Background
Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer death in American males. An estimated 161,360 new cases and 26,730 deaths from PCa were predicted for 2017 [
1]. Androgen is crucial for PCa development, and androgen deprivation therapy (ADT) is widely accepted as a first-line treatment for advanced and metastatic PCa [
2]. Although ADT is initially effective, most patients eventually progress to metastatic castration-resistant PCa (mCRPC) in 2 to 3 years. Despite the new generation of AR antagonists used alone or in combination with chemotherapeutic drugs for the treatment of mCRPC, mCRPC remains incurable with an average life span less than 19 months [
3]. New effective therapeutics is urgently required.
Poly ADP-ribose polymerase (PARP) is involved in the DNA damage response. The PARP inhibitor olaparib was recently approved by the FDA for mCRPC and ovarian cancer patients with
BRCA1/2 or
ATM gene mutations [
4‐
6].
BRCA1 and
BRCA2 are two critical tumor suppressor genes crucial for DNA double strand break (DSB) repair through homologous recombination (HR) pathways [
7], and play key roles in breast cancer [
8,
9]. Approximately 25 to 30% of mCRPC involves somatic mutations of the
BRCA1/2 genes, resulting in DNA repair deficiency [
10]. Aberrations of DNA repair genes have been associated with sensitivity to DNA damage drugs such as platinum, radiotherapy and PARP inhibitors [
4].
Veliparib is another PARP inhibitor developed by AbbVie USA [
11]. The FDA awarded veliparib orphan drug status in November 2016 for non-small cell lung cancer. As of 2017, 96 clinical trials involving veliparib were registered with the FDA based on its anticancer potential in several cancer types. A clinical trial combining abiraterone acetate and prednisone with or without veliparib in patients with metastatic castration-resistant prostate cancer is ongoing (NCT01576172,
ClinicalTrials.gov). Limited studies have been performed to directly compare the antitumor efficacy and mechanisms of olaparib and veliparib. It has been reported that oliparib have stronger catalytic inhibitory properties and the potency to trap PARP enzymes to the damage DNA than veliparib [
12]. The available data showed that olaparib and veliparib differ in their off-target effects. Olaparib reduced DNA damage repair activity via G2 cell cycle arrest in a p53-dependent manner, but veliparib did not have such an effect [
13].
Histone deacetylases (HDACs) play an important role in structural modification and gene expression regulation through induction of histone acetylation. Several HDAC inhibitors have been approved by the FDA to treat hematological malignancies [
14,
15]. Although they are not approved by the FDA, HDAC inhibitors have shown the anticancer potential for solid tumors such as PCa in preclinical studies [
16,
17]. HDAC1, 2 and 3 are highly expressed and excessively activated in PCa, especially in mCRPC [
18]. High expression of HDACs enhances the proliferation and metastatic potential of PCa cells [
18,
19], while HDAC inhibitors decrease the potential [
20,
21]. Importantly, HDAC is involved in HR DNA repair [
22]. HDAC1 and 2 are recruited to DNA break sites when DNA damage occurs, interact with PCNA, and localize to the sites of DNA replication [
23,
24]. SAHA, a pan-HDAC inhibitor, prevented DNA damage repair by down-regulating RAD50 and MRE11 [
25]. In addition, SAHA and valproic acid, another HDAC1 inhibitor, induced the downregulation of RAD51 expression [
26,
27]. Trichostatin A induces a DNA damage signaling pathway in an ATM-dependent manner [
28].
Since PARP and HDAC inhibitors prevent HR DNA repair, the combinationof two inhibitors has been tested to improve the anticancer efficacy [
29‐
31]. SAHA significantly improved the anticancer efficacy of olaparib in triple-negative breast cancer (TNBC) cells that expressed functional phosphatase and tensin homolog (PTEN) [
32]. By attenuating the levels of DNA damage response and HR proteins ATR, CHK1 and BRCA1, this pan-HDAC inhibitor induces ‘BRCAness’ and sensitizes TNBC cells lacking BRCA1 to the lethal effects of a PARP inhibitor [
33]. Additionally, SAHA and olaparib showed synergistic therapeutic effects on prostate cancer cell death, apoptosis and DNA damage by decreasing the protein levels of BRCA1 and RAD51, while have no effect on normal prostate epithelial cells [
31]. UHRF1 (Ubiquitin-like with PHD and ring-finger domain 1) is an important protein for DNA methylation maintenance, recognizing specific DNA hemimethylation and recruiting DNMT1 to catalyze methylation on the hemimethylated CpG motifs [
34]. UHRF1 is involved in drug resistance and DNA damage repair [
35]. UHRF1 recruits ERCC1 and MUS81 to repair DNA lesions [
36]. Additionally, when DNA damage occurs, BRCA1 recruits UHRF1 protein to DNA double-strand breaks in S phase, and then UHRF1 mediates K63-linked polyubiquitination of RIF1, resulting in its dissociation from 53BP1 and DSBs, thereby facilitating HR initiation [
37]. These findings suggested that UHRF1 plays a critical rolein the combinational therapy of PARP and HDAC inhibitors.
In this study we tested the anticancer efficacy of veliparib and SAHA alone or in combination, using cell viability, colony formation and apoptosis detection assays. We analyzed DNA damage when PCa cells were treated with SAHA and veliparib alone or in combination, and further explored the molecular mechanisms by which veliparib and SAHA co-target the UHRF1/BRCA1 complex to impair HR DNA damage repair. Eventually, the anticancer efficacy of drug combination was validated in xenograft models.
Methods
Reagents
PARP inhibitor veliparib (ABT-88) and HDAC inhibitor SAHA were purchased from Selleck, China (Shanghai, China). Both were dissolved in dimethylsulfoxide (DMSO). Stock solutions were 50 mM for SAHA and 100 mM for veliparib.
Cell culture
Human PCa cells LNCaP, VCaP, PC-3 and DU145 and non-malignant prostate epithelial cells were purchased from ATCC (Manassas, VA, USA). C4–2 and CWR22Rv1 were obtained from Dr. Chinghai Kao at the Indiana University School of Medicine. PCa cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Friendship, ME, USA) and penicillin/streptomycin antibiotics. RWPE-1 cells were maintained in defined Keratinocyte-SFM (1×) liquid (Invitrogen, Carlsbad, CA, USA). All cells were cultured with 5% CO2 in a 37 °C incubator.
Cell viability assay
Cell viability was assessed by crystal violet assay. Cells were plated in 24-well plates with a cell density of 4 × 104 cells per well, and incubated at 37°Cfor 24 h. Then the cells were treated with SAHA and veliparib alone or in combination. Three days after drug treatment, cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet solution for 20 min (300 uL/well). After thorough washing with tap water, plates were air-dried for at least 2 h at room temperature. Cells were lysed by shaking the cells in 1% SDS (400uL/well) for 30 min. Cell viability was evaluated by measuring the absorbance of each well at 570 nm with a VersaMax™ microplate reader.
The cells were plated in 6-well plates (500 cells/well) at 37 °C overnight and then treated with SAHA and veliparib alone or in combination. Cell culture media were replaced with fresh growth medium with drugs every 3 days. The cells were fixed and stained with 0.1% crystal violet solution 7 days after drug treatment. Clones with > 50 cells were counted under the microscope and the survival fractions were calculated as the average number of colonies± SD of three independent experiments.
Apoptosis assays
PCa cells were treated with SAHA and veliparib alone or in combination at the indicated concentrations. Cells were harvested 5 days after drug treatment, EDTA free trypsin and centrifugation at 2000 rpm for 5 min. After washing cells with PBS twice, cell apoptosis was analyzed by two methods: staining the cells with FITC-Annexin V/propidium iodide (PI) at room temperature avoiding light for 10 min, and assessing the sub-G1 cell sub-population after PI staining with flow cytometry. The positive cells were detected by flow cytometer.
Immunofluorescence
LNCaP cells were maintained in 24-well plates with coverslips and treated with SAHA and veliparib alone or in combination for 48 h. Cells were fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. The cells then were incubated with γH2AX antibody (phosphoS139, CST, Danvers, MA, USA) at 4 °C overnight. Cells were washed with cold PBS for three times, and then incubated with the second antibody. The nuclei were stained with DAPI. Images were captured and analyzed with Leica fluorescence microscopy.
Western blot analysis
Protein levels were assessed by western blot. Antibodies against HDAC, PARP, cleaved-PARP, H3, Acetyl-H3, and RAD51 were purchased from Cell Signaling Technology (CST). Anti-UHRF1, Ku-70, ERCC1, MSH2, MSH6, GAPDH and anti-α-tublin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRCA1 and phosphorylated-BRCA1(ser988) was purchased from ABclonal Technology (Upper Heyford, UK). Anti-PAR antibody was purchased from Trevigen (4335-MC-100, MD, USA).
Small RNA interference
Two specific siRNAs targeting 2 UHRF1 sequences were purchased from GenePharma(Shanghai, China). siRNAs were transfected into PCa cells using Dharmafect Transfection Reagents (Lafayette, CO, USA).
In vivo animal study
The animal experiment protocol had been approved by the Ethics Committee of Xiangya Hospital, Central South University. Twenty nude male mice (5 to 6 week-old) were purchased from the SLAC Laboratory, Shanghai, China. The tumor xenografts were induced by subcutaneously inoculating DU145 cells (5 × 106/100 uL) into the left flank region. Three weeks later, the nude mice bearing tumor xenografts were randomly divided into 4 groups, and received the following treatments for 3 continuous weeks: vehicle, SAHA (25 mg/kg.d,i.p.), veliparib(25 mg/kg.d,oralgavage), SAHA(25 mg/kg.d,i.p) plus veliparib(25 mg/kg.d,oral gavage). Tumor size and body weight were measured every 4 days, and tumor xenograft volume(V)was calculated using the following formula: V = ab2/2 (a: the long diameter and b: the short diameter). The tumor xenografs were isolated at the endpoint of experiment, and the tumor size and weight was compared by using the statistical analysis.
Statistics
All data were analyzed by Statistical Product and Service Solutions 17.0. Results were presented as Mean ± SD (Standard Deviation) or SEM (Standard error of the mean). One-way ANOVA was used to analyze the statistical difference of multiple groups. *P < 0.05, **P < 0.01 and ***P < 0.001. P < 0.05 was considered as significant.
Discussion
Veliparib is a PARP inhibitor now being tested for safety and anticancer efficacy in a number of clinical trials, including PCa trials. Although PARP inhibitors have shown great success for BRCA-mutated tumors, patients still develop acquired drug resistance. Several possible mechanisms have been proposed. Drug efflux through transporters decreases drug intake [
39]. The decline or loss of PARP1 expression decreases drug response. PARP1 has been reported the involvement in several DNA repair pathways [
40], including base excision repair (BER) [
41] or single-strand break repair (SSBR) by binding to apurinic/apyrimidinic (AP) sites [
42] or recruiting BER complex [
43], promotes nucleotide excision repair (NER) or mismatched repair by ADP-ribosylates XPA [
44] or MSH6 [
45], and promotes NHEJ by activating DNA-PKcs [
44,
45]. In addition, PARP1 promotes homologous recombination (HR) by activating ATM signaling [
46]. Cancer patients develop secondary genetic or epigenetic mutations restoring functional HR in tumors that were formerly HR deficient [
47,
48], or restore BRCA protein function [
49] after treatment. BRCA1/2 is involved in homologous recombination (HR) DNA damage repair by forming different protein complexes with CCDC98, RBBP8 and BACH1 at different stages of double strand DNA break repair (DBS) [
50,
51]. In BRCA-mutated cancer cells, PARP inhibitors further inhibit DNA damage repair, leading to cell death. In addition, somatic mutations of TP53BP1 induce partial restoration of HR [
52].
Improving the cell killing and cancer selectivity of veliparib apparently requires a novel drug combination. Recent studies showed that PCa with HR deficiency may be sensitive to PARP inhibitors and platinum chemotherapy [
53,
54]. In this study, we observed that SAHA or veliparib alone decreased cell viability and clonogenicity, and induced cell apoptosis and DNA damage. Importantly, co-administration of the two drugs synergistically decreased cell viability and clonogenicity and enhanced cell apoptosis and DNA damage, with no detectable toxicity to normal prostate epithelial cells at the tested dose window.
It has been reported that the PARP inhibition down-regulates BRCA1 in a pathway mediated by E2F4 and p130 [
55]. Other publication suggested that HDAC inhibition down-regulates BRCA1 mRNA level by decreasing the amount and recruitment of E2F1 transcription factor [
27]. In our present manuscript, we proposed a novel mechanism by which two drugs further induced BRCA1 protein degradation. UHRF1 is an important epigenetic regulator that has been implicated in treatment resistance [
56‐
58] and DNA damage repair [
59]. It is a binding factor for DNA interstrand crosslik lesions (ICL), and is involved in processing ICL lesions by recruiting structure-specific endonucleases [
36]. PARP1 interacts with UHRF1 protein [
60], suggesting that UHRF1 is involved in single strand DNA damage repair. UHRF1 plays a double-facet role in the regulation of BRCA1, i.e. UHRF1 silences BRCA1 gene transcription, and sustains BRCA1 protein stability. On one hand, UHRF1 as an epigenetic regulator, together with other enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 caused the epigenetic silencing of tumor suppressor genes including BRCA1 by inducing DNA methylation and histone post-translation modification changes [
61]. On the other hand, when DSB damage occurs, BRCA1 recruits UHRF1 to the damage site and mediates RIF degradation to help DNA repair [
37], and UHRF1 is required for the maintenance of protein stability of BRCA1. Deficiency of UHRF1 function promotes DNA damage sensitivity [
37]. Targeting UHRF1 may be an attractive strategy to improve the anticancer efficiency of PARP inhibitors. However, an effective UHRF1 inhibitor is still unavailable for pre-clinical and clinical studies [
34].
UHRF1 protein interacts with HDAC proteins in the epigenetic repression complex [
62]. The pan-HDAC inhibitor SAHA induced the acetylation of histone protein H3 (Fig.
6a), and induced the degradation of UHRF1 protein. We report for the first time that SAHA or veliparib alone decreased UHRF1 and BRCA1 protein levels to different extents, and that co-treatment with SAHA and veliparib consistently and synergistically decreased UHRF1 and BRCA1 protein levels. Since UHRF1 protein physically interacts with BRCA1, the depletion of UHRF1 decreased BRCA1 protein levels. Thus co-treatment with SAHA and veliparib decreased the protein levels of BRCA1 through UHRF1 (Fig.
7d). However, co-treatment did not reduce RAD51 protein levels. The synergistic reduction of BRCA1 protein in response to the combination of veliparib and SAHA is different from the previous report in which olaparib and SAHA induced a synergistic reduction of RAD51 but not BRCA1 [
31]. The mechanism for this difference is worth further study, though the PARP inhibitor per se certainly influences the pathways of DNA damage repair.