TGFBI promoter hypermethylation correlating with paclitaxel chemoresistance in ovarian cancer
© Wang et al; licensee BioMed Central Ltd. 2012
Received: 18 October 2011
Accepted: 16 January 2012
Published: 16 January 2012
The purpose of this study is to determine the methylation status of Transforming growth factor-beta-inducible gene-h3 (TGFBI) and its correlation with paclitaxel chemoresistance in ovarian cancer. The methylation status of TGFBI was examined in ovarian cancer and control groups by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). The TGFBI expression and cell viability were compared by Quantitative Real-Time PCR, Western Blotting and MTT assay before and after demethylating agent 5-aza-2'-deoxycytidine (5-aza-dc) treatment in 6 cell lines (SKOV3, SKOV3/TR, SKOV3/DDP, A2780, 2780/TR, OVCAR8). In our results, TGFBI methylation was detected in 29/40 (72.5%) of ovarian cancer and 1/10 (10%) of benign ovarian tumors. No methylation was detected in normal ovarian tissues (P < 0.001). No statistical correlation between RUNX3 methylation and clinicopathological characteristics was observed. A significant correlation between TGFBI methylation and loss of TGFBI mRNA expression was found (P < 0.001). The methylation level of TGFBI was significantly higher in paclitaxel resistant cell lines (SKOV3/TR and 2780/TR) than that in the sensitive pairs (P < 0.001). After 5-aza-dc treatment, the relative expression of TGFBI mRNA and protein increased significantly in SKOV3/TR and A2780/TR cells. However, no statistical differences of relative TGFBI mRNA expression and protein were found in other cells (all P > 0.05), which showed that re-expression of TGFBI could reverse paclitaxel chemoresistance. Our results show that TGFBI is frequently methylated and associated with paclitaxel-resistance in ovarian cancer. TGFBI might be a potential therapeutic target for the enhancement of responses to chemotherapy in ovarian cancer patients.
Epithelial ovarian cancer is the most lethal gynecologic malignancy, with 21 990 estimated new cases and 15 460 deaths in the USA in 2011 . Reasons for this high lethality include the advanced stage at which patients are diagnosed and the inherent aggressive biology of this cancer.
Maximal surgical cytoreduction followed by systemic chemotherapy with carboplatin and paclitaxel is the current standard treatment modality for advanced ovarian cancer . A key feature of ovarian cancer is its sensitivity to chemotherapeutic drugs such as paclitaxel, a prototype taxane, stabilizes microtubule polymers leading to mitotic arrest and apoptosis . Unfortunately, ovarian cancer cells, with their unstable genomes , are initially sensitive to these drugs, but long term utilization may result in the chemoresistance .
Epigenetic alterations play an important role in the initiation and progression of cancer [6–8]. Hypermethylation of CpG rich islands in promoter regions of genes has been characterized as a common epigenetic alteration for the silencing or inactivation of tumor suppressor genes and transcriptional repression in human malignancies [9, 10], including ovarian cancer [11–13]. In recent years, emerging evidence has also linked epigenetic changes to the development of drug resistance [14, 15].
Transforming growth factor-beta-inducible gene-h3 (TGFBI) is a secreted protein first identified in a human lung adenocarcinoma cell line treated with transforming growth factor-β . It has been shown to possess tumor suppressor function in vitro studies [17, 18], and to be correlated with specific sensitization to paclitaxel by inducing stabilization of microtubules via integrin-mediated signaling pathways . Recently, promoter hypermethylation of TGFBI was found in lung [20, 21] and prostate cancer . However, the role of TGFBI methylation in paclitaxel chemoresistance in ovarian cancer is unknown. Therefore, a better understanding of this epigenetic mechanism of TGFBI in ovarian cancer could facilitate the generation of new drugs that re-sensitize tumor cells to paclitaxel .
In this study, we examined the methylation status and expression of TGFBI in epithelial ovarian cancer tissues, paclitaxel-sensitive and -resistant ovarian cancer cell lines in order to determine whether the methylation of TGFBI is asscociated with paclitaxel chemoresistance.
Materials and methods
Ovarian cancer tissue samples and cell lines
From April 2008 to April 2009, 40 primary epithelial ovarian cancer(, 10 benign tumor and 10 normal ovarian tissues) were collected at the Department of Obstetrics and Gynecology, The Affiliated Hospital of Medical College, Qingdao University, China. The mean age of the patients was 43 years (range 21-77 years). The ovarian cancer patients have different histological types: serous papillary carcinoma (n = 20), mucinous carcinoma (n = 13), endometrioid carcinoma (n = 7). Six patients were in stage I, ten patients were in stage II, twenty-four patients were in stage III. Twenty-two patients had metastasis to pelvic lymph nodes. Eleven tumors were well-moderately differentiated, and 29 tumors were poorly differentiated. Ten benign tumor and 10 normal ovarian tissues were collected as control. All samples were obtained prior to chemotherapy or radiation therapy, which were placed in liquid nitrogen immediately after resection and stored at -80°C until use. The malignant and normal diagnosis was performed by pathologists. The study was performed after approval by our institute Medical Ethics Committee.
Human SKOV3, A2780 and OVCAR8 ovarian cancer cell lines were obtained from the bioengineering centre of The Affiliated Hospital of Medical College, Qingdao University, China. The chemoresistant cell lines (SKOV3/DDP, SKOV3/TR, and A2780/TR) were purchased from the China Center for Type Culture Collection (Wuhan, China). These cells were maintained in DMEM with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin at 37°C. SKOV3/TR and A2780/TR were cultured in RPMI-1640 medium containing 0.3 μmol/L paclitaxel to maintain the drugresistant phenotype.
Cells were grown to 70% confluence and treated with 10 μmol/L of demethylating agent (5-aza-2'-deoxycytidine, 5-aza-dc) (Sigma-Aldrich, St. Louis, MO, USA) for 3 days . After the treatment, cells were harvested and extracted for DNA, RNA and protein.
Nucleic acid isolation
The EZNA Tissue DNA Kit (Omega Corp, USA) was used to extract high purity DNA from different ovarian tissues and ovarian cancer cell lines. Total DNA content was quantified by UV absorbance value measured at A260 and A280, and diluted to a concentration of 1 μg/100 μl.
Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP)
Sequences of Primers for MSP and RT-PCR
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells with Trizol reagent (Invitrogen, San Diego, CA, USA), and it was reverse transcribed using miScript Reverse Transcription Kit (Qiagen, Hilden, Germany). The primers for mRNA are listed in Table 1. The quantification was performed with QuantiTect Probe RT-PCR (Qiagen, Hilden, Germany). The comparative threshold cycle method was used to determine gene relative expression.
Cells were washed twice with ice-cold phosphate-buffered saline and lysed using a modified RIPA buffer supplemented with 1 mM PMSF. The protein concentration was detected using BCA protein assay (Pierce, Rockford, IL, USA). Proteins were loaded onto 10% and 5% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk in PBS-Tween 20 for 2 h at room temperature, the membranes were incubated with anti-human monoclonal β-actin and anti-human TGFBI primary antibody overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody was added for 2 h at room temperature. The Detection was performed by chemiluminescence.
MTT Cell Proliferation Assay (Biosharp, USA) was used to measure cell viability. Before and after treated with 5-aza-dc, 1 × 104 cells/well were seeded in 96-well plates containing complete medium and incubated for 24 h. Then cells were exposed to serial dilutions of paclitaxel in a total volume of 200 μL in four replicate wells. After 48 hours, plates were added 20 μl of MTT reagent and incubated for 4 h, and then formazane crystals formed were dissolved in 150 μl of dimethyl sulfoxide (Wako, Tokyo, Japan). The optical density was measured at 490 nm on a microplate reader. The half maximal inhibitory concentration (IC50) value was assessed by different concentrations of paclitaxel (0.01, 0.1 and 1 μM).
All statistical analyses were performed using SPSS 15.0. Fisher's exact test or and χ 2 test were used to compare TGFBI methylation status among cases and between various clinicopathologic variables. Pearson correlation analysis was used to evaluate the relationship between TGFBI methylation status and mRNA expression. The differences of TGFBI mRNA and protein expression before and after 5-aza-dc treatment were analyzed by the Paired-Samples t test. P < 0.05 was considered statistically significant.
Frequency of TGFBI methylation in ovarian cancer tissues
Association of TGFBI methylation and clinicopathologic variables in 40 ovarian cancer patients
Age at diagnosis
< 50 years
Expression of TGFBI mRNA in ovarian cancer tissues
To examine whether TGFBI methylation results in the suppression of TGFBI expression, we examined TGFBI mRNA expression by qRT-PCR in 40 ovarian cancer tissues and 10 normal ovarian tissues. TGFBI mRNA expression was detected in all the normal ovarian tissues (10/10) and in most of the unmethylated ovarian cancer tissues (10/11). In contrast, TGFBI expression was not detected in the TGFBI-methylated ovarian cancer tissues (27/29), except for 2 tissues. We compared the TGFBI mRNA expression results of these ovarian cancer tissues with the TGFBI methylation data and found a significant correlation between TGFBI methylation and loss of TGFBI mRNA expression (P < 0.001). These results suggest that the inactivation of TGFBI expression is closely correlated with gene methylation in ovarian cancer tissues.
Demethylation and re-expression of TGFBI after treating with 5-aza-dc in ovarian cancer lines
In this study, we first detected the methylation status of the 5' CpG island of TGFBI in different ovarian tissues using MSP and BSP in order to determine whether TGFBI inactivation by DNA methylation is characteristic of human ovarian cancer. After repeated experiments, our results showed that the TGFBI is frequently methylated in ovarian cancer. Its methylation can be used as a novel epigenetic biomarker for ovarian cancer detection.
We further measured TGFBI mRNA and protein levels by RT-PCR and IHC in ovarian cancer tissues. Then we compared the TGFBI expression results with the TGFBI methylation data and found a significant inverse correlation between TGFBI methylation and TGFBI expression, which confirmed the important role of promoter methylation in regulating TGFBI expression. However, because 1 ovarian cancer tissue lacking TGFBI mRNA expression was not methylated, we presume that mechanisms of inactivating the gene other than methylation must exist.
Recently, Shah et al.  reported that TGFBI methylation was associated with tumor recurrence and metastasis, suggesting that TGFBI is required to suppress the aggressiveness of prostate and lung cancer. In our study, the methylation rate of carcinomas with poor differentiation was higher than those with well differentiation. Meanwhile, higher methylation rate was also found in late stage patients with ovarian cancers, though no significant correlation was found between TGFBI methylation status and clinicopathological characteristics, which was in accordance with the results of Kang et al . Our results showed that there were different patterns of mythylation according to the histology and the tumor grade, and revealed that hypermethylation of TGFBI in ovarian cancer might be associated with unfavourable prognosis. Further studies with large sample size and long-term follow-up are required to confirm the hypothesis.
Chemoresistance is the major cause of treatment failure for ovarian cancer. It is reported that DNA methylation may act as a potential cause of chemotherapy drug resistance [24–26]. In a recently study by Li et al. , cisplatin-sensitive and -resistant ovarian cancer cells were analyzed by methylation and mRNA expression microarray. Their results revealed that DNA hypermethylation may contribute to the onset of the chemoresistance in ovarian cancer.
In our study on cell lines, almost complete methylation pattern of the TGFBI promoter in 2 paclitaxel-resistant cell lines (SKOV3/TR and A2780/TR) was observed, with a complete loss or low level of TGFBI expression in these cell lines. In contrast, only sparsely methylated or unmethylated CpG sites were identified in cell lines with a rich level of TGFBI expression, including SKOV3, A2780, OVCAR8, and SKOV3/DDP ovarian cancer cell lines. Our results identified strong relation between TGFBI expression and response to chemotherapy. To our knowledge, this is the first evidence of TGFBI hypermethylation as a mechanism of paclitaxel chemoresistance in ovarian cancer.
Further, our results were confirmed by using DNA methylation inhibitors. The relative expression of TGFBI mRNA and protein increased significantly after treating with 5-aza-dc in palitaxel-resistant cells. However, no statistical differences of TGFBI expression were found after 5-aza-dc administration in other 4 cell lines. In addition, MTT assay showed that the rate of cell inhibition was significantly increased in SKOV3/TR and A2780/TR after 5-aza-dc treatment, which suggested that chemotherapy sensitivity to paclitaxel was enhanced and chemoresistance was reversed.
In conclusion, our study indicated that promoter hypermethylation of TGFBI is a frequent event in ovarian cancer. TGFBI methylation was associated with paclitaxel chemoresistance, and it can be used as a potential epigenetic biomarker and therapeutic target of paclitaxel resistance in ovarian cancer.
This work was supported by grants from National Natural Science Foundation of China (No. 81001167, No. 81172480/H1621, No. 81101973/H1621).
- Siegel R, Ward E, Brawley O, Jemal A: Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011, 61: 212-236. 10.3322/caac.20121.View ArticlePubMedGoogle Scholar
- Matei D: Novel agents in ovarian cancer. Expert Opin Investig Drugs. 2007, 16: 1227-1239. 10.1517/135437126.96.36.1997.View ArticlePubMedGoogle Scholar
- McGuire WP, Hoskins WJ, Brady MF, et al: Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Engl J Med. 1996, 334: 1-6. 10.1056/NEJM199601043340101.View ArticlePubMedGoogle Scholar
- Taniguchi T, Tischkowitz M, Ameziane N, et al: Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors. Nat Med. 2003, 9: 568-574. 10.1038/nm852.View ArticlePubMedGoogle Scholar
- Ferrandina G, Zannoni GF, Martinelli E, et al: Class III beta-tubulin overexpression is a marker of poor clinical outcome in advanced ovarian cancer patients. Clin Cancer Res. 2006, 12: 2774-2779. 10.1158/1078-0432.CCR-05-2715.View ArticlePubMedGoogle Scholar
- Yoshikawa H, Matsubara K, Qian GS, et al: SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet. 2001, 28: 29-35.PubMedGoogle Scholar
- Li QL, Ito K, Sakakura C, et al: Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell. 2002, 109: 113-124. 10.1016/S0092-8674(02)00690-6.View ArticlePubMedGoogle Scholar
- Momparler RL: Cancer epigenetics. Oncogene. 2003, 22: 6479-6483. 10.1038/sj.onc.1206774.View ArticlePubMedGoogle Scholar
- Feinberg AP, Tycko B: The history of cancer epigenetics. Nat Rev Cancer. 2004, 4: 143-153. 10.1038/nrc1279.View ArticlePubMedGoogle Scholar
- Esteller M: Epigenetics in cancer. N Engl J Med. 2008, 358: 1148-1159. 10.1056/NEJMra072067.View ArticlePubMedGoogle Scholar
- Yoon MS, Suh DS, Choi KU, et al: High-throughput DNA hypermethylation profiling in different ovarian epithelial cancer subtypes using universal bead array. Oncol Rep. 2010, 24: 917-925.PubMedGoogle Scholar
- Sellar GC, Watt KP, Rabiasz GJ, et al: OPCML at 11q25 is epigenetically inactivated and has umor-suppressor function in epithelial ovarian cancer. Nat Genet. 2003, 34: 337-343. 10.1038/ng1183.View ArticlePubMedGoogle Scholar
- Zhang H, Zhang S, Cui J, Zhang A, Shen L, Yu H: Expression and promoter methylation status of mismatch repair gene hMLH1 and hMSH2 in epithelial ovarian cancer. Aust N Z J Obstet Gynaecol. 2008, 48: 505-509. 10.1111/j.1479-828X.2008.00892.x.View ArticlePubMedGoogle Scholar
- Balch C, Huang TH, Brown R, Nephew KP: The epigenetics of ovarian cancer drug resistance and resensitization. Am J Obstet Gynecol. 2004, 191: 1552-1572. 10.1016/j.ajog.2004.05.025.View ArticlePubMedGoogle Scholar
- Tamura G: Hypermethylation of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia. World J Gastrointest Oncol. 2009, 1: 41-46. 10.4251/wjgo.v1.i1.41.PubMed CentralView ArticlePubMedGoogle Scholar
- Skonier J, Neubauer M, Madisen L, Bennett K, Plowman GD, Purchio AF: cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta. DNA Cell Biol. 1992, 11: 511-522. 10.1089/dna.1992.11.511.View ArticlePubMedGoogle Scholar
- Zhao YL, Piao CQ, Hei TK: Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells. Oncogene. 2002, 21: 7471-7477. 10.1038/sj.onc.1205891.View ArticlePubMedGoogle Scholar
- Shao G, Berenguer J, Borczuk AC, Powell CA, Hei TK, Zhao Y: Epigenetic inactivation of Betaig-h3 gene in human cancer cells. Cancer Res. 2006, 66: 4566-4573. 10.1158/0008-5472.CAN-05-2130.View ArticlePubMedGoogle Scholar
- Ahmed AA, Mills AD, Ibrahim AE, et al: The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel. Cancer Cell. 2007, 12: 514-527. 10.1016/j.ccr.2007.11.014.PubMed CentralView ArticlePubMedGoogle Scholar
- Shah JN, Shao G, Hei TK, Zhao Y: Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR. BMC Cancer. 2008, 8: 284-10.1186/1471-2407-8-284.PubMed CentralView ArticlePubMedGoogle Scholar
- Irigoyen M, Pajares MJ, Agorreta J, et al: TGFBI expression is associated with a better response to chemotherapy in NSCLC. Mol Cancer. 2010, 9: 130-10.1186/1476-4598-9-130.PubMed CentralView ArticlePubMedGoogle Scholar
- Ying J, Srivastava G, Hsieh WS, et al: The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors. Clin Cancer Res. 2005, 11: 6442-6449. 10.1158/1078-0432.CCR-05-0267.View ArticlePubMedGoogle Scholar
- Kang S, Dong SM, Park NH: Frequent promoter hypermethylation of TGFBI in epithelial ovarian cancer. Gynecologic oncology. 2010, 118: 58-63. 10.1016/j.ygyno.2010.03.025.View ArticlePubMedGoogle Scholar
- Staub J, Chien J, Pan Y, et al: Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance. Oncogene. 2007, 26: 4969-4978. 10.1038/sj.onc.1210300.View ArticlePubMedGoogle Scholar
- Zhang X, Yashiro M, Ren J, Hirakawa K: Histone deacetylase inhibitor, trichostatin A, increases the chemosensitivity of anticancer drugs in gastric cancer cell lines. Oncol Rep. 2006, 16: 563-568.PubMedGoogle Scholar
- Olopade OI, Wei M: FANCF methylation contributes to chemoselectivity in ovarian cancer. Cancer Cell. 2003, 3: 417-420. 10.1016/S1535-6108(03)00111-9.View ArticlePubMedGoogle Scholar
- Li M, Balch C, Montgomery JS, et al: Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer. BMC Med Genomics. 2009, 2: 34-10.1186/1755-8794-2-34.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.