif in the promoters of their target genes (7). Sp1 is ubiquitously expressed in various mammalian cells and plays an important role in the regulation of numerous genes involved in various cellular processes (8), such as cell differentiation, cell growth, and apoptosis. An increasing number of evidence shows that Sp1 is upregulated in many cancer tissues, including breast carcinomas (9), hepatocellular carcinomas (10), thyroid cancer (11), colorectal cancer (12), pancreatic cancer (13), gastric cancer (14), and lung cancer (15). Furthermore, Sp1 expression is also increased in the bladder epithelium of the mouse exposed to n-butyl-N-(4-hydroxybutyl)-nitrosamine, a well-characterized mouse carcinogen for invasive bladder cancer induction (16). Sp1 expression increases by 8- to 18-fold in malignant transformed fibroblasts, whereas knockdown of Sp1 expression blocks the tumorigenicity of transformed fibroblasts in xenografts athymic nude mouse model (17). It has been reported that the upregulation of Sp1 is also associated with poor clinical prognosis among patients with gastric and pancreatic cancer (14, 18, 19), suggesting that Sp1 may act as an oncoprotein in tumor development. The pro-oncogenic activity of Sp1 is primarily due to Sp1-regulated genes, which include several genes that play pivotal roles in cancer cell proliferation (Cyclin D1, EGFR), survival (survivin, bcl-2), angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)], and inflammation (NF-kB, p65; refs. 4, 20). Thus, Sp1 is considered as an important target for mechanism-based anticancer drugs. Our previous studies have revealed that ISO acts as a novel mechanism-based cancer therapeutic agent against human bladder cancer by inhibition of Sp1 transactivation in different human bladder cancer cell lines (4, 5). However, the anticancer effect of ISO in vivo and the molecular mechanisms underlying ISO inhibition of Sp1 expression has never been explored to the best of our knowledge. In current studies, we explored the ISO inhibition of human bladder tumor formation in xenografts athymic nude mouse model and the molecular mechanisms underlying ISO suppression of Sp1 expression both in vitro and in vivo.
Materials and Methods
Plasmids, antibodies, and reagents
The Sp1 expression construct, pEGFP-Sp1, and miR-137 expression construct, pcDNA3.2/V5-mmu-mir-137, were obtained from Addgene. Human Sp1 3′-untranslated region (UTR) luciferase reporter, being cloned into the pGL3-control luciferase assay vector, was kindly provided by Dr. Guido Marcucci from Department of Medicine, Ohio State University, Columbus, OH (21). Sp1 3′-UTR point mutation was amplified from wild-type (WT) template by overlap PCR using primers: forward: 5′-GATCTTTGCTAGGACATCCTAAATTTATATACTT-3′; reverse: 5′-AAGTATATAAATTTAGGATGTCCTAGCAAAGATC-3′. The miR-145 expression construct, pBluescript-miR-145 (hsa-miR-145), was kindly provided by Dr. Renato Baserga from Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA (22). The miR-137 inhibitor expression plasmid (HmiR-AN0175-AM03) was purchased from Genecopoeia. The antibody against β-actin was bought from Cell Signaling Technology. The antibodies against Cyclin D1, Sp1, Sp4, and GAPDH were bought from Santa Cruz Biotechnology. ISO with purity more than 99% was purchased from Roche Pharma and was dissolved in DMSO to make a stock concentration at 20 mmol/L.
Cell culture and transfection
Human bladder cancer cell line UMUC3 was provided by Dr. Xue-Ru Wu (Departments of Urology and Pathology, New York University School of Medicine, New York, NY) in 2010 as described in our previous studies (5). T24T was kindly provided by Dr. Dan Theodorescu (Department of Urology, University of Virginia, Charlottesville, VA) in 2010 and used in our previous studies (4, 5, 23). The UMUC3 cells were cultured in DMEM supplemented with 10% FBS, 2 mmol/L l-glutamine, and 25 μg/mL of gentamycin, and the T24T cell was cultured in a 1:1 mixture of DMEM/Ham's F12 medium supplemented with 5% FBS, 2 mmol/L l-glutamine, and 25 μg/mL of gentamycin. All cell lines were subjected to DNA tests and authenticated in our previous studies (4). Both cell lines are regularly authenticated on the basis of viability, recovery, growth, morphology, and chemical response as well, and were most recently confirmed 4 to 6 months before use by using a short tandem repeat method. The transfections were carried out using PolyJet DNA In Vitro Transfection Reagent (SignaGen Laboratories) according to the manufacturer's instructions. The stable transfection selection of Sp1, miR-137, and miR-145 in UMUC3 and T24T cells was subjected to neomycin selection for 4 to 6 weeks, whereas miR-137–specific inhibitor stable transfectants were selected by hygromycin for 4 to 6 weeks. The survived stable transfectants were pooled as stable mass culture as described in our previous studies (24, 25).
Cells were extracted with cell lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 1% SDS, and 1 mmol/L Na3VO4), and protein concentrations were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific). The cell extracts were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membranes (Bio-Rad), probed with the indicated primary antibodies, and incubated with the alkaline phosphatase (AP)-conjugated secondary antibody. The protein band specifically bound to the primary antibody was detected by Typhoon FLA 7000 (GE Healthcare) using an alkaline phosphatase–linked secondary antibody and an enhanced chemifluorescence Western blotting system as described in our previous studies (5, 23).
Anchorage-independent growth assay
Anchorage-independent growth in soft agar (soft-agar assay) was performed as described in our earlier studies. Briefly, the 1 × 104 cells mixed with ISO at final concentration of 10 μmol/L or vehicle control in 10% FBS Basal Medium Eagle (BME) containing 0.33% soft agar and were seeded over the basal layer containing 0.5% agar containing 10% FBS/BME in each well of 6-well plates. The plates were incubated in 5% CO2 incubator at 37°C for 3 weeks. Colonies were captured under a microscope, and only colonies with over 32 cells were counted. The results were presented as mean ± SD obtained from three independent experiments.
The cells were treated with ISO at 10 μmol/L or control vehicle, and the cells were then harvested and fixed in 75% ethanol overnight. The cells were suspended in the staining buffer containing 0.1% Triton X-100, 0.2 mg/mL RNase A, and 50 mg/mL propidium iodide at 4°C. DNA content was then determined by a flow cytometry Epics XL flow cytometer (Beckman Coulter Inc.), and the results were analyzed with EXPO32 software.
Cells were treated with 10 μmol/L of ISO and were then extracted for total RNA using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. The cDNAs were synthesized with the Thermo-Script RT-PCR system (Invitrogen). The mRNA was evaluated by semiquantitative RT-PCR. The primers for human sp1 were 5′-ATTAACCTCAGTGCATTGGGTA-3′ and 5′-AGGGCAGGCAAATTTCTTCTC-3′. The primers for human β-actin were 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′-AGGGGCCATCCACAGTCTTC-3′. The PCR products were separated on 2% agarose gels and stained with ethidium bromide, and the results were imagined with Alpha Innotech SP Image system (Alpha Innotech Corporation).
Quantitative RT-PCR for miRNA assay
Total miRNAs were extracted using the miRNeasy Mini Kit (Qiagen). Total RNA (1 μg) was used for reverse transcription, and the miRNA expression was determined by the 7900HT Fast Real-time PCR system (Applied Biosystems) using the miScript PCR Kit (Qiagen). The primer for miRNA was purchased from Invitrogen, and U6 was used as a control. Cycle threshold (CT) values was determined, and the relative expression of miRNAs was calculated by using the values of 2−ΔΔCT.
(35S) methionine pulse assays
T24T cells were cultured in each well of 6-well plate till 70% to 80% confluence, and the cell culture medium was replaced with 0.1% FBS DMEM and incubated for another 24 hours. The cells were then treated with 10 μmol/L of ISO diluted in 2% FBS methionine/cysteine-free DMEM containing 35S-labeled methionine/cysteine (250μ Ci per dish, Trans 35S-label, ICN) for the indicated time periods. The cells were extracted with lysis buffer (Cell Signaling Technology) containing complete proteinase inhibitor mixture (Roche). Total lysate of 500 mg was incubated with anti-Sp1 antibody-conjugated agarose beads (R&D Systems) at 4°C overnight. The immunoprecipitate was washed with the cell lysis buffer five times and heated at 100°C for 5 minutes after final washing. The protein samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. 35S-labeled Sp1 protein was imaging captured with the PhosphorImager (Molecular Dynamics).
Cancer tissue specimens
Twenty-six pairs of primary invasive bladder cancer specimens and their paired adjacent nontumorous bladder tissues were obtained from patients who underwent radical cystectomy at Department of Urology of the Union Hospital of Tongji Medical College between 2012 and 2013. All specimens were immediately snap-frozen in liquid nitrogen after surgical removal. Histologic and pathologic diagnoses were confirmed by a pathologist based on the 2004 World Health Organization Consensus Classification and Staging System for bladder neoplasms. All specimens were obtained with appropriate informed consent from the patients, and the approval was obtained from the Medical Ethics Committee of Tongji Medical College, China.
For the determination of Sp1 3′-UTR luciferase reporter activity, the cells were transiently cotransfected with Sp1 3′-UTR luciferase reporter and TK. The transient transfectants were seeded into each well of 96-well plates (1 × 104 cells per well) and cultured for 24 hours. The cells were treated with ISO (10 μmol/L) for the indicated times and then extracted with lysis buffer (25 mmol/L Tris-phosphate, pH 7.8, 2 mmol/L EDTA, 1% Triton X-100, and 10% glycerol), and the luciferase activity was determined by the microplate luminometer (Microplate Luminometer LB 96V; Berthold GmbH & Co.) using the luciferase assay Kit (Promega Corp.).
Tumor xenografts and in vivo ISO treatment
All animal studies were performed in the animal institute of Wenzhou Medical University according to the protocols approved by the Medical Experimental Animal Care Commission of Wenzhou Medical University. The 12 female athymic nude mice (3–4 weeks old) were purchased from Shanghai Silaike Experimental Animal Company, Ltd. (license No. SCXK, Shanghai 2010—0002), and the mice at age of 5 to 6 weeks were randomly divided into two groups and were then subcutaneously injected with 0.2 mL of T24T cells (2 × 106 suspended in 100 μL PBS) in the axillary region. The mice of ISO group received i.p. injection of 150 mg/kg ISO every other day, starting at day one after cell inoculation, whereas control mouse received vehicle only. The nude mice were maintained under sterile conditions according to the protocol of the American Association for the Accreditation of Laboratory Animal Care. These mice were evaluated twice a week for the appearance and size of tumors, and tumors were measured with calipers to estimate the volume. Tumor sizes were evaluated using the formula: Volume (mm3) = [width2 (mm2) × length (mm)]/2. Six weeks after ISO treatment, the mice were sacrificed and the tumors were surgically removed, photographed, weighed, and used for further pathologic and histopathologic evaluation. No mouse died or was sacrificed before the end of the in vivo experiment.
Tumor tissues obtained from the sacrificed mice were formalin-fixed and paraffin-embedded. For IHC staining, we used antibodies specific against Sp1 (1:30; Santa Cruz Biotechnology) or Cyclin D1 (1:200; Santa Cruz Biotechnology). The resultant immunostaining images were captured using the Axio Vision Rel.4.6 computerized image analysis system (Carl Zeiss). Protein expression levels were analyzed by calculating the integrated optical density per stained area using Image-Pro Plus version 6.0 (Media Cybernetics). More detailed procedure was described in our previous published studies (26).
Genomic DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instruction. Genomic DNA (2 μg) was treated with sodium bisulfite using the EpiTect Bisulfite Kit (Qiagen). Methylation-specific PCR was performed using 20 ng of bisulfite-converted DNA and the specific primers. Methylated primer and unmethylated primers for miR-137 promoter were designed according to Shimizu and colleagues (27). PCR products were run on 2% agarose gel and visualized after ethidium bromide staining. Bisulfite-converted methylated and unmethylated DNA from EpiTect PCR Control DNA Set (Qiagen) was used as positive and negative controls.
The Student t test was used to determine the significance of differences between different groups. The differences were considered to be significant at P ≤ 0.05. The Spearman correlation test was chosen for examining the correlations between Sp1 expression and Cyclin D1 expression and tumor weight.
ISO represses tumor growth via downregulation of Sp1 protein expression both in vivo and in vitro
Our previous in vitro studies have well demonstrated that ISO treatment induces cell-cycle G0–G1 arrest and inhibits cancer cell anchorage-independent growth through targeting Sp1/Cyclin D1 axis in bladder cancer cells (4). To further determine the potent anticancer activities of ISO in vivo, we established a xenograft model in mice using human bladder cancer T24T cells, and then treated these mice with ISO. As shown in Fig. 1A and B, ISO treatment resulted in a dramatic inhibition of T24T xenograft tumor growth as compared with vehicle control group (P < 0.01, n = 6). ISO treatment also impaired the expression of Sp1 and Cyclin D1 in the tumor tissues obtained from the tumor-bearing mice (Fig. 1C–E), which is consistent with our prior report in vitro (4). In addition, quantitative analysis of Sp1 expression intensity showed that Sp1 was positively associated with tumor weight (r = 0.825, P < 0.01) and Cyclin D1 expression (r = 0.916, P < 0.01) in tumor tissues obtained from xenograft nude mice (Fig. 1F and G). In combination with our previous report, these findings strongly suggest that downregulation of Sp1 protein is an important event responsible for anticancer activities of ISO. To further test this notion, we used human bladder cancer T24T and UMUC3 cells to establish stable transfectants with GFP-Sp1 or scramble control vector, respectively. As expected, ISO treatment significantly suppressed the expression of endogenous Sp1, but not endogenous Sp4, accompanied by a marked decrease in Cyclin D1 expression in a time-dependent manner (Fig. 2A). However, ISO treatment had no observable effects on exogenous expression of GFP-Sp1, ectopic expression of which attenuated the suppressive effects of ISO treatment on Cyclin D1 expression in T24T and UMUC3 (Fig. 2B). More interestingly, ectopic expression of Sp1 pronounced abolished the G0–G1 phase arrest and anchorage-independent growth inhibition in T24T and UMUC3 cells upon ISO treatment (Fig. 2C–F). Taken together, these findings not only showed that anticancer activities of ISO both in vitro and in vivo were associated with the downregulation of Sp1 and Cyclin D1 in human bladder cancer cells, but also emphasized the crucial role for Sp1 downregulation in ISO anticancer effects.
ISO inhibits Sp1 protein translation
Because ISO treatment specifically inhibited endogenous Sp1 expression without altering ectopic GFP-Sp1 protein expression in T24T and UMUC3 cells, we anticipated that the downregulation of Sp1 expression by ISO treatment might occur at levels of transcriptional, posttranscriptional, or translational. We, therefore, examined the potential effects of ISO on Sp1 mRNA expression. The results indicated that ISO treatment did not have any observable effects on Sp1 mRNA expression level, thereby excluding the possibility that ISO treatment modulates Sp1 expression at transcriptional or posttranscriptional levels (Fig. 3A). To further test the notion that ISO treatment might affect Sp1 expression at translational level, we determined the effects of ISO on new Sp1 protein synthesis using short-term 35S-methionine/cysteine pulse-labeling assay in T24T cells following ISO treatment. As expected, the incorporation of 35S-methionine/cysteine into newly synthesized Sp1 protein was gradually elevated along with the incubation time periods in T24T cells, whereas synthesis rate of new Sp1 protein was markedly attenuated in T24T cells that were treated with ISO (Fig. 3B). This result demonstrates that ISO treatment inhibits Sp1 protein translation.
miR-137 induction was crucial for inhibiting Sp1 protein translation by binding to the 3′-UTR of Sp1
A large number of regulatory elements in either 3′-UTR or 5′-UTR of mRNA have been identified and characterized for their regulation of protein translation (28). To elucidate the mechanisms leading to ISO inhibition of Sp1 protein translation, Sp1 3′-UTR luciferase reporter was transiently cotransfected with pRL-TK into T24T and UMUC3 cells, respectively. The transfectants were used to evaluate the effect of ISO on 3′-UTR luciferase reporter activity. As shown in Fig. 4A, ISO treatment resulted in a dramatic reduction of Sp1 3′-UTR activity in a time-dependent manner in both T24T and UMUC3 cells, indicating that Sp1 mRNA 3′-UTR might be regulated by ISO for its inhibition of Sp1 protein translation. MiRNAs have been reported to bind to mRNA 3′-UTR and suppress protein translation (29). Therefore, we used the miRcode, miRWalk, and TargetScan database to screen the possible miRNAs that could potentially target Sp1 mRNA 3′-UTR. The results obtained from comprehensive analysis indicated that miR-29a, miR-29b, miR-29c, miR-137, and miR-145 could have potential binding to 3′-UTR of Sp1 mRNA (Fig. 4B). To identify which of these miRNAs was responsible for regulation of Sp1 protein translation, we evaluated these miRNA's expression in both cell lines treated with ISO. These data showed that ISO treatment induced the expression of miR-145 and miR-137, without affecting the others in both T24T and UMUC3 cells (Fig. 4C and D), indicating that miR-137 and miR-145 might be involved in downregulation of Sp1 protein translation followed ISO treatment.
To test whether miR-137 and/or miR-145 played a role in regulation of Sp1 protein translation, miR-145 and miR-137 were stably transfected into T24T cells, respectively, and the ectopic expression levels of miR-145 and miR-137 were evaluated by real-time PCR as shown in Fig. 5A and B. Overexpression of miR-137 blocked Sp1 3′-UTR luciferase reporter activity in a dual-luciferase reporter assay, whereas ectopic expression of miR-145 did not show the observable effect under same experimental conditions (Fig. 5C). Consistently, stable overexpression of miR-137 in UMUC3 also dramatically decreased the Sp1 3′-UTR luciferase reporter activity (Fig. 5D and E). To define whether miR-137 inhibition was due to its specific binding to potential miR-137 binding site at Sp1 mRNA 3′-UTR, we constructed mutant of Sp1 3′-UTR luciferase reporter as displayed in Fig. 5F. Both WT and mutant of Sp1 3′-UTR luciferase reporter were stably transfected into T24T (vector) and T24T (miR-137) transfectants, respectively. As shown in Fig. 5G, miR-137 overexpression significantly reduced WT Sp1 3′-UTR luciferase reporter activity, whereas mutation of miR-137 binding site at Sp1 3′-UTR luciferase reporter completely attenuated miR-137 inhibition of Sp1 3′-UTR luciferase reporter activity, indicating that miR-137 is likely to bind to Sp1 3′-UTR directly and regulate Sp1 protein translation. Consistent with miR-137 inhibition of Sp1 3′-UTR luciferase reporter activity, overexpression of miR-137 also impaired Sp1 and Cyclin D1 protein expression in both T24T and UMUC3 cells (Fig. 5H), and it did not show any inhibitory effect on exogenous GFP-Sp1 protein expression and GFP-Sp1–mediated Cyclin D1 expression (Fig. 5I).
miR-137 was downregulated in human bladder cancer tissues, and ectopic expression of miR-137 suppressed bladder cancer cell monolayer growth, anchorage-independent growth, and induced G0–G1 cell growth arrest in human bladder cancer cells
miR-137 gene locates on chromosome 1p22 and has been reported to be downregulated in some cancer tissues, such as breast cancer (30), colorectal cancer (31), and non–small cell lung cancer (32). However, the association of miR-137 with human bladder cancer has not been reported yet to the best of our knowledge. To explore this possibility, the expression level of miR-137 in bladder cancer tissues was determined and compared with that in the paired adjacent nontumorous bladder tissues. The results indicated that miR-137 expression was almost completely impaired in human bladder cancer tissues as compared with that in adjacent normal bladder tissues (Fig. 6A, n = 26). To assess the biologic role of miR-137 in regulation of human bladder cancer cell growth, we stably transfected miR-137 into T24T cells, and the effect of miR-137 overexpression on monolayer growth, anchorage-independent growth, and cell cycles was evaluated in comparison with scramble vector transfectants. As shown in Fig. 6B, overexpression of miR-137 could mimic ISO treatment and reduce bladder cancer monolayer growth. Furthermore, overexpression of miR-137 also profoundly inducted G0–G1 growth arrest accompanied with attenuation of anchorage-independent growth in T24T cells, and these biologic effects of miR-137 could be reversed by ectopic expression of GFP-Sp1 (Fig. 6C–E).
Above results from in vitro human bladder cancer cells demonstrate that miR-137 is induced by ISO treatment, which was crucial for ISO inhibition of Sp1 protein translation by binding to Sp1 mRNA 3′-UTR. We next evaluated the ISO effect on miR-137 expression in mouse tumor nodules obtained from in vivo animal studies. The results revealed that miR-137 expression was significantly increased in tumor nodules from mice that were treated with ISO in comparison with these that were treated with control vehicle (Fig. 7A). To provide a direct evidence showing the critical role of miR-137 in ISO inhibition of Sp1 and Cyclin D1 expression as well as in ISO anticancer activity, the specific miR-137 inhibitor was stably transfected into T24T cells, and the stable transfectants were used to evaluate its role in miR-137 induction by ISO in its inhibition of Sp1 protein expression, Sp1 mRNA 3′-UTR activity, Cyclin D1 expression, anchorage-independent growth, as well as the induction of G0–G1 growth arrest in human bladder cancer cells. As illustrated in Fig. 7B, miR-137 inhibitor expression did attenuate miR-137 induction followed by ISO treatment. Consistently, the ectopic expression of miR-137 inhibitor also reversed ISO downregulation of Sp1 3′-UTR activity, Sp1 protein expression as well as Cyclin D1 protein expression (Fig. 7C and D). Moreover, the inhibition of ISO-induced miR-137 expression by miR-137 inhibitor also attenuated ISO induction of G0–G1 cell growth arrest and ISO inhibition of anchorage-independent growth in human bladder cancer T24T cells (Fig. 7E and F). Collectively, our results clearly demonstrate that ISO-induced miR-137 expression acted as a tumor suppressor by binding to Sp1 mRNA 3′-UTR and inhibiting Sp1 protein translation, by which Cyclin D1 attenuates expression, subsequently resulting in cell growth arrest and anchorage-independent growth inhibition in the bladder cancer cells.
To acquire more evidences for further translational application of ISO in the management of clinical patients, the in vivo animal verification and extensive mechanistic in vitro studies were carried on in current study. First, the in vivo animal studies demonstrated that the antitumor activity of ISO in the subcutaneously transplanted tumor of human bladder cancer in nude mouse model was in line with our previous in vitro studies. Second, we consistently highlighted a crucial role of ISO downregulation of Sp1 protein expression as a key factor mediating its anticancer activity both in vivo and in vitro. Our extensive in vitro studies revealed that the anticancer effect of ISO was mediated by its downregulation of Sp1 protein translation via induction of miR-137, which directly binds to Sp1 mRNA 3′-UTR region. Although miR-137 expression is reported in a few types of tumors, including colorectal (33), gastric (34), lung (32), and glioblastoma (35), its expression and function in human bladder cancers have not been explored yet to the best of our knowledge. Our studies indicated that miR-137 expression was impaired in human bladder cancer tissues, and it acted as a tumor-suppressive miRNA that suppresses the anchorage-independent growth and induces cell-cycle G0–G1 arrest in human bladder cancer cells.
Chinese herb G. cleistostachyum has been used as a traditional Chinese medicine for treatment for arthritis, bronchitis, cardiovascular system disease, and several cancers including bladder cancer almost for a century (36). ISO, a new derivative of stilbene compound, isolated from G. cleistostachyum and its chemical structure is a 4-methoxyresveratrol (37). Our most recent studies have explored anticancer activity of ISO by inducing cell-cycle G0–G1 arrest and inhibiting cancer cell anchorage-independent growth through downregulating Sp1/Cyclin D1 axis in vitro human bladder cancer cells, suggesting that ISO has a potential being a novel mechanism-based cancer therapeutic agent against human bladder cancer in vitro. This provides a basis for possible clinical utilization of ISO as a preventive and therapeutic agent against bladder cancers in clinical patients (4). However, the research lacked in vivo animal verification and extensive in vitro studies. In present studies, the xenograft nude mouse model was used for the intensive studies on the anticancer effect of ISO in bladder cancers. Consistent with the findings in vitro, our new results obtained from current studies revealed that ISO is a potent agent for its inhibition of the tumor growth in the xenograft nude mouse model. We also observed that Sp1 and its regulated Cyclin D1 expression were also downregulated in transplanted tumor nodules in mice followed with ISO treatment. The further analysis revealed that Sp1 expression, Cyclin D1 expression, and tumor growth were very well positively correlated. Current in vivo animal studies together with our early in vitro studies demonstrate that ISO is a novel mechanism-based cancer therapeutic agent that mainly targets Sp1/Cyclin D1 axis in human bladder cancers.
Sp1 is an important transcription factor that is involved in the regulation of many gene expressions and cellular functions (6) and is overexpressed in various cancer cell lines and tumor tissues (9–15). Sp1-regulated genes and oncogenes play an important role in cancer cell proliferation, survival, angiogenesis, and inflammation (38–41). The transcription factor is ideal for development of mechanism-based drugs because Sp1 expression is associated with aging (42–44). Several drugs that target Sp1 have been identified, and these include the NSAIDs tolfenamic acid, COX-2 inhibitors, and the nitro-NSAID GT-094, and several natural products, including betulinic acid (BA), celastrol, and the synthetic triterpenoids methyl 2-cyano-3, 12-dioxooleana-a-dien-28-oate (CDDO-Me) and methyl,2-cyano-3,4-dioxo-18β-olean-1,12-dien- 30-oate (CDODA-Me; refs. 41, 45–47). Our previous studies reveal that Sp1 downregulation is essential for ISO anticancer effect on human bladder cancer cells. However, the mechanisms underlying ISO downregulation of Sp1 were still unknown. It is reported that clinically used and mechanism-based anticancer drugs downregulate Sp1 proteins in cancer cell lines through multiple pathways that are dependent on the drug and cell context (48). For example, curcumin induces proteasome-dependent downregulation of Sp1 in bladder cancer cells (40), whereas in pancreatic cancer cells, the effects of curcumin on decreased expression of Sp1 are ROS dependent (39, 48). In pancreatic cancer cells, tolfenamic acid induced degradation of Sp1 (49), but differently, curcumin induced ROS-dependent downregulation of Sp1 (39, 48). Our current studies demonstrated that ISO treatment specifically inhibited Sp1 protein translation without affecting sp1 mRNA level via its induction of miR-137. MiRNA, approximately 22 nucleotides noncoding RNAs, has been reported to be negatively regulator mediating gene expression by modulating mRNA stability or suppressing protein translation by binding to its targeting mRNA 3′-UTR (29). It is estimated that the expression of at least 30% of human genes is regulated by miRNAs (50). For example, miR-29b inhibits Sp1 expression in tongue squamous cell carcinoma (51). Therefore, we speculated that miRNAs might involve in the ISO downregulation of Sp1 expression. The induction of miRNAs, including miR-145 and miR-137, was observed in T24T and UMUC3 cells treated with ISO. Ectopic expression of miR-137, but not miR-145, showed suppression of Sp1 mRNA 3′-UTR activity and protein expression in human bladder cancer T24T and UMUC3 cells, whereas mutation of miR-137 binding site in Sp1 mRNA 3′-UTR luciferase reporter attenuated miR-137 inhibition of Sp1 mRNA 3′-UTR activity, indicating that miR-137 specifically targets Sp1 mRNA 3′-UTR for its inhibition of Sp1 protein translation, further revealing the identification of Sp1 being a novel miR-137–targeted gene.
miR-137 is located on human chromosome 1p22 and has been implicated to act as a tumor suppressor in several cancer types. Increasing numbers of miR-137 target genes have been documented and have shown to play important roles in various human cancers. For example, Liu and colleagues report that miR-137 regulates epithelial-mesenchymal transition (EMT) and inhibits cell migration via downregulation of Twist1 in gastrointestinal stromal tumor (52). Chen and colleagues demonstrate that miR-137 suppresses tumor progression and metastasis in colorectal cancer (53). Liu and colleagues recently also report that miR-137 suppresses tumor growth and metastasis in human hepatocellular carcinoma by targeting AKT2 (54). The studies from Shimizu and colleagues showed that ectopic expression of miR-137 suppresses bladder cancer cell proliferation (27), whereas another studies indicated that overexpression of miR-137 promotes cell proliferation, migration, and invasion of bladder cancer cells (55). In our studies presented here, we found that miR-137 expression level in primary human bladder cancer tissues was dramatically downregulated as compared with their paired adjacent nontumorous tissues. Further intensive in vitro studies displayed that miR-137 induction was able to induce G0–G1 cell growth arrest and suppress monolayer cancer cell growth and anchorage-independent growth in bladder cancer cell lines via inhibiting Sp1/Cyclin D1 axis. It is reported that miR-137 promoter region is frequently methylated in primary bladder tumors than in normal urothelium (27). Thus, we propose that miR-137 downregulation in human bladder cancer tissues might be due to the hypermethylation of its promoter region, while induction of miR-137 in human bladder cancer cells upon ISO treatment might be associated with reduction of miR-137 promoter methylation followed by ISO treatment. However, the results obtained from methylation-specific PCR showed that miR-137 promoter methylation was not affected upon ISO treatment (Fig. 7G), revealing that ISO-induced miR-137 expression was through miR-137 promoter methylation-independent manner. Further investigation of the mechanisms underlying ISO upregulation of miR-137 will be highly significant for providing deep insight into understanding anticancer effect of ISO, and is now ongoing project in our research program.
In summary, our studies demonstrated that ISO exhibited anticancer effect on human bladder cancer experimental system both in vivo and in vitro via its upregulation of miR-137 expression, which in turn inhibited Sp1 protein translation and subsequently resulted in Cyclin D1 protein expression, leading to cell-cycle G0–G1 arrest and inhibiting anchorage-independent cell growth of human bladder cancer cells as illustrated in Fig. 7H. The comprehensive studies including in vivo and in vitro studies are crucial for potential translational application of ISO in the management of clinical patients. Our studies not only provide a novel insight into understanding the anticancer activity of ISO, but also reveal that ISO could be used as a therapeutic drug for treatment of human bladder cancer with miR-137 downregulation.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: H. Jin, C. Huang
Development of methodology: Z. Xu, J. Li, G. Jiang
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Zeng, J. Gu, H. Huang, G. Gao, X. Zhang, J. Li, G. Jiang, H. Sun
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Zeng, Z. Xu, J. Gu, H. Huang, C. Huang
Writing, review, and/or revision of the manuscript: X. Zeng, H. Jin, C. Huang
Study supervision: H. Huang, C. Huang
The authors thank Dr. Guido Marcucci from the Department of Medicine, Ohio State University, for the gift of human Sp1 3′-UTR luciferase reporter and Dr. Renato Baserga from the Department of Cancer Biology, Thomas Jefferson University, for the gift of miR-145 expression construct pBluescript-miR-145.
This work was partially supported by grants from NIH/NCI [CA112557, CA177665, and CA165980 (to C. Huang)], NIH/NIEHS ES000260 (to C. Huang), and NSFC 81229002 (to C. Huang) as well as Key Project of Science and Technology Innovation Team of Zhejiang Province (2013TD10; to H. Huang).
The costs of publication of this article were defrayed in part by the payment 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.