Background
Prostate cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related death, in men in the developed world [
1]. Since Huggins, Stevens and Hodges [
2] demonstrated that prostate epithelial cells require androgens for growth and survival, the mainstay of treatment for men with metastatic prostate cancer has been suppression of testosterone production by surgical or medical castration, a strategy termed androgen deprivation therapy (ADT). Whilst these treatment modalities are initially effective (reviewed in [
3]), most patients eventually relapse with castrate-resistant prostate cancer (CRPC), which is incurable and the primary cause of mortality associated with this disease.
It is now well established that the mediator of androgen action, the androgen receptor (AR), plays a key role in the progression of prostate cancer following ADT, despite castrate levels of circulating testosterone. A number of mechanisms, including increased levels of the AR mRNA or protein [
4‐
8], mutation of the
AR gene to produce more active or promiscuous forms of the receptor [
9‐
14], altered levels of AR coregulators (reviewed in [
15]), the expression of constitutively active AR splice variants [
16‐
18], and adrenal and intratumoral biosynthesis of androgens [
19‐
23], explain continued AR signaling during ADT. As many of these mechanisms are refractory to conventional ADT, there is considerable impetus to develop new and more potent agents targeting the androgen signaling axis. Two such agents are enzalutamide (MDV-3100), a novel AR antagonist that has demonstrated clinical activity in men who have failed both ADT and docetaxel-based chemotherapy [
24], and abiraterone acetate, which targets an enzyme required for adrenal and intratumoral androgen biosynthesis. Phase III clinical trials demonstrated that these agents extend median survival of men with advanced CRPC by several months and both have received FDA approval [
25].
Despite the success of enzalutamide and abiraterone, it is accepted that treatment with these agents remains essentially palliative, and that combinatorial treatment strategies targeting multiple cellular pathways in addition to androgen signaling are more likely to improve outcomes for men with CRPC. One such combination therapy comprises the AR antagonist bicalutamide and the histone deacetylase (HDAC) inhibitor vorinostat, which act synergistically together to cause death of cell line models of prostate cancer [
26]. Vorinostat has a global effect on the acetylation of histones and other proteins within the cell but also reduces AR levels and activity and thereby directly targets androgen signaling [
26]. The aim of this study was to interrogate the molecular mechanisms underlying the synergistic action of bicalutamide and vorinostat in prostate cancer. Through expression profiling and functional studies, we identified
NFKBIA (IκBα) as a critical mediator of this therapy, and in doing so provided novel insight into AR signaling and how this might be effectively targeted in prostate cancer.
Methods
Cells and reagents
LNCaP human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA), maintained in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS) and used within a range of 20–40 passages. VCaP human prostate cancer cells were purchased from the ATCC, maintained in DMEM supplemented with sodium pyruvate, non-essential amino acids and 10 % FBS, and used within 60–70 passages. Vorinostat was obtained from Merck (New Jersey, USA) and dissolved in DMSO. Bicalutamide was obtained from Astra Zeneca (London, UK) and dissolved in ethanol. Cycloheximide was obtained from Sigma (St. Louis, MO, USA) and dissolved in DMSO. Anti-AR (N-20), anti-prostate specific antigen (PSA; C-19) and anti-heat shock protein 90 (HSP90; H-114) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-IκBα antibody was obtained from Cell Signaling Technology Inc (Danvers, MA, USA). Anti-αtubulin antibody was obtained from Merck Millipore (Billerica, MA, USA). Horseradish peroxidase conjugated anti-rabbit, anti-mouse, and anti-sheep/goat secondary antibodies were obtained from DAKO (Botany, NSW, Australia). Non-specific, scrambled siRNA and ON-TARGETplus siRNAs targeting NFKBIA were purchased from Dharmacon (Lafayette, CO, USA) and the NFKBIA-IRES-eGFP lentiviral ORF plasmid was purchased from GeneCopoeia (Rockville, MD, USA). The pLV410 eGFP lentiviral ORF plasmid was kindly provided by Dr. Philip Gregory (University of Adelaide, Adelaide, Australia).
Cell viability assays
LNCaP or VCaP cells were seeded in triplicate in 24-well plates, and allowed to attach overnight before the growth medium was replaced with medium containing vehicle control or the indicated concentrations of vorinostat, bicalutamide, or the two agents in combination. Doses were calculated based on individual dose-response curves for each agent and cell line to ensure consistency in the antiproliferative response between different cell lines ([
26], Additional file
1: Figure S1). Cells were counted every 2 days using a hemocytometer and cell viability was assessed using Trypan blue dye exclusion. For sequential treatments, cells were treated with drug one for 24 h, at which point the treatment medium was removed and replaced with drug two for 48 h. To assess the effect of cycloheximide, cells were pre-treated with 10 μM cycloheximide for 1 h, which was then removed and replaced with treatment medium. Wash out experiments were performed by allowing the treatment medium to remain on the cells for 1, 2, 4, 6, 8, 16, or 24 h, at which point it was removed and replaced with drug-free medium. At each end-point cells were counted using a hemocytometer and viability assessed as above.
Microarray analysis
LNCaP cells were cultured with vehicle control, 1 μM vorinostat, 5 μM bicalutamide, or the two agents in combination for 6 h. Adjustment of the dose of bicalutamide compared to the initial cell viability assays was necessary to ensure consistency in terms of cell death between the two experiments, due to variation in the sensitivity of LNCaP cells to this agent over time. Total RNA was extracted from the cells using Trizol reagent (Life Technologies, Carlsbad, CA, USA), and RNA integrity was analyzed on an Agilent Systems Bioanalyzer. RNA from cells treated with the combination of vorinostat and bicalutamide was compared with RNA from cells treated with either vehicle control or either of the agents individually using Affymetrix Human GeneChip ST 1.0 arrays at the Adelaide Microarray Centre, as described previously [
27]. Differential gene expression was assessed by ANOVA with the p-value adjusted using a step-up multiple test correction to control the false discovery rate (FDR) [
28]. Adjusted
p-values < 0.05 were considered to be significant.
Quantitative real-time PCR
Independent RNA samples used to validate the microarray data were generated by culturing LNCaP cells with vehicle control, 1 μM vorinostat, 2.5 μM bicalutamide or the two agents in combination for 3, 6, 9 and 12 h. Total RNA (1 μg) was DNAse treated with Turbo DNA Free (Ambion, Austin, TX, USA), and then reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed with a 1:10 dilution of the cDNA using SYBR green (Bio-Rad) on a CFX Real-Time System (Bio-Rad). geNORM analysis was used to determine appropriate housekeeper genes for each sample set. Microarray RNA was normalized to HPRT1 and RPL19, and the independent sample set was normalized to GUSB and HPRT1. Primer sequences are listed in Additional file
2: Table S1.
Immunoblotting
LNCaP cells cultured with 2.5 μM bicalutamide or 1 μM vorinostat, individually and in combination, were lysed in radioimmunoprecipitation assay lysis buffer (10 mM Tris–HCl, 125 mM NaCl, 1 mM EDTA, 1 % Triton X-100) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates (20 μg) were electrophoresed through 7.5–15 % SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked overnight (4 °C) in 3 % non-fat milk powder in Tris-buffered saline containing 0.05 % Tween-20 (TBST). Immunodetection was performed overnight at 4 °C in 3 % non-fat milk powder in TBST using an anti-AR (1:1000) rabbit polyclonal antibody, anti-IκBα (1:1000) mouse monoclonal antibody, or anti-PSA (1:500) goat polyclonal antibody. Antibodies against HSP90 (1:1000, rabbit polyclonal) and α-tubulin (1:1000, mouse monoclonal) were used to assess loading. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies and visualized on autoradiography film using enhanced chemiluminescence detection (GE Healthcare).
Pathway analysis
Enriched gene pathways were identified using Ingenuity Pathway Analysis (IPA), the Database for Annotation, Visualization and Integrated Discovery (DAVID), and Gene Set Enrichment Analysis. Significantly regulated genes (
p < 0.05) were uploaded into IPA software v9.0 (Ingenuity Systems, CA, USA) in separate lists for the combination vs. bicalutamide alone and the combination vs. vorinostat alone. Each gene was mapped to its corresponding molecule in the Ingenuity pathways knowledge base, and core analysis identified enriched pathways and networks in the dataset against a background of the Affymetrix Human GeneChip ST 1.0 array. Lists of genes significantly regulated by the combination compared to either vehicle control or either of the individual agents were uploaded to the Functional Annotation Tool available through DAVID (
https://david.ncifcrf.gov/; [
29,
30]), converted to DAVID default IDs, and analyzed against a background of the microarray platform. Genes significantly regulated by the combination, either uniquely or when compared with the individual agents, were analyzed against a background of all genes significantly regulated by the combination over vehicle control. Enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified using DAVID. Gene Set Enrichment Analysis [
31] was implemented using the Broad Institute’s public GenePattern server (
http://genepattern.broadinstitute.org/gp/pages/index.jsf), with default parameters.
Transfection of siRNA and expression constructs
For siRNA transfection, LNCaP or VCaP cells were seeded directly into transfection medium containing phenol red free (PRF) RPMI 1640, lipofectamine 2000 (Life Technologies), and reconstituted scrambled siRNA control or siRNA targeting NFKBIA at a concentration of 10 nM. Four siRNAs were tested, and #1 and #4 were found to be the most effective at achieving knockdown. After 4 h of culture, additional PRF-RPMI medium containing FBS and L-glutamine was added to the LNCaP transfection mixture, while DMEM containing FBS, L-glutamine and non-essential amino acids was added to the VCaP transfection mixture. Cells were harvested for counting and assessment of cell death using Trypan blue dye exclusion, and then lysed in RIPA buffer for immunoblot analysis three days post-treatment (LNCaP) or six days post-treatment (VCaP). The two timepoints used reflect the different growth kinetics between the two cell lines.
For transient transfection of lentiviral constructs expressing green fluorescent protein (GFP) or co-expressing NFKBIA and GFP, LNCaP cells were seeded at ~40 % confluency and allowed to attach overnight. Growth medium was removed and replaced with transfection medium containing PRF-RPMI, lipofectamine 2000, and 1.5 μg plasmid DNA. As for siRNA transfection, additional medium containing FBS, L-glutamine and either vehicle control or combination therapy was added to the transfection mix after 4 h of culture. At three days post-treatment, fluorescent cells were visualized using a fluorescent microscope, and transfection efficiency was estimated at between 40 and 50 %. Cells were then harvested and assessed for death using Trypan blue dye exclusion, after which they were lysed in RIPA buffer for subsequent immunoblot analysis.
Discussion
Metastatic prostate cancer inevitably becomes resistant to current hormonal therapies and, consequently, combinatorial therapeutic approaches that may be more efficacious and less prone to resistance-associated failure have garnered significant interest. We have previously shown that combining the AR antagonist bicalutamide with the HDAC inhibitor vorinostat synergistically induces growth arrest and cell death in prostate cancer cells. Importantly, this combination approach uses doses of both agents that are individually sub-effective [
26], implying that it would minimize dose-related toxicity. The current study provides new insight into the molecular mechanism by which these disparate agents interact synergistically to induce death of prostate cancer cells, and implicates IκBα, a regulator of the NF-κB and p53 pathways, as a critical factor for prostate cancer cell viability and treatment response.
Given that vorinostat and bicalutamide both target AR, we initially hypothesized that the combination of these two agents would enhance blockade of androgen signaling, a pathway that promotes growth and survival of prostate cancer cells. This hypothesis was reinforced by previous work from our laboratory demonstrating that the combination treatment induced death only in cells with a functional AR signaling axis, and that addition of excess dihydrotestosterone (DHT) to the system prevented cell death [
26]. The genome-wide microarray expression data generated in the current study also supported this hypothesis. Specifically, many androgen-regulated genes were significantly altered by the combination compared to individual agents, and pathway analysis demonstrated deregulation of androgen signaling and prostate cancer networks by the combination treatment. Interestingly, for six known androgen regulated genes, we did not observe consistently greater downregulation by the combination therapy in an independent set of RNA samples. This could suggest that enhanced androgen blockade is not the substantive mechanism by which the combination exerts its effect. However, this subset of genes only represents approximately 10 % of the genes significantly regulated by both DHT and the combination (when compared with the individual agents), and 1 % of the total genes significantly regulated by both DHT and the combination (when compared with vehicle control). It is possible that small changes to a large number of androgen regulated genes is an important factor in the mechanism of action of the combination therapy, or that these cumulative changes are able to sensitize the prostate cancer cells to HDAC inhibition.
While the importance of enhanced blockade of androgen signaling by the combination treatment remains ambiguous, the expression data revealed that this treatment also modulates a multitude of other critical cellular processes. For example, p53 signaling and other pathways involved in cell cycle arrest and cell death were highly enriched in genes modulated by the combination. In considering potential mediators of the synergistic interaction between vorinostat and bicalutamide, we noted that our pathway analyses consistently implicated an inhibitor of NF-κB signaling,
NFKBIA, in this phenomenon. Interestingly,
NFKBIA is an androgen regulated gene, and the protein encoded by this gene, IκBα, is best known as an inhibitor of NF-κB signaling. However, IκBα can also inhibit p53 signaling and thereby functions dichotomously to either block p53-mediated cell death or NF-κB-mediated cell growth [
34‐
37], with the final phenotypic outcome likely depending on the relative levels of NF-κB and p53 within a given cell. At a mechanistic level, IκBα sequesters NF-κB or p53 in the cytoplasm in an inactive complex; following various stimulatory events, IκBα is phosphorylated and targeted for degradation via the ubiquitin-proteasome pathway, relieving inhibition of these factors. With respect to prostate cancer cell death, we observed rapid downregulation of
NFKBIA in cells treated with the combination of bicalutamide and vorinostat that was associated with increased expression of
TP53INP1 and CDKN1A, two commonly known p53-inducible genes, and induction of cell death. Taken together, this data suggests that, in the context of the combination therapy, loss of IκBα results in cell death, which may be facilitated by the induction of p53 signaling. This hypothesis is supported by the observation that knockdown of
NFKBIA in the absence of drug treatment resulted in a similar level of cell death to that observed with the combination in two independent prostate cancer cell lines. Moreover, co-treatment with
NFKBIA siRNA and the combination therapy caused significantly more cell death than either individual treatment. Importantly, overexpression of NFKBIA almost completely negated the effect of the combination treatment on cell death.
Two other observations arising from the current study are worth noting for their clinical ramifications. First, the combination of bicalutamide and vorinostat was efficacious in models with a mutant (LNCaP) or amplified wild-type (VCaP) AR gene. Given that many clinical prostate cancers are characterized by aberrant AR signaling, and that intra-tumoral heterogeneity may result in foci that each potentially have structurally different androgen receptors, this is a promising feature of the combination therapy. Second, both vorinostat and bicalutamide are required simultaneously in culture for induction of cell death, indicating that if sensitization is happening it occurs rapidly. This finding indicates that future clinical testing will require the agents to be dosed together and not sequentially.
Conclusion
In summary, we have defined a novel mechanism of action by which bicalutamide and vorinostat, when used in combination, mediate death of prostate cancer cells. While enhanced blockade of androgen signaling is potentially important, we have demonstrated that other cellular pathways also play critical roles. Specifically, IκBα was identified as a critical regulator of therapy-mediated cell death; this factor may have potential either as a new therapeutic target and/or a marker of drug response. The ability to monitor molecular markers of apoptotic response to such therapeutic strategies will aid in the clinical development of this combinatorial approach for treatment of prostate cancer.
Acknowledgements
This work was supported by the National Health and Medical Research Council of Australia (627185 to WDT & LMB), Cancer Australia (627229 and 1012337 to LMB & WDT), the Royal Adelaide Hospital Research Committee (to MMC, LMB & WDT), an infrastructure grant from the Prostate Cancer Foundation of Australia (2011/0452 to WDT), Cancer Council of South Australia Senior Research Fellowship (to LMB), a Future Fellowship from the Australian Research Council (FT130101004 to LMB), Young Investigator grants from the Prostate Cancer Foundation of Australia (LAS, YI 0810; MMC, YI 0412), a Young Investigator Award from the Prostate Cancer Foundation (the Foundation 14 award; LAS) and the Lions Medical Research Foundation Post-Graduate Scholarship (SLC). We thank Ms Natalie Ryan for her assistance in generating material for the microarray study, Ms Joanna Gillis for her assistance in generating material for the VCaP knockdown studies, Ms Heather Armstrong for technical assistance, and Dr Paul Neilsen for expert advice on p53 signaling.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SLC carried out the molecular studies, participated in the microarray profiling and analysis, participated in the design of the study and drafted the manuscript. MMC participated in the microarray profiling studies and the manuscript drafting. WDT participated in the design of the study and manuscript drafting. LAS participated in microarray analysis, design of the study, and manuscript drafting. LMB conceived of the study, designed the study and participated in manuscript drafting. All authors read and approved the final manuscript.