Histone deacetylase inhibitors enhance Ad5-TRAIL killing of TRAIL-resistant prostate tumor cells through increased caspase-2 activity
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- VanOosten, R.L., Earel, J.K. & Griffith, T.S. Apoptosis (2007) 12: 561. doi:10.1007/s10495-006-0009-9
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Interest in TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic has been high since its first description. Recently, the use of histone deacetylase inhibitors (HDACi) to treat cancer has progressed from the laboratory to the clinic, and the combination of HDACi and TRAIL is very powerful in killing human tumors. Using a panel of prostate tumor cell lines (ALVA-31, DU-145, and LNCaP) with varying TRAIL sensitivity, we examined their sensitization to a recombinant adenovirus encoding TRAIL (Ad5-TRAIL) by sodium butyrate and trichostatin A. HDACi treatment increased coxsackie-adenovirus receptor (CAR) expression, resulting in increased adenoviral infection, and increased TRAIL-mediated killing. In TRAIL-resistant DU-145 cells, HDAC inhibition also decreased protein kinase casein kinase (PKCK) 2 activity, leading to caspase-2 activation. The importance of PKCK2 and caspase-2 in DU-145 sensitization was demonstrated with the PKCK-2-specific inhibitor, which enhanced Ad5-TRAIL-induced death, or the caspase-2-specific inhibitor, zVDVAD, which blocked Ad5-TRAIL-induced death. Thus, our data highlight the connection between HDAC inhibition of PKCK2 activity and tumor cell sensitivity to TRAIL-induced apoptosis. Specifically, HDAC inhibition leads to decreased PCKC2 activity, which is followed by caspase-2 activation and partial cleavage of caspase-8 that sensitizes the tumor cell to TRAIL.
The administration of genes into tumor sites in situ through the use of various gene delivery systems, such as non-replicative viral vectors, is becoming a viable alternative therapy for treating cancer. Recombinant adenoviral vectors infect a wide range of proliferating and quiescent cell types, making this system a suitable tool for studying diseases, vaccine therapy, and potential clinical use . Adenoviral-mediated gene transfer has been used in a variety of experimental conditions that include transfers to the liver, lung, central nervous system, and cancer cells [2–7]. Most gene therapy protocols currently being investigated for cancer are designed to eradicate tumor cells directly with toxic genes or indirectly by using genes that elicit antitumor immune responses. There are increasingly more clinical gene therapy trials underway and, although many investigations have shown great promise in pre-clinical studies, the efficient and accurate delivery of therapeutic genes remains a challenge in all solid tumor oncology. Moreover, clinical gene therapy protocols involving local delivery of tumoricidal genes also have to deal with the potential of multifocal lesions, making it difficult to have the injected agent come in contact with every tumor cell.
Since the principal viral vector for cancer gene therapy is adenovirus, the success of adenoviral-based therapies is, therefore, primarily dictated by coxsackie-adenovirus receptor (CAR) recognition . Adenoviral-based gene therapy is being evaluated for prostate cancer [9, 10], but since there is decreased CAR expression on human prostate cancer cell lines and in carcinoma-containing prostates compared to normal prostate cells and tissues [11, 12], adenoviral uptake by the cancer cells may be decreased in situ. Although targeting strategies are being investigated to circumvent CAR dependence, all clinical adenoviral gene therapy approaches reported to date have been CAR-dependent. If CAR expression on target cells could be increased, it is predicted that the clinical efficacy of adenoviral-based gene transfer therapy for cancer could also be increased. Previous studies on histone deacetylase inhibitors (HDACi) have demonstrated their ability to increase CAR expression on tumor cells . HDACi are a growing field as they not only augment adenoviral gene transfer, but also to sensitize tumor cells to apoptotic death [14-17].
This report describes the use of a recombinant adenoviral vector encoding the cDNA for human TNF-related apoptosis-inducing ligand (Ad5-TRAIL), which was first developed and described by our laboratory . TRAIL induces apoptosis in tumor or transformed cells, but not in normal cells [19, 20]. The difficulty with therapies involving recombinant TRAIL is that large quantities need to be delivered in vivo to inhibit tumor outgrowth [21, 22]. Therefore, the use of an adenoviral vector to administer TRAIL permits the local expression of elevated levels of TRAIL protein. This study examines the tumoricidal potential of combining Ad5-TRAIL with histone deacetylase inhibitors (HDACi) against the TRAIL-resistant prostate tumor cell line DU-145. Our data indicate that HDACi sensitize these cells by activating caspase-2, which “primes” the cells for the TRAIL death signal.
Materials and methods
Cells and reagents
The human prostate tumor cell lines, ALVA-31, DU-145, and LNCaP, were obtained from Dr. Timothy Ratliff (University of Iowa, Iowa City, IA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, sodium pyruvate, non-essential amino acids, and HEPES (hereafter referred to as complete RPMI). Normal human prostate epithelial cells were obtained from Cambrex (San Diego, CA) and cultured as directed. Sodium butyrate and trichostatin A were purchased from Sigma (St. Louis, MO). The PKCK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), was purchased from EMD Biosciences (San Diego, CA).
Cells were treated for 16 h with HDACi at 105 cells/well in a 24-well plate. Cells were harvested and stained with an unlabeled anti-CAR antibody (Upstate Cell Signaling Solutions, Lake Placid, NY) at a 1:100 dilution for 20 min. Cells were washed and then stained with a PE-conjugated, Fc-specific, goat anti-mouse F (ab’)2 for 20 min, washed and analyzed on a FACScan (Becton-Dickson, San Jose, CA). To measure TRAIL-R1 and —-R2 expression, vehicle or HDACi-treated cells were incubated with the PE-conjugated DJR1 (eBioscience, San Diego, CA; 1 μg/ml), DJR2-4 (eBioscience), specific for TRAIL-R1 or TRAIL-R2, respectively, or a PE-conjugated mouse IgG1 isotype control mAb (eBioscience) for 1 h at 4°C. After washing, cells were analyzed immediately on a FACScan.
Production of Ad5-TRAIL
A replication-deficient adenovirus encoding human TNFSF10 (Ad5-TRAIL  expressed from the cytomegalovirus (CMV) promotor was generated using the RAPAd.I system  at the University of Iowa Gene Transfer Vector Core (Iowa City, IA). Recombinant adenovirus encoding green fluorescent protein (Ad5-GFP) was used as a reporter virus.
In vitro killing of human prostate tumor cells
Cell sensitivity to Ad5-TRAIL was assayed as follows. Cells were added to 24-well plates (5 × 105 cells/well) in complete medium, and pretreated with HDACi or vehicle control for 16 h before infection with Ad5-TRAIL at the indicated concentrations (pfu/cell). Apoptotic cell death was determined after 5 h by flow cytometry using FITC-conjugated annexin V (R&D Systems, Minneapolis, MN) and propidium iodide (PI; Sigma, St. Louis, MO) as described . In some cases, the caspase-2-specific pentapeptide inhibitor, zVDVAD-fmk [20 μM; BioVision Research Products, Mountain View, CA), was added at the same time as the HDACi, or the PKCK2 inhibitor, TBB (30 μM) was added to the cells at the same time as the Ad5-TRAIL, and then the cells were examined for apoptotic death by annexin V/PI staining.
Cells (5 × 105 cells/well in 6-well plates) were treated with HDACi for 24 h. Cells were lysed in PBS containing 1% Nonidet P-40 Complete Mini protease inhibitor tablet (1 tablet/10 ml; Roche, Indianapolis, IN). Lysate fractions were mixed with a 6X reducing SDS-PAGE loading buffer, heated for 5 min at 100°C, separated by SDS-PAGE, and transferred to nitrocellulose membrane. Following an overnight block in 5% nonfat dry milk in PBS-Tween-20, the membrane was incubated with the anti-p21 antibody (diluted 1:500, Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membrane was incubated with an anti-rabbit HRP antibody (diluted 1:5000, Jackson ImmunoResearch) for 1 h. Following several washes, the blots were developed by chemiluminescence. To measure caspase-8 cleavage, DU-145 cells (5 × 105 cells/well in 6-well plates) were treated with HDACi for 24 h. Cell lysates were prepared, and then mixed with a 6X reducing SDS-PAGE loading buffer, heated for 5 min at 100°C, separated by SDS-PAGE, and transferred to nitrocellulose membrane. Following overnight block in 5% nonfat dry milk in PBS-Tween-20, the membrane was incubated with an anti-caspase-8 mAb (provided by Dr. Marcus Peter, University of Chicago) for 1 h. After washing, the membrane was incubated with an anti-mouse HRP antibody (diluted 1:5000, Jackson ImmunoResearch) for 1 h. Following several washes, the blots were developed by chemiluminescence.
Cell cycle analysis
Cells (2 × 106 cells/well in a 6-well plate) were treated for 16 h with HDACi. Cells were removed and stained with PI staining solution (0.2% Triton X-100, 0.1 % sodium citrate (w/v), 50 μg/ml propidium iodide, 0.2 mg/ml RNase A).
Quantitative real time PCR
Cells were plated at 5 × 105 cells/well in 24 well plates, and pretreated for 16 h with HDACi or vehicle control. Total RNA was then isolated with TRIzol reagent (Invitrogen, Carlsbad, CA), and 2 μg was reverse-transcribed using Superscript II. The real-time quantitative RT-PCR primers and probes for TRAIL-R1 and —-R2 were designed to cross an intron using published sequences (human genome project BAC clone RP11-1149023 and RP11-875011, respectively). Sequences of the real-time quantitative RT-PCR primers and probes used were:TRAIL-R1 (forward:5′-TGTACGCCCTGGAGTGACAT-3′; reverse:5′-CACCAACAGCAACGGAACAA-3′; probe:5′-6FAM-TGTCCACAAAGAATCAGGCAATGGACATAAT-TAMRA-3′) and TRAIL-R2 (forward: 5′-CACTCACTGGAATGACCTCCTTT-3′; reverse: 5′-GTGCAGGGACTTAGCTCCACTT-3′; probe: 5′-6FAM-TCACACCTGGTGCAGCGCAAGCAG-TAMRA-3′). The real-time quantitative RT-PCR primer/probe sets for rRNA was purchased from PE Applied Biosystems (Foster City, CA). 250 ng of cDNA was used as a template for TaqMan assay for CAR transcripts and the internal control of rRNA. The TaqMan PCR reaction was carried out as described previously .
Protein Kinase Casein Kinase 2 activity
PKCK2 activity was measured using a Casein Kinase 2 Assay Kit (Upstate Cell Signaling Solutions, Lake Placid, NY). Cells (106 cells/well in a 6 well plate) were treated with HDACi for 24 h. Cell lysates were prepared using RIPA buffer, and immediately run with the Casein Kinase 2 Assay Kit as per manufacturer's instructions.
Measurement of caspase-2 activity
CAR is the primary receptor for adenoviral recognition and internalization [8, 26], making its expression critical for the success of adenoviral-based gene transfer therapies for cancer. Among the many technical challenges associated with human gene therapy is the need to develop more effective vectors. One way to do this could be accomplished by increasing the endogenous CAR expression, especially in cancers where CAR expression is commonly decreased (such as urological cancers) . In recent years, the inclusion of HDACi in gene therapy protocols has dramatically increased the success of the intended treatment. HDACi epigenetically modify chromatin , which leads to a variety of outcomes, including cell cycle and angiogenesis inhibition and induction of apoptosis. HDACi also increase CAR expression on many different tumor cell types , making it possible to investigate the potential of gene therapy in settings that would normally be unsuccessful. We were interested in examining the potential of combining HDACi with Ad5-TRAIL as a therapy for prostate cancer. Analysis of CAR expression on our panel of prostate tumor cells revealed both HDACi tested, sodium butyrate (SB) and trichostatin A (TSA), increased CAR expression on the cell surface of both ALVA-31 and DU-145 cells, but not LNCaP (Fig. 1(A)). Since CAR is important for adenovirus infection, we predicted the increase in CAR levels after HDACi treatment would correlate with increased adenoviral transgene expression. To investigate this, we treated each cell line with either vehicle or HDACi, followed by the addition of an adenoviral vector encoding green fluorescent protein (Ad5-GFP). There was a significant increase in the mean fluorescent intensity of each HDACi treatment group over the untreated or vehicle-treated cells, including LNCaP (Fig. 1(B)). Previous results from our laboratory have demonstrated that HDACi increases adenoviral entry/infection of tumor cells, as well as increases CMV promotor driven transgene expression , providing two different methods to achieve increased transgene expression. We believe this explains the increase in GFP expression in LNCaP, despite the lack of CAR upregulation. More importantly, these results also suggest that Ad5-TRAIL transgene expression and killing of the each of the prostate tumor cell lines will also be enhanced following HDACi treatment.
HDACi increase the tumoricidal activity of Ad5-TRAIL
Having determined that the HDACi-induced increase in killing by Ad5-TRAIL occurred with each of the prostate tumor cell lines tested, it became necessary to determine if the HDACi-induced increase in tumor cell death was the result in a change in the amount of regulation on the TRAIL apoptosis signaling pathway. We focused our attention on DU-145 to investigate the mechanism of the HDACi-induced sensitization, as we believed the HDACi-induced alterations would be most obvious in this cell line. Thus, we next tested whether the increased cell death observed with the combination Ad5-TRAIL and HDACi was the consequence of the cells becoming more susceptible to TRAIL-mediated apoptosis. To do this, we examined tumor cell death after treatment with HDACi and recombinant TRAIL (rTRAIL) protein. Indeed, there was a profound increase in rTRAIL-induced tumor cell death after either SB or TSA pretreatment compared to untreated or vehicle-treated cells (Fig. 2(B)), indicating that HDACi provide a multifaceted benefit when combined with Ad5-TRAIL —- that is, increasing adenoviral infectivity and transgene expression and altering tumor cell sensitivity to TRAIL-mediated apoptosis.
HDACi alter cell cycle progression in DU-145 cells
HDACi sensitize DU-145 cells to TRAIL by activating caspase-2
Since its discovery in 1995 , numerous studies have investigated the potential of using TRAIL as a cancer therapeutic because it is a potent inducer of apoptosis in tumor cells but not in normal cells and tissues. Early reports demonstrated the safety of large, systemic doses of recombinant TRAIL protein, leading to further studies that evaluated the antitumor activity in vivo [22, 42]. For these studies, immunocompromised mice were subcutaneously injected with human tumor cells, followed by intraperitoneal or intravenous injections of soluble TRAIL starting at various days after tumor implantation [22, 42]. Multiple doses of TRAIL beginning the day after tumor implantation suppressed tumor outgrowth, with many animals becoming tumor-free. One major drawback to these findings, however, was that large amounts of TRAIL were required to inhibit tumor formation, since most of the protein was cleared within 5 hours , potentially making it problematic to administration of equivalent doses of recombinant TRAIL protein into humans. Thus, we were the first to report the development of a nonreplicative recombinant adenoviral vector encoding the TRAIL gene  that can be administered locally at the site of the tumor , as an alternative to systemic administration of recombinant TRAIL protein.
Localized therapy of solid tumors has been successful in a number of settings. The treatment of prostate cancer with local (intraprostatic) regimens is common practice [43–45]. As demonstrated in the studies performed to date, transfer of the TRAIL gene by Ad5-TRAIL into human prostate tumor cells in vitro and in vivo led to the rapid transcription of the transferred TRAIL gene and production of functional full-length TRAIL protein that induced apoptotic death in TRAIL-sensitive tumor cell targets but not normal cells [18, 30]. Despite the initial success with Ad5-TRAIL, there are a number of limitations that can significantly decrease the efficacy of using this type of adenoviral-based, gene transfer therapy regimen for any cancer treatment. Perhaps the most important necessity for adenoviral gene therapy is CAR expression at the tumor site. CAR is the cellular receptor for most adenoviruses, specifically binding to the fiber knob [8, 26], and secondary interactions are mediated by αv integrins on the cell surface with the adenovirus penton base for subsequent endocytosis of the viral particles [46, 47]. Loss of CAR expression correlates with increased growth of tumor cells and their invasiveness [48-50], suggesting its expression may also serve as a tumor suppressor in certain malignancies. Recent reports have determined CAR expression is epigenetically regulated, where the activation of the CAR promoter in urogenital tumor cells is largely modulated by histone acetylation . Determining that CAR expression is modulated by the level of histone acetylation provides an explanation for the reports demonstrating that HDACi enhance adenoviral infection and transgene expression in a variety of tumor cell lines .
HDACi are a novel class of chemotherapeutic agents, initially identified by their ability to reverse the malignant phenotype of transformed cells. HDAC inhibition leads to the accumulation of marked amounts of acetylated histone species that result in profound effects on tumor cells, such as inhibiting cell cycle, or inducing differentiation or apoptosis [52–54]. We were interested in the potential use of HDACi to enhance the tumoricidal activity of Ad5-TRAIL in prostate tumor cells by either increasing vector infection or altering the level of TRAIL sensitivity of the tumor cells, or both. The combination of HDACi and Ad5-TRAIL proved to significantly increase the amount of prostate tumor cell death compared to either agent alone. Our results clearly indicate that either sodium butyrate or trichostatin A treatment of prostate tumor cells not only increases adenoviral infectivity (Fig. 1), but also increases the susceptibility of the cells to TRAIL-mediated apoptosis (Fig. 2).
While the proximal signaling events have been extensively studied for Fas and TNFR1, initial evaluation of the TRAIL receptor signaling pathway proved difficult, yielding contradictory results from different laboratories. It was not until several years after their cloning that the molecular signal transduction events leading to apoptosis induced via TRAIL/TRAIL receptor interaction were elucidated. Death receptor, be it TRAIL-R1/R2, Fas, or TNF-R1, crosslinking leads to the formation of a multiprotein structure called the death-inducing signaling complex (DISC ) that includes the death receptor, the death adapter protein Fas-associated death domain protein (FADD ), and the proteolytic cysteine protease procaspase-8. All death receptors share a homophilic protein/protein intracellular domain, called the DD [57, 58], which is required to attract specialized apoptosis signaling molecules that often contain a DD themselves. FADD interacts directly with the DD of TRAIL-R1, TRAIL-R2, Fas, and indirectly with the DD of TNF-R1 through TRADD (TNF Receptor-associated death domain protein). In a homotypic interaction, the DD of FADD binds to the DD of TRAIL-R1 or —-R2. The death effector domain (DED) of FADD, in turn, interacts with the DED of procaspase-8 . Procaspase-8 is proteolytically cleaved and activated at the DISC, which then initiates the apoptosis executing caspase cascade. The downstream executioner caspases, caspase-3, -6, and -7, are then activated to cleave the numerous structural and regulatory proteins that maintain cellular integrity.
Within the context of the apoptotic signal transduction pathway outlined, the location of caspase-2 in this pathway has been historically lacking. It was not until recently that it was determined caspase-2 functions to partially cleave procaspase-8, facilitating the recruitment of caspase-8 to the TRAIL receptor DISC upon TRAIL ligation . Caspase-2 can only function in this capacity when intracellular PKCK2 activity is low, or when high PKCK2 activity is downregulated by a specific inhibitor. We observed that untreated DU-145 cells have high PKCK2 activity and low levels of active caspase-2. In contrast, sodium butyrate- or trichostatin A-treated DU-145 cells had low PKCK2 activity and detectable levels of caspase-2 (Fig. 5). Moreover, HDACi-treatment also led to the appearance of the 43/41 kDa fragment of procaspase-8. Addition of a caspase-2-specific inhibitor protected HDACi-treated DU-145 cells from Ad5-TRAIL-induced apoptosis. The connection between HDAC inhibition and alterations in PKCK2 activity has been previously reported . In addition, Izeradjene et al.. demonstrated that inhibition of PKCK2 phosphorylation events resulted in a dramatic sensitization of tumor cells to TRAIL-induced apoptosis [36, 37]. Our results demonstrating HDACi-mediated downregulation of PKCK2 activity, increased activity of caspase-2, and the modulation of DU-145 cells from TRAIL-resistant to TRAIL-sensitive are the first, to our knowledge, to connect the above-mentioned observations together. Thus, agents that target the activation of caspase-2 may serve as potent companions to cancer therapeutics designed to initiate tumor cell apoptosis by binding to death receptors.
The development of TRAIL as a cancer therapeutic will undoubtedly encounter a number of obstacles. Identifying ways to enhance the tumoricidal activity of TRAIL is important because the number of identified TRAIL-resistant tumors will only continue to increase. Thus, identifying agents to use in combination with TRAIL is essential for the potential long-term success of TRAIL as a therapeutic molecule. Our studies with HDACi are especially important in the evolving development of the Ad5-TRAIL vector.