Transcriptional regulation of human osteopontin promoter by histone deacetylase inhibitor, trichostatin A in cervical cancer cells

Abstract

Background: Trichostatin A (TSA), a potent inhibitor of histone deacetylases exhibits strong anti-tumor and growth inhibitory activities, but its mechanism(s) of action is not completely understood. Osteopontin (OPN) is a secreted glycoprotein which has long been associated with tumor metastasis. Elevated OPN expression in various metastatic cancer cells and the surrounding stromal cells often correlates with enhanced tumor formation and metastasis. To investigate the effects of TSA on OPN transcription, we analyzed a proximal segment of OPN promoter in cervical carcinoma cells.

Results: In this paper, we for the first time report that TSA suppresses PMA-induced OPN gene expression in human cervical carcinoma cells and previously unidentified AP-1 transcription factor is involved in this event. Deletion and mutagenesis analyses of OPN promoter led to the characterization of a proximal sequence (-127 to -70) that contain AP-1 binding site. This was further confirmed by gel shift and chromatin immunoprecipitation (ChIP) assays. Western blot and reverse transcription-PCR analyses revealed that TSA suppresses c-jun recruitment to the OPN promoter by inhibiting c-jun levels while c-fos expression was unaffected. Silencing HDAC1 followed by stimulation with PMA resulted in significant decrease in OPN promoter activity suggesting that HDAC1 but not HDAC3 or HDAC4 was required for AP-1-mediated OPN transcription. TSA reduces the PMA-induced hyperacetylation of histones H3 and H4 and recruitment of RNA pol II and TFIIB, components of preinitiation complex to the OPN promoter. The PMA-induced expression of other AP-1 regulated genes like cyclin D1 and uPA was also altered by TSA. Interestingly, PMA promoted cervical tumor growth in mice xenograft model was significantly suppressed by TSA.

Conclusions: In conclusion, these findings provide new insights into mechanisms underlying anticancer activity of TSA and blocking OPN expression at transcriptional level by TSA may act as novel therapeutic strategy for the management of cervical cancer.

Figure 1

TSA suppresses PMA-induced OPN transcription in HeLa cells. A. HeLa cells were preincubated with 0-1 μM TSA for 1 h followed by treatment with PMA (50 ng/ml) for 6 h. Whole cell lysates were analyzed by western blot using anti-OPN antibody. B. HeLa cells were pretreated with TSA followed by PMA under similar conditions as described above. Total RNA was isolated and OPN mRNA levels were detected by semiquantitative RT-PCR and analyzed by agarose gel electrophoresis. Actin was used as control. The data represents three experiments exhibiting similar results. C. HeLa cells were cotransfected with hOPN promoter deletion constructs (-500/+20, -267/+20, -127/+20, -70/+20 and -20/+20) containing luciferase reporter gene along with renilla luciferase vector, pRL followed by stimulation with PMA. The luciferase activity was measured in cell lysates and normalized to Renilla luciferase activity. Fold-changes in luciferase activity with respect to control were calculated. Columns, mean of triplicate determinations; bar, S.D. *, p < 0.05. D. HeLa cells were cotransfected with various hOPN promoter mutation constructs (-500/+20, CM, AM and OM) containing luciferase gene along with pRL vector and treated with PMA. The luciferase activity was measured and normalized. Fold-changes in luciferase activity with respect to control were calculated. Columns, mean of triplicate determinations; bar, S.D. *, p < 0.05.

Figure 2

PMA induces AP-1 DNA binding to the OPN promoter in HeLa cells. A. HeLa cells were treated with PMA (50 ng/ml) for 2 h. Nuclear extracts were prepared and incubated with ³²P-labeled probe containing AP-1 binding site of OPN promoter and analyzed by EMSA. B. For supershift assay, nuclear extracts from PMA-treated HeLa cells were incubated with anti-c-Jun or anti-c-Fos antibody and then analyzed by EMSA. C. HeLa cells were pretreated with TSA (0.5 μM) for 1 h and then with PMA (50 ng/ml) for 2 h. Cross-linked chromatin fragments were immunoprecipitated with anti-p-c-Jun antibody and was PCR amplified using specific primers derived from the region of OPN promoter containing AP-1 binding site. For negative controls, normal mouse IgG was used or specific antibody was omitted.

Figure 3

TSA blocks PMA-induced c-Jun but not c-Fos expression in HeLa cells. A and C. HeLa cells were pretreated with TSA (0-1 μM) for 1 h followed by treatment with PMA (50 ng/ml) for 2 h. Cell lysates (50 μg) containing equal amount of total proteins were analyzed by western blot using either anti-c-Jun or anti-c-Fos antibody. B and D. HeLa cells were pretreated with TSA and then with PMA under similar conditions as described above. Total RNA was isolated and the levels of c-jun and c-fos mRNAs were detected by semiquantitative RT-PCR. Actin was used as control.

Figure 4

TSA inhibits PMA-induced hyperacetylation of histones H3 and H4 and recruitment of RNA pol II and TFIIB to OPN promoter in HeLa cells. A-D. HeLa cells were pretreated with TSA for 1 h and then with PMA for 2 h. Cross-linked DNA-protein complexes were immunoprecipitated with anti-acetyl H3, anti-acetyl H4, anti-RNA pol II or anti-TFIIB antibody and PCR amplified using specific primers derived from the region of OPN promoter containing AP-1 binding site. For negative controls, normal mouse IgG was used. The data represents three experiments exhibiting similar results.

Figure 5

HDAC1 is involved in regulation of PMA-induced OPN promoter activity in HeLa cells. A. panels I-III. HeLa cells were transfected with either siRNAs to HDAC1, HDAC3 and HDAC4 or control siRNA. The expressions of various HDACs were analyzed by western blot using their specific antibodies. Actin served as loading control. B. HeLa cells were transfected with siRNAs to HDAC1, HDAC3 and HDAC4. These transfected cells were cotransfected with hOPN promoter (-500/+20) containing luciferase reporter gene along with Renilla luciferase, pRL vector and then treated with PMA. The luciferase activity was measured in cell lysates and normalized to Renilla luciferase activity. Fold-changes in luciferase activity with respect to control were calculated. Columns, mean of triplicate determinations; bar, S.D. *, p < 0.05.

Figure 6

Effect of TSA on cyclin D1 and uPA expression at protein and RNA levels in HeLa cells. A and C. HeLa cells were pretreated with TSA (0-1 μM) for 1 h followed by treatment with PMA (50 ng/ml) for 6 h. Cell lysates were analyzed by western blot using anti-cyclin D1 or anti-uPA antibody. B and D. Total RNA was isolated from HeLa cells treated under similar conditions and cyclin D1 and uPA mRNA levels were detected by semi-quantitative RT-PCR. Actin was used as loading control for both western blot and RT-PCR.

Figure 7

TSA suppresses cervical tumor growth in response to PMA in NOD/SCID mice model. A. HeLa cells (1 × 10⁶/0.2 ml) were mixed with matrigel (1:1 ratio) and injected subcutaneously into the flanks of mice. Six mice were kept in each group. Either PMA alone or in combination with two doses of TSA was injected intratumorally. After 4 weeks, mice were sacrificed and tumors were excised. The tumor volumes were calculated and the growth kinetics in tumor volume in fold change vs time in weeks is represented in the form of graph. The error bars represent the standard error of mean (SEM). *, p < 0.05. B and C. Tumor lysates were analyzed by western blot using anti-OPN, anti-c-Jun, anti-cyclin D1 and anti-uPA antibodies. Tumors were also analyzed by semiquantitative RT-PCR using gene specific primers. Actin was used as control. D. Schematic representation of PMA-induced OPN transcription through c-Jun leading to enhanced cervical tumor growth and TSA suppresses this process.