Potential conflict of interest: Nothing to report.
Hepatocellular carcinoma (HCC) is a particularly lethal form of cancer, yet effective therapeutic options for advanced HCC are limited. The poly(ADP-ribose) polymerases (PARPs) and histone deacetylases (HDACs) are emerging to be among the most promising targets in cancer therapy, and sensitivity to PARP inhibition depends on homologous recombination (HR) deficiency and inhibition of HDAC activity blocks the HR pathway. Here, we tested the hypothesis that cotargeting both enzymatic activities could synergistically inhibit HCC growth and defined the molecular determinants of sensitivity to both enzyme inhibitors. We discovered that HCC cells have differential sensitivity to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and PARP inhibitor olaparib, and identified one pair of cell lines, termed SNU-398 and SNU-449, with sensitive versus resistant phenotype to both enzyme inhibitors, respectively. Coadministration of SAHA and olaparib synergistically inhibited the growth of SNU-398 but not SNU-449 cells, which was associated with increased apoptosis and accumulated unrepaired DNA damage. Multiple lines of evidence demonstrate that the hepatic fibrosis/hepatic stellate cell activation may be an important genetic determinant of cellular sensitivity to both enzymatic inhibitors, and coordinate activation or inactivation of the aryl hydrocarbon receptor (AhR) and cyclic adenosine monophosphate (cAMP)-mediated signaling pathways are involved in cell response to SAHA and olaparib treatment. Conclusion: These findings suggest that combination therapy with both enzyme inhibitors may be a strategy for therapy of sensitive HCC cells, and identification of these novel molecular determinants may eventually guide the optimal use of PARP and HDAC inhibitors in the clinic. (HEPATOLOGY 2012;55:1840–1851)
Poly (ADP-ribose) polymerases (PARPs) are a family of enzymes that share a catalytic PARP homology domain and the ability to poly(ADP-ribosyl)ate protein substrates.1, 2 Among them, PARP1 and PARP2 are activated by single- and double-strand breaks (SSB and DSB, respectively) and play a critical role in the base excision repair pathway by binding to DNA breaks and recruiting DNA repair proteins to the site of damage.1-3 Inhibition of PARPs induces accumulation of large numbers of unrepaired SSBs, leading to the collapse of replication forks during S phase and the consequent generation of DSBs. Normally, these replication-associated DSBs would be repaired by the error-free homologous recombination (HR) repair pathway with no deleterious effect seen.4 However, in cells in which HR is defective, such as the BRCA-mutated cells, DSBs can be repaired by a more error-prone nonhomologous end-joining (NHEJ) pathway, resulting in chromosome aberrations and cell lethality.3, 5 Indeed, accumulating evidence has proposed that PARP inhibition might be a useful therapeutic strategy for the treatment of a wider range of tumors bearing a variety of deficiencies in the HR pathway or displaying properties of “BRCAness.”3, 5, 6
DSB repair occurs within chromatin and can also be modulated by chromatin-modifying enzymes. In this context, histone deacetylases (HDACs) are critically important to enable functional HR by controlling the expression of HR-related genes and promoting the proper assembly of HR-directed subnuclear foci.7 In contrast, inhibition of HDAC enzymes down-regulates the expression of HR DNA repair proteins and impairs recruitment of these key HR proteins to the site of DNA damage, resulting in a decrease in homology-directed repair of DSBs.7-10 These findings indicate a potential therapeutic utility of HDAC inhibitors in cancer patients with tumors who have overactive HR or in combination with antitumor agents that induce damage repaired by HR.
Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide, yet effective therapeutic options for advanced HCC are limited.11, 12 Emerging evidence demonstrating development of HCC association with perturbed DNA damage response and repair pathway11 opens the possibility of targeting the components of DNA damage response pathway for the treatment of HCC. In this study, we investigated the hypothesis that cotargeting the enzymatic activities of PARPs and HDACs could synergistically inhibit the growth of HCC and defined the molecular determinant of sensitivity to both enzymatic inhibitors.
The human HCC cell lines HepG2, PLC/PRF/5, SNU-398, and SNU-449 were obtained from the American Type Culture Collection and the Huh-7 cell line was purchased from the Japanese Collection of Research Bioresources. The pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the PARP inhibitor olaparib were obtained from Merck and Selleck Chemicals, respectively. All experimental assays, including cell viability assay, flow cytometric analysis of apoptosis, pulsed field gel electrophoresis assay, colony formation assay, antibodies, and western blot analysis, microarray gene expression arrays and data analysis, quantitative real-time polymerase chain reaction (PCR), and statistical analyses are described in the Supporting Information in detail.
Differential Sensitivity of HCC Cells to SAHA and Olaparib Treatment.
To test whether cotargeting the enzymatic activities of PARPs and HDACs could inhibit the growth of HCC cells, five well-characterized human HCC cell lines were treated with different concentrations of SAHA or olaparib alone for 96 hours and their growth was subsequently assayed using the standard XTT assay. The results showed that SAHA inhibited the proliferation of HepG2, Huh7, PLC/PRF/5, and SNU-398 but not SNU-449 cells in a dose-dependent manner (Fig. 1A). In contrast, olaparib effectively inhibited the proliferation of HepG2, Huh7, and SNU-398 but not PLC/PRF/5 and SUN-449 cells in a dose-dependent manner (Fig. 1B). These results suggest that HCC cells have differential sensitivity to SAHA and olaparib, probably due to the heterogeneous genetic background (Supporting Table S1). Given that the genetic background of SNU-398 and SNU-449 cell lines are relatively comparable (Table S1) and that both cell lines exhibit striking sensitive (SNU-398) versus resistant (SNU-449) phenotype to both enzyme inhibitors (Fig. 1A,B), we chose this pair of cell lines as a model to investigate the synergistic action of SAHA and olaparib in HCC cells and to define the underlying mechanisms.
We next investigated the growth inhibitory effects of a combination of SAHA and olaparib at very low concentrations in HCC cells by XTT assays. As shown in Fig. 1C, incubation of SNU-398 cells with 0.5 μM SAHA or 3 μM olaparib alone for 96 hours did not significantly alter cell viability, whereas the simultaneous treatment with SAHA and olaparib at the same concentrations resulted in a significant reduction of cell viability. In contrast, SAHA had no detectable effect on cell viability when combined with olaparib at the same concentrations in resistant SNU-449 cells (Fig. 1C). Colony-forming assays further confirmed these results, showing a greater inhibition of clonogenicity in SNU-398 but not SNU-449 cells following SAHA and olaparib treatment (Fig. 1D; Supporting Fig. S1). Together, these findings suggest that HCC cells have differential sensitivity to SAHA and olaparib and coadministration of both inhibitors had a synergistic antiproliferative effect in the sensitive SNU-398 but not the resistant SNU-449 cells.
Synergistic Induction of Apoptosis in the Sensitive but Not Resistant Cells Following SAHA and Olaparib Treatment.
To investigate the underlying mechanisms of the synergistic antiproliferative effect of SAHA and olaparib, cells were treated with 0.5 μM SAHA, 3 μM olaparib alone or in combination for 24 hours, and then subjected to flow cytometry analysis of DNA stained with propidium iodide, which has been widely used for the evaluation of apoptosis by determination of the percentage of events that accumulated in the sub-G1 position in different experimental models. As shown in Fig. 2A and Supporting Fig. S2A, combination of SAHA with olaparib resulted in 12.4% apoptosis in SNU-398 cells as compared with 4.32% and 2.21% for individual agent alone, respectively. In contrast, in SNU-449 cells the combination of SAHA with olaparib resulted in 1.57% apoptotic cells compared with 1.08% and 1.50% for each agent alone. The effects of SAHA and olaparib treatment on apoptosis were further determined by FITC Annexin V staining, which is used to quantitatively determine the percentage of apoptotic cells within a population based on the property of cells to lose membrane asymmetry in the early phases of apoptosis. Results showed that coadministration of SAHA and olaparib significantly increased apoptosis (12.8%) in SNU-398 cells compared with treatment with individual agent alone, whereas only a minor effect was observed in SNU-449 cells (Fig. 2B, Supporting Fig. S2B). These results suggest the synergism in inducing apoptosis by SAHA and olaparib in sensitive SNU-398 but not resistant SNU-449 cells.
We next analyzed the expression of apoptosis-related proteins in both cell lines by western blot analysis. The results showed that coadministration of SAHA and olaparib resulted in a synergistic increase in the levels of the cleaved caspase-3 and cleaved PARP in SNU-398 cells as compared with SAHA or olaparib treatment alone (Fig. 2C, Supporting Fig. S3A, Table S2). In contrast, the same effect in SNU-449 cells was only modest (Fig. 2C, Supporting Fig. S3A, Table S2), indicating that SNU-398 cells are more sensitive to the combination of both agents than SNU-449 cells do. Together, these results suggest that the anti-proliferative activity of SAHA and olaparib in SNU-398 cells was, at least in part, attributable to apoptosis induction. In addition, it is noteworthy that treatment of cells with SAHA down-regulated the expression of HDAC1 and HDAC2 proteins in SNU-398 cells (Figs. 2C, Supporting Fig. S3A, Table S3).
Combination of SAHA and Olaparib Leads to an Accumulation of Unrepaired DNA Damage and to a Decrease in HR DNA Repair Proteins.
To test whether coadministration of SAHA and olaparib causes a decrease in DSB repair capacity of HCC cells, we performed a pulsed field gel electrophoresis (PFGE) to assess for DNA repair ability of HCC cells upon HDAC and PARP inhibition. The results revealed that a combination of SAHA and olaparib resulted in a significant increase in the levels of unrepaired DNA damage in SNU-398 cells (Fig. 2D,E). In contrast, no significant increase in DSBs was detected in SNU-449 cells after treatment with SAHA and olaparib. These results indicate a possible mechanism by which SAHA enhances cellular sensitivity to olaparib through abrogation of the DSB repair pathway.
We next investigated the levels of repair proteins in both cell lines by western blot analysis and found that cotreatment with SAHA and olaparib synergistically down-regulated the protein levels of checkpoint protein 1 (Chk1), Chk2, and fanconi anemia group D2 protein (FANCD2) in SNU-398 but not SNU-449 cells (Fig. 2F, Supporting Fig. S3B, Tables S2, S4). Interestingly, the expression levels of p53-binding protein 1 (53BP1), a mediator of DNA damage checkpoint, were significantly down-regulated in SNU-449 cells as compared with SNU-398 cells following treatment with both inhibitors (Figs. 2F, Supporting Fig. S3B). In contrast, other repair proteins such as ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and H2AX remain unaffected (Fig. 2F, Supporting Fig. S3B). It has been shown that silencing Chk1, Chk2, and FANCD2 proteins leads to a reduction in HR repair and an increased sensitivity to PARP inhibitor.6 In contrast, loss of 53BP1 partially restores the HR defect and decreases sensitivity of BRCA1-deleted cells to DNA-damaging agents.13 Thus, activation of diverse DNA repair pathway by SAHA- and olaparib-induced DNA damage in SNU-398 and SNU-449 cells might contribute to the distinct sensitivity to both enzyme inhibitors.
Hepatic Fibrosis/Hepatic Stellate Cell Activation May Be an Important Genetic Determinant of Sensitivity to Both Enzyme Inhibitors.
To understand the molecular basis of the differential sensitivity of SNU-398 and SNU-449 cells to SAHA and olaparib, we next performed genome-wide expression profile analysis to identify the genetic differences between both cell lines using the Affymetrix Human Exon 1.0 ST arrays. Statistical analysis of the data generated a set of 1,147 differentially expressed genes with P < 0.05 and fold change expression ≥ ± 1.5, and the top 50 differentially expressed genes are shown in Table S5. Among them, 53.90% were up-regulated, whereas 46.10% were down-regulated in SNU-449 cells as compared with SNU-398 cells (Fig. 3A). We next performed hierarchical clustering analysis of the differentially expressed genes and the normalized log2 ratio values were used to obtain the heatmap (Fig. 3B). The gene leaf nodes were optimized in the heat maps representing the differential expression of genes between both cell lines.
To investigate the functions of these differentially expressed genes, we next performed gene ontology (GO) analysis on all the sets of genes with a P-value cutoff set to 0.1 and found that 43.16% of these differentially expressed genes are related to a cell component, followed by 37.85% to molecular function, and 18.89% to a biological process (Fig. 3C). The details with GO terms for the above-mentioned comparisons are shown in Table S6. We next carried out ingenuity pathways Analysis (IPA) on all these differential expressed genes and Fischer's exact test was applied with P < 0.05. The top 10 significant functions and canonical pathways, in which these differentially expressed genes are involved, are shown in Fig. 3D,E, respectively. The most likely function and significant canonical pathway of these differentially expressed genes are cancer and hepatic fibrosis / hepatic stellate cell activation, respectively.
Hepatic fibrosis represents the consequences of a sustained wound-healing response to chronic liver disease, in which hepatic stellate cell (HSC) activation is an initial event. Following liver injury of any etiology, HSCs undergo a response known as “activation”, which is the transition of quiescent cells into proliferative, fibrogenic and contractile myofibroblasts.14 Activated HSCs are resistant to most proapoptotic stimuli including serum deprivation, doxorubicin, etoposide, and oxidative stress mediators15 as supported by an increased expression of a number of prosurvival factors.16 As shown in Table S7, we discovered that a number of progrowth and prosurvival factors were significantly up-regulated in SNU-449 cells as compared with SNU-398 cells. Most of them are growth factors and cytokines that promote cell proliferation, angiogenesis, and prevent apoptosis.
To test whether these survival factors are involved in cell sensitivity to SAHA and olaparib for HCC, we next validated the expression of several survival factors, including chemokine (C-C motif) ligand 2 (CCL2), endothelin 1 (EDN1), fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor beta polypeptide (PDGFB) in both cell lines by quantitative (q)PCR. Consistent with the microarray data, we repeatedly found that all five genes were up-regulated in resistant SNU-449 cells as compared with sensitive SNU-398 cells (Fig. 4A, Table S8). Given that EDN1 is most highly up-regulated gene in resistant SNU-449 cells and has been shown to promote tumor cell proliferation, survival, angiogenesis, invasion, and metastasis,17 we next tested the hypothesis that induced expression of EDN1 in sensitive SNU-398 cells could convert its sensitive phenotypes to a more resistant one. To this end, SNU-398 cells were transfected with pCMV6-AC/hEDN1 expression plasmid and assessed the effect of EDN1 on cellular sensitivity to both enzyme inhibitors. As shown in Fig. 4C, induced expression of EDN1 in sensitive SNU-398 cells (Fig. 4B, Table S9) significantly increases cell viability following SAHA and olaparib treatment as compared with empty vector control. In addition, we noticed that SAHA and olaparib treatment can still significantly inhibit cell growth as compared with dimethyl sulfoxide (DMSO) control in EDN1-overexpressed cells (Fig. 4C, right panel), raising the possibility that other prosurvival factors may also contribute to HCC sensitivity to both inhibitors. Collectively, these results suggest the hepatic fibrosis/hepatic stellate cell activation may be an important genetic determinant of sensitivity to both enzymatic inhibitors due to up-regulation of numerous survival factors.
Activation of the Aryl Hydrocarbon Receptor (AhR) Pathway Is an Important Feature of the Antiproliferative Effects of Both Enzyme Inhibitors in Sensitive HCC Cells.
To further identify markers that may predict a response to SAHA and olaparib treatment, we next performed expression profiling analysis in both cell lines following treatment with 0.5 μM SAHA, 3 μM olaparib alone or in combination for 24 hours, and the larger number of transcript expression changes was observed in SNU-398 cells than SNU-449 cells. The normalized log2 ratio values of the differentially regulated genes in each comparison were used to obtain the heat maps (Supporting Fig. S4). In this context, 68 and 21 genes were found to be induced by SAHA treatment in SNU-398 and SNU-449 cells, respectively (Fig. 5A, Tables S10, S11). When cells were treated with olaparib and compared with DMSO control, a total of 28 and 5 genes were specifically induced in SNU-398 and SNU-449 cells, respectively (Fig. 5B, Tables S12, S13). Interestingly, a total of 165 and 45 genes were specifically induced in SNU-398 and SNU-449 cells, respectively, following cotreatment with SAHA and olaparib (Fig. 5C, Tables S14, S15).
To further investigate the functions of these regulated genes by SAHA and olaparib treatment, we performed GO analysis on all the sets of genes with a P-value cutoff set to 0.1. As shown in Fig. 5D, 51.71% of these induced genes in SNU-398 cells related to molecular function, followed by 32.68% to the biological process and 15.61% to the cellular component. In contrast, 42.11% of differentially expressed genes induced by SAHA and olaparib in SNU-449 cells were linked to the cellular component, 31.58% to the biological process, and 26.32% to molecular function. The details with GO terms for all the above-mentioned comparisons are shown in Tables S16 and S17, respectively. We next performed Ingenuity pathway analysis on all the genes that were influenced by SAHA and olaparib treatment. Excitedly, we found that the most significant canonical pathway, in which the induced genes might be involved, is the aryl hydrocarbon receptor (AhR) signaling in SNU-398 cells (Fig. 6A, Table S18), and that the most likely functions of the genes regulated by SAHA and olaparib in SNU-398 cells are cancer, cellular development, cell death, cell growth, and proliferation (Fig. 6B).
The AhR is a ligand-activated basic helix-loop-helix transcription factor that mediates gene activation events induced by environmental contaminants. Previous studies have suggested that AhR signaling is involved in normal liver growth and development18 and modulates the susceptibility of hepatocytes to the proapoptotic effects of tumor necrosis factor-alpha and Fas stimulation.19 To further test whether AhR signaling is involved in the growth inhibitory effect of SAHA and olaparib in sensitive SNU-398 cells, we next carried out qPCR analysis to validate the altered expression of several representative genes that are involved in AhR signaling, including the aldehyde dehydrogenase 3 family, member B1 (ALDH3B1), cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), CYP1B1, and p21 (Table S18). In agreement with the microarray data, we found that ALDH3B1 was down-regulated, whereas CYP1A1, CYP1B1, and p21 were up-regulated in SNU-398 cells following SAHA and olaparib treatment (Fig. 6C, Table S19).
Aldehyde dehydrogenases (ALDHs) are critical enzymes in the metabolism of endogenous and exogenous aldehydes,20 which are up-regulated in a variety of human cancer tissues and cell lines resulting in resistance to anticancer drugs.21 ALDH3B1 belongs to the ALDH3 family20 and is highly expressed in liver and has an important role in the defense against oxidative stress.20 Given that ALDH3B1 was down-regulated in sensitive SNU-398 cells following SAHA and olaparib treatment (Fig. 6C, Table S19), we hypothesized that restoration of ALDH3B1 in sensitive SNU-398 cells could reverse its sensitive phenotype to both inhibitors. Interestingly, we found that expression of ALDH3B1 (Fig. 6D, Table S9) significantly increases cell viability following inhibition of both SAHA and PARP (Fig. 6E), probably by protecting cells from the damaging effects of oxidative stress.20 Thus, we concluded that activation of the AhR signaling pathway in sensitive SNU-398 cells may be an important feature of the antiproliferative effects of both enzyme inhibitors.
Dysregulation of the cAMP-Mediated Signaling May Contribute to Resistance to Both Enzyme Inhibitors.
In contrast, the genes induced by SAHA and olaparib in SNU-449 cells are primarily involved in the cAMP-mediated signaling (Fig. 7A). cAMP is a signaling molecule important for a variety of cellular functions and exerts its effects by activating the cAMP-dependent protein kinase, which transduces the signal through phosphorylation of different target proteins. These representative genes involved in the cAMP-mediated signaling are shown in Table S20. Ingenuity pathway analysis revealed that the functions of these genes are involved in genetic disorder, hematological disease, and carbohydrate metabolism (Fig. 7B). qPCR analysis further demonstrated that cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and regulator of G-protein signaling 4 (RGS4) were up-regulated, whereas calcium/calmodulin-dependent protein kinase II beta (CAMK2B) was down-regulated in SNU-449 cells after SAHA and olaparib treatment (Fig. 7C, Table S21). Interestingly, a recent study using a synthetic lethal small-interfering RNA (siRNAs) screen of cancer cell lines found that silencing of PRKAR2B by siRNAs resulted in a 62% decrease in cell growth following treatment with PARP inhibitor as compared with control siRNA-transfected cells,22 indicating that PARKR2B has a potential role in cell resistance to PARP inhibitor. In light of our results that cotreatment of SAHA and olaparib resulted in an over 20-fold increase in the levels of PRKAR2B in resistant SNU-449 cells (Fig. 7C, Table S21) and a previous report from other laboratory,22 we next tested the possibility that overexpression of PRKAR2B in sensitive SNU-398 cells could decrease sensitivity to both enzyme inhibitors. As expected, we found that exogenous expression of PRKAR2B (Fig. 7D, Table S9) in SNU-398 cells significantly increases cell viability in the presence of SAHA and olaparib (Fig. 7E). Thus, these results suggest that up-regulation of cAMP-mediated signaling, especially PRAKR2B, may be an important marker of cell resistance to SAHA and olaparib for HCC. Interestingly, PRKAR2B has been documented to be down-regulated in human cancer cells and its overexpression induces growth inhibition.23 One possibility is that, like other tumor suppressor genes Chk1, Chk2, and BRCA1, PRKAR2B has a potential role in DSB repair and can enable HCC cells to survive DNA damage that is induced by SAHA and olaparib.
PARP expression is significantly increased in human HCC compared with adjacent nontumor tissues24, 25 and inhibition of PARP-1 decreases HCC growth.26 Interestingly, HDAC inhibitors have been shown to exert its antitumor activity against HCC in preclinical models.12 The unanswered questions in this field are whether cotargeting both enzymatic activities could synergistically inhibit HCC growth and what is the genetic determinant of cellular sensitivity to both enzyme inhibitor therapies. In this study, we report that cotargeting the enzyme activities of PARPs and HDACs synergistically inhibited the growth of sensitive HCC cells. The possible mechanism could be that inhibition of PARP induces the generation of DSBs, which is normally repaired by the error-free HR repair pathway (Fig. 8A). When coadministered with HDAC inhibitor that blocks the HR-mediated repair pathway,7-10 cells exhibit a mimic HR-deficient phenotype, resulting in PARP hyperactivation and PARP inhibitor sensitivity27 (Fig. 8B). Our findings also highlight the notion that a deficiency in HR is a determinant of sensitivity to PARP inhibition and not BRCA1/2 deficiency per se, because both SNU-398 and SNU-449 cell lines express wild-type BRCA1.28 In support of our findings, it has been shown that PARP inhibitors can inhibit the growth of breast cancer cells irrespective of their BRCA1 status,29 and olaparib monotherapy resulted in responses in patients with high-grade serous ovarian carcinoma without germline BRCA1 or BRCA2 mutation.30
Another novel finding is that the differentially expressed genes between the sensitive and resistant cell lines are primarily involved in hepatic fibrosis/hepatic stellate cell activation (Fig. 3E), which is characterized by up-regulation of numerous progrowth and prosurvival factors. One key question is whether hepatic fibrosis / hepatic stellate cell activation signaling is connected to the DNA repair pathway, which could explain the differential sensitivity of both cell lines to SAHA and olaparib. Indeed, most of these genes, such as Met proto-oncogene and epidermal growth factor receptor, can promote DNA repair by up-regulating proteins involved in DNA repair,31, 32 which may be one of the mechanisms by which SNU449 cells are resistant to SAHA and olaparib treatment. In support of this notion, we demonstrate that induced expression of EDN1 converts the sensitive phenotype of SNU-398 cells to a more resistant one (Fig. 4C), suggesting a potential role of EDN1 in cell resistance to both inhibitors. EDN1 has been shown to activate various kinases that are known to be involved in the survival of cell signaling and proliferation and to inhibit apoptosis of cancer cells by various mechanisms.17 Moreover, a recent study demonstrated that EDN1 reduces DNA damage and/or enhances DNA repair in human melanocytes following ultraviolet radiation.33 As EDN1 is up-regulated in SNU-449 cells relative to SNU-398 cells, it could be one of the genes that protect SNU-449 cells from DNA damage induced by SAHA and olaparib treatment. Thus, we conclude that the hepatic fibrosis / hepatic stellate cell activation may be a novel determinant to the sensitivity of HCC cells to SAHA and olaparib treatment.
In summary, the findings presented here establish that HCC cells have a heterogeneous response to HDAC and PARP inhibitors and the hepatic fibrosis / hepatic stellate cell activation may be a genetic determinant of cellular sensitivity to HDAC and PARP inhibitors. Combination therapy with a selective HDAC and PARP inhibitor may be a strategy for therapy of sensitive HCC cells, in which the coordinate activation or inactivation of the AhR and cAMP-mediated signaling pathway is an important feature of the antiproliferative and proapoptotic effects of both inhibitors. Thus, treatment targeting DNA repair mechanisms seems to provide new hope for treatment of HCC and identification of these novel determinants may eventually guide the optimal use of PARP and HDAC inhibitors in the clinic.
We thank all members of the Kumar Laboratory for fruitful discussions and technical help and Ms. Amanda J. Lyon for critical reading of the article.