Abstract
Background/Aim: Cabazitaxel is an approved second-line treatment for docetaxel-refractory metastatic castration-resistant prostate cancer. However, the median time to progression on cabazitaxel is 2.8 months. We aimed to determine whether DNA methylation plays a role in cabazitaxel resistance. Materials and Methods: DU145 cells, resistant to docetaxel and cabaxitaxel (DU145 10DRCR), were generated from cells resistant to 10 nM docetaxel (DU145 10DR). The effect of pre-treatment with 5-azacytidine was determined with regards to cabazitaxel sensitivity. Gene expression profiling was carried-out on DU145 10DR, DU145 10DRCR and DU145 10DRCR treated with 5-azacytidine. Results: Pre-treatment of cells with 5-azacytidine resulted in enhanced sensitivity to cabazitaxel. Gene expression profiling identified a subset of genes that may be regulated by DNA methylation. Conclusion: Our results indicate that DNA methylation of pro-apoptotic and cell-cycle regulatory genes may contribute to cabazitaxel resistance and pre-treatment with 5-azacytidine may restore sensitivity to cabazitaxel in prostate cancer cells.
Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death in American men. There will be an estimated 220,800 new prostate cancer cases and 27,540 estimated prostate cancer-related deaths in the United States in 2015 (19). After an initial response period, metastatic prostate cancer progresses to castration resistance. Most prostate cancer-related deaths occur in patients with metastatic castration-resistant prostate cancer (CRPC). The median survival in patients with metastatic CRPC is only 12-18 months. Based on results from two randomized control studies (RCTs), TAX327 and SWOG-99-16 (13, 22), the Food and Drug Administration (FDA) approved the use of docetaxel in combination with prednisone for the treatment of metastatic CRPC in 2004. However, after an initial response to docetaxel, approximately 80% of patients demonstrate PSA relapse within 12 months and median time to progression is approximately 6 months (13). For over 6 years, there were no other treatment options for patients who progressed, on or after docetaxel chemotherapy. In 2010, a novel taxane, cabazitaxel was approved as a second-line chemotherapy treatment in these patients by the FDA. However, the median time to progression on cabazitaxel is 2.8 months (2). The molecular mechanisms of cabazitaxel resistance are presently not fully understood. One possible mechanism may involve epigenetic silencing of pro-apoptotic genes and genes involved in cell-cycle regulation.
We have previously shown that growth arrest and DNA damage inducible-alpha (GADD45a), a pro-apoptotic gene, is frequently inactivated by methylation in prostate cancer and contributes to docetaxel sensitivity (15). Furthermore, our results from the Phase I study on azacitidine, docetaxel and prednisone in treatment of metastatic CRPC who progressed on or after docetaxel chemotherapy showed that this combination is well-tolerated and shows an exciting response in a recently completed Phase I study in patients with prior docetaxel treatment (20). In the present study, we aimed to identify pathways of resistance to cabazitaxel and determine whether epigenetic gene silencing contributes to cabazitaxel resistance in prostate cancer cells.
Materials and Methods
Cell culture. DU145 prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were routinely cultured in RPMI-1640 medium (Mediatech, Manassas, VA, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine (Invitrogen, Carlsbad, CA, USA) and 100 μg/ml penicillin-streptomycin (Invitrogen) in a humidified incubator at 37°C with 5% CO2. Docetaxel and 5-azacytidine were procured from LC laboratories (Woburn MA, USA) and Sigma Aldrich (St. Louis MO, USA) respectively. Cabazitaxel was provided by sanofi-aventis (Bridgewater, NJ, USA).
Comparison of sensitivity of DU145 wt and DU145 10DR cells to cabazitaxel. DU145 wt and DU145 cells resistant to 10 nM docetaxel (DU145 10DR) were seeded in 96-well plates and treated under varying concentrations of cabazitaxel ranging from 0.6 nM to 20 nM for 72 h. Control cells were left untreated. Cell viability was assayed by Cell-titer blue assay.
Drug-resistant cells. DU145 cells resistant to 10 nM docetaxel (DU145 10DR) were obtained by culturing in docetaxel in a dose-escalating manner. After cells sensitive to the drug were no longer present and the surviving DU145 cells had re-populated the flask and continued to divide through four passages, the concentration of drug in the medium was increased. This was continued in a step-wise manner until a final concentration of 10 nM docetaxel was reached. DU145 10DR cells were maintained in medium containing 10 nM docetaxel. Using a similar strategy, DU145 10DR cells resistant to cabazitaxel (DU145 10DRCR) were also generated. These cells were maintained in medium containing 10 nM docetaxel and 10 nM cabazitaxel.
Drug treatment. Wild-type and drug-resistant DU145 cells were treated with different concentrations of cabazitaxel and cell viability was measured 72 h following treatment using Cell Titer Blue (Promega, Madison, WI, USA). Cells were treated with different concentrations of 5-azacytidine for 72 h, after which RNA was extracted. For combination treatment, wild-type and drug-resistant cells were seeded in 96-well plates and treated with 5-azacytidine for 72 h followed by treatment with cabazitaxel for 72 h. Cell viability was measured after 24, 48 and 72 h using Cell Titer Blue.
Cell viability assay. Cells were incubated with RPM1 medium containing Cell Titer Blue for 5 h and fluorescence (560Ex/590Em) was measured in a Synergy HT multi-task plate reader (Biotek, Winooski, VT, USA).
RNA extraction. RNA was extracted from cells using the Masterpure RNA purification kit (Epicentre, Madison, WI, USA) as per the manufacturer's instructions and reverse transcribed using MMLV Reverse Transcriptase (USB Corporation, Cleaveland, OH).
Gene expression profiling. Gene expression profiling in DU145 10DRCR and DU145 10DR with and without 5-azacytidine treatment was permormed using the Illumina HumanHT-12 Expression BeadChip (Illumina, San Diego, CA, USA). Differential gene expression analysis was performed using the Partek Genomic Suite v.6.6 software (St. Louis, MO, USA). Only probes with a detection p-value <0.5 for at least one sample were selected for analysis. After subtraction of background, data were normalized using the quantile method. Values <0.01 were converted to 0.01 to avoid deletion to zero. Differentially expressed probes were identified by performing Analysis of Variance (ANOVA).
Evaluation of resistance of DU145 10DRCR cells to cabazitaxel. DU145 cells resistant to docetaxel (DU145 10DR) and DU145 10DR cells resistant to 10 nM cabazitaxel (DU145 10DRCR) were seeded in 96 well plates and treated with 10 nM cabazitaxel for 72 h. Control cells were left untreated. Cell viability was assayed by Cell-titer blue assay.
Results
DU145 cells resistance to docetaxel and cabazitaxel. Firstly, we evaluated the sensitivity of DU145 wild-type (wt) and DU145 cells resistant to 10 nM docetaxel (DU145 10DR) to cabazitaxel. DU145 wt cells were found to be more sensitive to cabazitaxel compared to DU145 10DR cells (Figure 1). Next, we generated DU145 10DR cells resistant to cabazitaxel (DU145 10DRCR) by culturing cells in cabazitaxel in a dose-escalating manner. We then evaluated the sensitivity of DU145 10DRCR cells to cabazitaxel compared to that of DU145 10DR cells. After 72 h of treatment with 10 nM cabazitaxel, there was 8% cell death in DU145 10DRCR cells compared to 80% cell death in DU145 10DR cells (Figure 2).
Pre-treatment with 5-azacytidine enhances sensitivity to cabazitaxel in DU145 cells. DU145 wild-type cells (DU145 wt Panel A), DU145 cells resistant to docetaxel (DU145 10DR Panel B) and DU145 10DR cells resistant to 10 nM cabazitaxel (DU145 10DRCR Panel C) were seeded in 96-well plates and treated with 1 μM 5-Azacytidine for 72 h followed by 10-nM cabazitaxel for 72 h. Control cells were left untreated. Cell viability was assayed by Cell-titer blue assay 24, 48 and 72 h after cabazitaxel treatment.
Pre-treatment with azacitidine reverses resistance to cabazitaxel in cabazitaxel-resistant prostate cancer cells. DU145 wild-type and drug-resistant cells were seeded in 96-well plates and treated with 1 μM 5-azacytidine for 72 h followed by treatment with cabazitaxel for 72 h. Cell viability was measured after 24, 48 and 72 h using Cell Titer Blue. Pre-treatment with 5-azacytidine resulted in increased cytotoxicity of cabazitaxel. DU145 wt cells treated with a combination of 5-azacytidine and cabazitaxel showed 18.43% increase in cell death compared to cells treated with 10 nM cabazitaxel alone. DU145 10DR cells treated with a combination of 5-azacytidine and cabazitaxel showed 26.19% increase in cell death compared to cells treated with 10 nM cabazitaxel with no 5-azacytidine pre-treatment. DU145 10DRCR cells treated with 5-azacytidine alone showed 21% cell death compared to cells that were left untreated. DU145 10DRCR cells pre-treated with 5-azacytidine showed a 31% increase in cell death when treated with 10 nM cabazitaxel compared to cells with no pre-treatment (Figure 3). This shows that pre-treatment with 5-azacytidine enhances sensitivity to cabazitaxel and reverses resistance to cabazitaxel, to some extent, in DU145 10DRCR cells.
Venn diagram showing epigenetically-regulated genes in DU14510DRCR cells compared to DU14510DR cells. Gene expression profiling in DU145 10DRCR and DU145 10DR with and without AzaC treatment were done using the Illumina HumanHT-12 Expression BeadChip. The intersect comprising of 183 genes represents genes that are potentially epigenetically regulated that is derived from the overlap of genes having reduced expression in DU145 10DRCR cells compared to DU145 10DR cells (719 genes) and the genes whose expression is increased upon azacitidine treatment in DU145 10DRCR cells (1,111 genes).
Gene expression profiling to identify epigenetically-regulated genes in cabazitaxel-resistant cells. To identify the genes that are epigenetically regulated and may contribute to cabazitaxel resistance, gene expression profiling in DU145 10DRCR and DU145 10DR with and without 5-azacytidine treatment was performed using the Illumina HumanHT-12 Expression BeadChip. Differential gene expression analysis was performed using Partek Genomic Suite v.6.6 software. Probes filtered for False Discovery Rate (FDR)-corrected p-value <0.05 and fold change >1.5 and <-1.5 were used to generate the Venn Diagram (Figure 4). The intersect, comprising of 183 genes represents genes that are potentially epigenetically regulated and is derived from the overlap of genes that have reduced expression in DU145 10DRCR cells compared to DU145 10DR cells (719 genes) and genes whose expression is increased upon azacitidine treatment in DU145 10DRCR cells (1,111 genes). Transcripts from the intersect of the Venn Diagram were imported into WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/) and KEGG pathways enriched (FDR<0.1) in the list of transcripts were identified (Table I).
List of genes potentially regulated by DNA methylation that may contribute to cabazitaxel resistance in Du145 prostate cancer cells.
We observed that pre-treatment with 5-Azacytidine enhances sensitivity of DU145 10DRCR cells to cabazitaxel indicating the contribution of methylation-mediated regulation of genes in cabazitaxel resistance in these cells. Through gene expression profiling, we identified 183 epigenetically regulated genes in cabazitaxel resistance. Pathway analysis showed that these genes were involved in MAPK signaling, p53 signaling, GnRH signaling, Gap junction, cytokine-cytokine receptor interaction, focal adhesion, cell cycle, Wnt signaling pathways etc.
Discussion
To our knowledge, this is the first report on cabazitaxel resistance in prostate cancer cells. To date, there exist only published reports on genes involved in docetaxel resistance in prostate cancer. We found that pre-treatment of DU145 10DRCR cells with 5-azacytidine enhances sensitivity to cabazitaxel. This result suggests that DNA methylation-mediated silencing of genes may play a role in resistance of DU145 cells to cabazitaxel. Previous studies from our lab have demonstrated the role of epigenetic silencing of pro-apoptotic and tumor suppressor genes in development of resistance to chemotherapeutic drugs. We showed that GADD45a, a gene involved in apoptosis and cell cycle regulation, plays a role in docetaxel sensitivity in DU145 prostate cancer cells. GADD45a is silenced by DNA methylation in DU145 cells as well as in prostate cancer tissues. Up-regulation of GADD45a either by recombinant gene expression or by treatment with 5-azacytidine resulted in increased sensitivity to docetaxel chemotherapy (15). Following this, we conducted a phase I/II clinical trial to check the safety and efficacy of 5-azacytidine, docetaxel and prednisone in patients with docetaxel refractory metastatic castration resistant prostate cancer. Our results showed that this combination is active in these patients (20). In a recently published study, we showed that GADD45a is frequently methylated in serum of prostate cancer patients compared to patients with benign prostatic disease and can be a useful marker in distinguishing benign from prostate cancer patients (17). We have also demonstrated the role of TMS1, a pro-apoptotic gene, in sensitivity of bladder cancer cells (16) and breast cancer cells (5) to chemotherapeutic agents.
Our results indicated that DU145 wt cells were more sensitive to cabazitaxel compared to DU145 10DR cells. For instance, at a concentration of 0.6 nM, we observed 80% cell death in DU145 wt cells compared to only 29% cell death in DU145 10DR cells. Similarly, when treated with 5 nM cabazitaxel, we found 92% cell death in DU145 wt cells compared to 83% in DU145 10DR cells. Hence, there seems to be an inherent resistance to cabazitaxel in docetaxel-resistant cells compared to DU145 wt. Since docetaxel and cabazitaxel are both taxane drugs, possible mechanism of the cross-resistance could be the involvement of same pathways and genes in sensitivity to docetaxel and cabazitaxel.
Gene expression profiling revealed potential genes and pathways involved in cabazitaxel resistance in DU145 cells. Although we found several genes that were over-expressed and under-expressed in DU145 10DR CR cells compared to parent DU145 10DR cells, we primarily focused on genes that were regulated by DNA methylation. For this reason, we analyzed the overlap of genes having reduced expression in DU145 10DRCR cells compared to DU145 10DR cells (719 genes) and the genes whose expression is increased upon azacitidine treatment in DU145 10DRCR cells (1,111 genes). We found 183 genes that were potentially epigenetically regulated based on this analysis. Out of these KEGG pathways were identified for 45 genes. The pathways included prostate cancer, MAPK signaling, metabolism, p53 signaling, gap junction, toll-like receptor signaling, Wnt signaling etc. DVL1, the human homolog of the Drosophila dishevelled gene (dsh) is a cytoplasmic mediator of the Wnt/b-catenin signaling pathway, that is critical for embryonic development, stem-cell maintenance, and oncogenesis (24). DVL cascade is related to apoptosis in several cell types, and is linked to the aberrant activation of Wnt/b-catenin signaling (23). It has also been shown that DVL1 contributes to cyclosporine-induced apoptosis in cardiomyoblast cells (26). Our results showed that DVL1 is re-activated by azacitidine treatment in DU145 10DRCR cells indicating that DNA methylation may be a possible mechanism of regulation of DVL1 and may contribute to cabazitaxel resistance in DU145 cells. DVL1 has been reported to be methylated in ovarian cancer and increased methylation is associated with an increased risk of disease progression and poor response (1). Another gene of interest is CDK6, that encodes a member of the cyclin-dependent protein kinase (CDK) family, which are known to be important regulators of cell-cycle progression (4). This kinase is a catalytic subunit of the protein kinase complex that is important for cell-cycle G1 phase progression and G1/S transition. This kinase has been shown to phosphorylate, and thus regulate the activity of the tumor suppressor protein Rb (10). Sun et al. reported that down-regulation of CDK6 results in cell-cycle arrest in lung cancer cells (21). Consistent with this, Huang et al. showed that selective and reversible inhibition of CDK4/CDK6 inhibits proliferation and enhances bortezomib-induced cytotoxic killing of cancer cells and suggested that reversible inhibition of CDK4/CDK6 in sequential combination therapy, thus, represents a novel mechanism-based cancer therapy (6). However, a recent study showed that overexpression of CDK6 causes p53-dependent apoptosis (8). Since CDK6 expression is restored by treatment with 5-azacytidine indicating that the gene may be down-regulated by CpG methylation and may confer cabazitaxel resistance in DU145 cells. IL8 and TUBB2B have been found to be under-expressed in docetaxel-resistant DU145 and PC3 cells and may have a role in docetaxel resistance in these cells (11). Interestingly, we present indirect evidence that IL8 and TUBB2B may be regulated by DNA methylation in cabazitaxel-resistant cells indicating that they may confer cross-resistance to docetaxel and cabazitaxel. De Larco et al. showed that IL8 gene expression is regulated by methylation of two CpG sites upstream in the gene in breast cancer. Contrary to the common epigenetic paradigm in which methylation of promoter CpG islands silences gene expression, the authors found that increased methylation of these 2 sites resulted in overexpression of IL8 (3).
Another gene that may confer cross-resistance to docetaxel and cabazitaxel is GADD45a. As mentioned above, we have previously shown the role of GADD45a in docetaxel sensitivity in prostate cancer (15, 16). Other genes of interest include ATF4, DDIT3, DUSP5 and DUSP1 that play a role in MAPK signaling and may play role in apoptosis in response to cabazitaxel treatment. DDIT3 is hypermethylated in A2780 ovarian cancer cells and thought to contribute to cisplatin resistance (25). It has been shown that silencing of DUSP5 by promoter hypermethylation causes increased maintenance of phosphorylated ERK1/2, drives cell proliferation and contributes to gastric carcinogenesis (18). Khor et al. showed that DUSP1 was hypermethylated in oral squamous cell carcinoma compared to normal tissues and was a potential diagnostic, prognostic and therapeutic target (9). PMAIP1 is a pro-apoptotic member of the Bcl-2 protein family. The expression of PMAIP1 is regulated by the tumor suppressor p53 and has been shown to be involved in p53-mediated apoptosis (12). Our results showed that PMAIP1 may have a role in cabazitaxel sensitivity in prostate cancer. Interestingly, Putnik et al. found that although PMAIP1 expression increased with treatment of breast cancer cells with 2-azadeoxycytidine, the promoter was not methylated in untreated cells (14). This suggested that 5-aza-deoxycytidine regulates the expression of these genes either via de-methylation of other methylated DNA regions, such as CpG shores, shelves and open seas (7), or indirectly, through de-methylation of other genes. We have previously reported a similar finding on GADD45a in prostate cancer. The promoter region of GADD45a is unmethylated. However, gene expression is regulated by the methylation of 4 CpGs situated ~700 bp upstream of the transcription start site.
Our results indicate that one of the mechanisms of cabazitaxel resistance in prostate cancer is methylation-mediated silencing of tumor suppressor and pro-apoptotic genes. Furthermore, resistance to cabazitaxel can be reversed by treatment with a de-methylating agent such as 5-Azacytidine. Further studies on genes identified in the present study may lead to a better understanding of mechanisms of cabazitaxel resistance in prostate cancer. Although the scenario of management of metastatic castration-resistant prostate cancer has changed considerably over recent years with the availability of several treatment options, patients eventually stop responding to these treatments. A clinical trial to test the efficacy of combination treatment with 5-azacytidine and cabazitaxel may be useful as an alternative option for these patients.
Acknowledgements
This work was funded by a grant from Sanofi-Aventis to RS.
Footnotes
Conflicts of Interest
The Authors have no conflicts of interest to disclose.
- Received September 15, 2015.
- Revision received October 20, 2015.
- Accepted October 23, 2015.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
