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DNA-PKcs: A promising therapeutic target in human hepatocellular carcinoma?
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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Review
DNA-PKcs: A promising therapeutic target in human hepatocellular carcinoma? Rosa M. Pascale a,1 , Christy Joseph b,1 , Gavinella Latte a , Matthias Evert c , Francesco Feo a , Diego F. Calvisi a,b,∗ a
Department of Clinical and Experimental Medicine, University of Sassari, 07100 Sassari, Italy Institute of Pathology, Universitätsmedizin Greifswald, 17489 Greifswald, Germany c Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany b
a r t i c l e
i n f o
Article history: Received 25 August 2016 Accepted 11 October 2016 Available online xxx Keywords: Hepatocellular carcinoma DNA repair Signaling pathways Targeted therapies
a b s t r a c t Hepatocellular carcinoma (HCC) is a frequent and deadly disease worldwide. The absence of effective therapies when the tumor is surgically unresectable leads to an extremely poor outcome of HCC patients. Thus, it is mandatory to elucidate the molecular pathogenesis of HCC in order to develop novel therapeutic strategies against this pernicious tumor. Mounting evidence indicates that suppression of the DNA damage response machinery might be deleterious for the survival and growth of the tumor cells. In particular, DNA dependent protein kinase catalytic subunit (DNA-PKcs), a major player in the non-homologous end-joining (NHEJ) repair process, seems to represent a valuable target for innovative anti-neoplastic therapies in cancer. DNA-PKcs levels are strongly upregulated and associated with a poor clinical outcome in various tumor types, including HCC. Importantly, DNA-PKcs not only protects tumor cells from harmful DNA insults coming either from the microenvironment or chemotherapeutic drug treatments, but also possesses additional properties, independent from its DNA repair activity, that provide growth advantages to cancer cells. These properties (metabolic and gene reprogramming, invasiveness and metastasis, resistance to apoptosis, etc.) have started to be elucidated. In the present review, we summarize the physiologic and oncogenic roles of DNA-PKcs, with a special emphasis on liver cancer. In particular, this work focuses on the molecular mechanism whereby DNA-PKcs exerts its pro-tumorigenic activity in cancer cells. In addition, the upstream regulator of DNA-PKcs activation as well as its downstream effectors thus far identified are illustrated. Furthermore, the potential therapeutic strategies aimed at inhibiting DNA-PKcs activity in HCC are discussed. © 2016 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The repair mechanims of double-strand breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA-PKCS: a key component of the NHEJ machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The final phase of NHEJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA repair-independent functions of DNA-PKCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mechanistic and prognostic role of DNA-PKCS in human hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Molecular mechanisms responsible for DNA-PKCS activation in hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA-PKCS inhibitors for the treatment of liver cancer: preliminary evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Abbreviations: CHIP, chromatin immunoprecipitation; DNA-PKcs, DNA dependent protein kinase catalytic subunit; DSBs, double strand breaks; GI, genomic instability; HCC, hepatocellular carcinoma; NHEJ, non-homologous end-joining. ∗ Corresponding author at: Department of Clinical and Experimental Medicine, University o Sassari, via P. Manzella 4, 07100 Sassari, Italy. E-mail address: [email protected] (D.F. Calvisi). 1 Pascale RM and Joseph C equally contributed to this work. http://dx.doi.org/10.1016/j.dnarep.2016.10.004 1568-7864/© 2016 Elsevier B.V. All rights reserved.
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Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction
2. The repair mechanims of double-strand breaks
Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer, with 0.25-1 million new cases per year and a life expectancy of about 6 months from the time of the diagnosis [1,2]. HCC incidence changes with age, sex, ethnic group, and geographic region and is rapidly rising in Western countries [3,4]. Major risk factors associated with HCC development include chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, alcoholic and nonalcoholic steatohepatitis, food contamination by Aflatoxin B1, and some inherited metabolic disorders [1,5]. In addition, HCC incidence shows differences within the human population in response to risk factors, thus suggesting the implication of a polygenic control in HCC incidence [6–8]. The interaction of genetic and environmental risk factors creates a wide genotypic and phenotypic heterogeneity within human HCC [7–9]. Partial liver resection, radiofrequency ablation, and liver transplantation potentially curative options for HCC [10]. However, only a small subset of HCC cases is amenable to these treatments due the late diagnosis of the disease [1,10,11]. Therapies with pharmacological agents, trans-arterial chemo-embolization or administration of yttrium-90 microspheres, and percutaneous ethanol injection do not improve substantially the prognosis of patients with advanced disease [1,10,11]. Thus, the study of the mechanisms involved in HCC progression could provide opportunities for more efficacious targeted therapies. Different etiologic stimuli may induce hepatocarcinogenesis, which is preceded in most cases by the development of hepatitis and cirrhosis in humans and/or in animals (Fig. 1). Hepatocarcinogenesis is a multiphasic process: the interaction of DNA with carcinogens, oxidative damage and reactive radicals, produced during carcinogen metabolism and/or inflammation, and compensatory hepatocyte regeneration in early phases of the process, induce lesions in the DNA, including the highly toxic double strand breaks (DSBs). This extensive DNA damage leads to chromosomal aberrations, resulting in loss of heterozygosity and genomic instability (GI) [12]. This process is followed by the development of foci of altered hepatocytes (Fig. 1), which partly evolve to low-grade and high-grade dysplastic nodules, and then to HCC with increasingly lower levels of differentiation (Fig. 1). During these events, GI progressively increases in liver lesional cells, with consequent chromosome aberrations as well as oncogene and oncosuppressor mutations and progressive deregulation of multiple signaling pathways, reaching their maximum in poorly differentiated, highly metastatic HCC [13–16]. Recent studies focused on strategies to directly interfere with DNA repair mechanisms in cancer, such as non-homologous end joining (NHEJ), a DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and, according to recent findings, a potential chemo-sensitization target in numerous cancer types including ovarian cancer [17], breast cancer [18], non-small cell lung cancer [19], colon cancer [20], glioblastoma [21]. Recent evidence implicating a critical role of DNA-PKcs in HCC [22–24] suggest that this protein could be a potential candidate target for new personalized therapies against liver cancer. The present review is principally devoted to an interpretive analysis of recent finding on DNA-PKcs in HCC as a potential target of new therapeutic approaches.
Living organisms have developed refined systems of genome stability maintenance to prevent cancer [25], by recognition of broken DNA sites and repair of the DNA lesions [26]. In mammalian cells, DSBs may be repaired by two main processes: homologous recombination and non-homologous end-joining (NHEJ). In the homologous recombination process, the repair of the damaged DNA strand is achieved by using a homologous stretch of DNA from the sister chromatid. NHEJ consists instead in the re-ligation of the broken DNA molecule [26–28]. NHEJ is a multiphasic process (Fig. 2), which includes: (a) the recognition of DSB by the Ku70/80 ring-shaped protein heterodimer; (b) the recruitment, assembly, and stabilization of the NHEJ complex at the site of damage; (c) the binding to the ends of the broken chains and their stabilization; (d) the activation of the catalytic subunit od DNA-PKcs and the ligation of broken ends [28]. NHEJ is initiated by the recognition and binding of the Ku70/80 dimer to the sugar backbone at the site of DNA damage. Bound Ku70/80 recruits the NHEJ apparatus to the DNA lesion by directly linking the canonical NHEJ proteins: DNA-PKcs (DNA dependent protein kinase catalytic subunit), XRCCA (X-ray cross complementing protein 4), XLF (XRCC4-like factor), and DNA Ligase IV. This protein complex ties the DNA ends and activates DNA-PKcs. The latter process is then followed by the ligation of the broken ends, which is mediated by DNA Ligase IV, and the disintegration of the NHEJ complex [26–30]. The active NHEJ complex may require other components, such as DCLRE1C (DNA cross-link repair protein 1C or Artemis), DNA polymerase and , TDT (terminal dinucletidyltrasferase), PNKP (polynucleotide kinase-phosphatase), APTX (aprataxin), and APLF (aprataxin-PNKP-like factor) [31]. 3. DNA-PKCS: a key component of the NHEJ machinery DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3) kinase-like kinase family (PIKK), which includes other two proteins responsive to DNA damage: ATM (ataxia-telengectasia mutated) and ATR (Rad3-related protein) [32,33]. The N-terminal region of DNA-PKcs is composed of HEAT (Huntington-elongation-factor 3, regulatory subunit A of PP2A, TOR1) repeats and other ␣-helical regions, constituting the 74% of DNA-PKcs. The C region of the protein contains the PI3 kinase domain, which is involved in phosphorylation of other proteins as well as auto-phosphorylation, and is flanked N-terminally by the FAT (FRAP, ATM, TRRAP) domain and C-terminally by the FATC (FAT C-terminal) domain [34–36] (Fig. 3). Structural studies have shown that the N-terminal portion of DNA-PKcs has a pincer-shaped structure forming a central channel that probably binds electrostatically to double strand-DNA (DSDNA) and the C-terminal domains, containing the kinase domain, forms an head/crown placed on top of the pincer-shaped structure [37,38] (Fig. 3). However, according to recent studies, DS-DNA seems to interact closer to the base of DNA-PKcs [39,40]. Following the recognition of the DSB ends by the Ku (Ku70/80) complex, during initiation of NHEJ, the binding of DNA-PKcs to KuDNA complex, involving the final 13 aminoacidic residues of Ku80, induces a conformational change in the FAT and FATC domains, surrounding the PIK3 kinase domain, followed by the activation of the kinase activity [41]. Further, the N-terminus region of DNAPKcs also plays a role in modulating its enzymatic activity [42]. However, the mechanisms whereby the interaction of DNA-PKcs
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interaction with PP6c is required for DNA-PKcs activation after DNA damage [48], and this interaction occurs primarily through PP6R1 [49]. 4. The final phase of NHEJ
Fig. 1. Schematic representation of liver carcinogenesis. Hepatitis/cirrhosis induced in humans and/or animals by different carcinogenic stimuli, are followed by the development of hepatocellular carcinoma. The clonal expansion of cells initiated by carcinogens leads to the development of foci of altered hepatocytes (FAH), which may evolve to dysplastic nodules (DN). This is followed by the development of well-differentiated hepatocellular carcinomas (WDH) that further progress to moderately-differentiated (MDH) and poorl-differentiated (PDH) carcinomas. During this process, progressively increase of genomic instability (GI) occurs.
with Ku and DNA leads to DNA-PKcs activation are not yet completely understood. Available evidence indicates a role of the kinase domain [43]. Surprisingly, only a few phosphorylation events are required for NHEJ in vivo, most of which consist of auto-phosphorylation, although DNA-PKcs may be also phosphorylated by different kinases, such as PIKKs, ATM, and ATR (36). Proteomics studies have also revealed that DNA-PKcs is modified by acetylation [44] and ubiquitination in vivo [45], but it is not known how these posttranslational modifications affect its function. Interestingly, various proteins have been shown to interact with DNA-PKcs. Among these proteins is phosphatase 6 (PP6), composed of catalytic (PP6c) and regulatory subunits (PP6R1, PP6R2 and PP6R3) [46,47]. In particular, it has been reported that DNA-PKcs
The mechanism whereby Ku complex is removed from the DNA ends during the repair process remains poorly defined. Ku heterodimer ubiquitination has been proposed as a mechanism to remove the Ku complex from DNA ends [50]. To complete NHEJ, DNA synthesis fills-in the gaps, and end joining is carried out by DNA Ligase IV, which is stabilized and activated by XRCC4 that promotes its adenylation [51]. DNA Ligase IV capacity to ligate non-cohesive ends of DNA is enhanced by XLF [52,53]. An important role is also played by APLF, which stimulates the ligation by XRCC4-DNA Ligase IV, facilitates the assembly of the NHEJ complex, and promotes the retention of XRCC4 in association with PARP3 and APLF [54,55]. In conclusion, the basic steps of NHEJ include: (1) DSB detection by the Ku heterodimer with subsequent formation of the DNAPKcs-Ku-DNA complex, (2) lesion processing, (3) DNA end ligation by DNA Ligase IV in complex with XRCC4 and XLF. The results of structural studies suggest that the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular helixes suitable to store/release conformational energy to regulate NHEJ complexes, and DNA end protection, processing, and ligation [56]. 5. DNA repair-independent functions of DNA-PKCS Recent experimental findings convincingly indicate that DNAPKcs possesses additional functions that are not dependent on its DNA repair activity. In particular, a mounting body of data points to a crucial role of DNA-PKcs during mitosis. This hypothesis was generated following the observation that siRNA-induced depletion of DNA-PKcs or suppression of DNA-PKcs kinase activity by the chemical inhibitor NU7441, results in mitotic defects [57]. The molecular mechanisms responsible for the activation of DNA-PKcs in mitosis are not completely understood. Present evidence suggests that they differ from the Ku-dependent activation of DNA-PKcs after
Fig. 2. Schematic representation of non-homologous end-joining (NHEJ) and protein concerned with the different steps. Abbreviations: APLF, aprataxin-PNKP-like factor; XLF, XRCC4-like factor; XRCCA, X-ray cross complementing protein 4. Other details are in the text.
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Fig. 3. Molecular structure of DNA-PKcs. Details are in the text.
DNA damage, and that PLK1 (polo-like kinase 1) and PP6 regulate DNA-PKcs phosphorylation during mitosis [58]. Other important functions of DNA-PKcs are the protection of telomeres [59–61] and the regulation of inflammatory and immune responses [62,63]. DNA-PKcs promotes the phosphorylation of RNA polymerase II C-terminal domain, thus controlling transcriptional elongation [64,65]. It is also involved in directing transcriptional factors like AIRE (Autoimmune Regulator) to the promoter regions of Tolllike receptor (TLR) genes [66]. In hormone-dependent cancers, a circuit of reciprocal regulation between the Androgen Receptor (AR) and Estrogen Receptor (ER) with DNA-PKcs has been identified [67–69]. Furthermore, several studies indicate that DNA-PKcs is a major, positive regulator of lipogenic genes. Indeed, it has been found that DNA-PKcs catalyzes the phosphorylation of the upstream transcription factor 1 (USF-1) gene, which is responsible for coordinating changes in lipogenesis and gluconeogenesis during the fasting–feeding cycle [70,71] The functions of DNA-PKcs independent of its DNA repair activity are schematically illustrated in Fig. 4. Together with aberrant fatty acid biosynthesis, considered a hallmark of malignancy [71], DNA-PKcs is involved also in the upregulation of a specific set of genes, including PREX1, ROCK2, Integrin b4, and VAV3, that are primarily responsible for other cancer traits, namely invasion and metastasis [72,73]. The importance of DNA-PKcs in mediating the metastatic properties of cancer cells was underscored by in vivo experiments on mouse models as well as by the finding that levels of DNA-PKcs predict clinical disease recurrence and metastasis development in prostate cancer [72,73]. Besides its activity in the nucleus, DNA-PKcs is also involved in the regulation of cytoplasmic organelles, such as the Golgi apparatus [74]. Specifically, following DNA damage, DNA-PKcs phosphorylates GOLPH3 (Golgi phosphoprotein 3) and increases its binding to Myosin XVIIIA (MYO18A), which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival [74].
6. Mechanistic and prognostic role of DNA-PKCS in human hepatocellular carcinoma Cumulating evidence points to an important role played by DNA-PKcs along HCC development and progression. Among different histotypes of liver tumors, hepatocellular carcinoma was found to be the one with the highest expression of DNA-PKcs, followed by cholangiocarcinoma and biliary cystadenocarcinoma [22]. In addition, it has been described that the expression and activity of DNA-PKcs are concurrently increased with hypoxia-inducible factor 1-alpha (HIF-1alpha) levels under hypoxic conditions in the HepG2 human hepatoma cell line [76]. In these conditions, DNAPKcs can both directly interact with and phosphorylate HIF-1alpha, thus suggesting that DNA-PKcs promotes HIF-1alpha stabilization [76]. These important findings envisage the possibility that the DNA-PKcs/HIF-1alpha complex could significantly contribute to therapy resistance in hypoxic tumor cells. Using the same hepatoma cell line, An et al. found that DNA-PKcs contributes to the stabilization of c-Myc oncoprotein via the Akt/GSK-3beta signaling cascade [77]. In HepG2 cells, similar to that described in the HeLa cell line, DNA-PKcs inhibited the proteolysis of c-Myc by suppressing its proteasomal degradation [77]. The importance of DNA-PKcs as a main modulator of c-Myc oncoprotein was further confirmed by the same authors in the LO2 normal liver cell line. In these cells, indeed, overexpression of DNA-PKcs led to increased protein levels of c-Myc as well as upregulation of AKT [77]. Taken together, these data indicate that DNA-PKcs induction is associated with pro-oncogenic functions in the liver, via its ability to positively regulate important oncogenes, such as AKT and c-Myc, and to stimulate cell survival under hypoxic conditions via its interaction with HIF-1alpha. Following these pioneering and promising studies, our group has further investigated the importance of DNA-PKcs in human HCC. In a recent study using a large collection of human liver tumor specimens, we showed that DNA-PKcs levels and activity are
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Fig. 4. Functions of DNA-PKcs independent of DNA-repair activity. (A) Induction by DNA damage of DNA-PKcs interaction with polo-like kinase 1 (PLK1), followed by activation of phosphorylation of checkpoint kinase 2 (Chk2) in mitosis and phosphorylation of BRCA1, which facilitates DNA repair and regulates mitotic spindle formation. (B) Maintenance of telomere endcapping by telomerase action and homologous recombination favored by DNA-PKcs. (C) Role of DNA-PKcs in activation of immune system and inflammatory response upon recognition of foreign DNA. (D) Control of the transcriptional initiation and elongation through phosphorylation of RNA polymerase II by DNA-PKcs. (E) Circuit of activation of androgen receptors (ARs). ARs activated by dihydrotestosterone (DHT) promote DNA-PKcs expression, which coactivates ARs. (F) The succession fasting/feeding stimulated phosphorylation of USF-1 (upstream transcription factor 1) by DNA-PKcs, leading to fatty acid synthetase (FAS) up-regulation and lipogenesis.
progressively induced from non-neoplastic surrounding liver tissues to HCC. In particular, the highest levels of DNA-PKcs were detected in the biologically most aggressive liver tumors [23]. Subsequently, when investigating the possible relationship(s) between DNA-PKcs and the clinic-pathological features of the patients, we found that DNA-PKcs activity directly correlates with genomic instability, proliferation and angiogenic (microvessel density) indices, and inversely with apoptosis and patients’ survival length [23]. These findings suggest a role of DNA-PKcs in the acquisition of malignant properties of liver cancer cells. In accordance with our data, the prognostic role of DNA-PKcs in human HCC has been subsequently confirmed and substantiated in an independent
investigation by Cornell et al. [24]. In the latter study, DNA-PKcs activity was identified as a marker of poor histological tumor grade and an independent indicator of short survival on HCC patients. In addition, DNA-PKcs levels were found to be positively and strongly associated with resistance to palliative doxorubicin treatment, as detected by the time to radiological progression of the tumor [24].
7. Molecular mechanisms responsible for DNA-PKCS activation in hepatocellular carcinoma The upstream effectors responsible for activation of DNA-PKcs in HCC have recently started to be elucidated. At the molecular level,
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Fig. 5. Mechanisms of DNA-PKcs upregulation. Transcriptional activation of DNA-PKcs gene by (A) amplification and (B) HSF1 stimulation of RAS/ERK signaling leading to AP-1 activation and DNA-PCcs overexpression. Post-transcriptional regulation of DNA-PKcs by (C) stabilization (hyper-folding) of DNA-PKcs by Reptin/RUVBL2 protein, and (D) physical interaction of TNKS1BP1 protein with DNA-PKcs and PARP1, followed by DNA-PKcs auto-phosphorylation and activation.
we found that upregulation of DNA-PKcs mRNA and protein levels is induced by the heat shock factor 1 (HSF1) transcription factor in HCC cell lines [23]. In these cell lines, however, a direct upregulation of DNA-PKcs by HSF1 could not be demonstrated. Indeed, although a putative binding site for HSF1 was identified by transcription binding site prediction tools, the subsequent Chromatin ImmunoPrecipitation (ChIP) experiment conducted in human hepatoblast WRL-68 cells as well as in HuH6 and HLE HCC cell lines did not confirm the interaction between HSF1 and DNA-PKcs [23]. On the other hand, several putative binding sites for AP-1, a pivotal effector of HSF1 downstream of MAPK and JNK pathways, were detected in the DNA-PKcs promoter. Importantly, when HSF1 forced overexpression in the SNU-423 HCC cell line was coupled to treatment with specific MAPK and JNK soluble inhibitors, HSF1-induced upregulation of DNA-PKcs mRNA and protein was abolished. In addition, ChIP analysis showed a functional interaction between c-Jun and c-Fos (members of the AP-1 complex) and the DNA-PKcs promoter [23]. Altogether, these data strongly suggest that HSF1 induces DNA-PKcs in HCC cell lines indirectly, via activation of members of the AP-1 complex [78]. Additional mechanisms responsible for DNA-PKcs upregulation in HCC have been subsequently identified. In particular, DNA-PKcs induction in this tumor type might be the consequence of genomic aberrations, as amplification of the DNA-PKcs gene locus (8q11.21) was detected in 55% of a large collection of HCC specimens, where it correlated significantly with DNA-PKcs gene expression [24]. Furthermore, recent evidence indicates that DNA-PKcs is also regulated at the post-transcriptional level in HCC. Indeed, it has been described that Reptin/RUVBL2, a member of the ATPase protein
family and a presumed liver oncogene, concurs to stabilize the levels of the DNA-PKcs protein in HCC cells via direct interaction [77]. Importantly, in the same study, the authors showed that Reptin/RUVBL2 promotes the stabilization of ATM kinase as well, thus implying a crucial role of Reptin/RUVBL2 in DSB repair in HCC cells. Finally, in another investigation, tankyrase 1 binding protein 1 (TNKS1BP1), which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily, was found to physically interact with DNAPKcs and PARP-1 as well as to promote the association between DNA-PKcs and PARP-1 in various cell lines, including HepG2 [78]. In particular, it was shown that overexpression of TNKS1BP1 induces the auto-phosphorylation of DNA-PKcs at Ser2056 in a PARP-1 dependent manner, which contributes to an increased activity of the DNA DSB repair system [78]. Altogether, the present data indicate that multiple molecular events might contribute to unrestrained induction of DNA-PKcs in liver cancer. The main molecular mechanisms responsible for DNAPKcs activation thus far identified in HCC are summarized in Fig. 5.
8. DNA-PKCS inhibitors for the treatment of liver cancer: preliminary evidence It is well established that DNA repair pathways are crucial for normal cell survival, and that defects in the execution and control of these mechanisms have been linked with the development of many cancer types [79]. However, although an effective DNA repair system possesses beneficial effects for normal cells, it is plausible to hypothesize that the same system plays a role in carcinogenesis by both protecting tumor cells from stress-induced
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death and promoting proliferation in the presence of a potentially lethal microenvironment [79]. Noticeably, only some DNA repair modules are retained by cancer cells, thus rendering the latter cells dependent on a few pathways for survival [79]. Therefore, the use of inhibitors against essential components of the retained DNA repair system might represent a promising path to eradicate specifically cancer cells. In support of the latter hypothesis, it has been demonstrated that breast cancer cells from Breast Cancer 2 Early Onset (BRCA2)-deficient tumors display a strong dependency on the DNA stress protein PARP-1, as PARP-1 inhibition impairs the DNA-damage response to the point of lethality [80]. Interestingly, mutations in the BRCA2 gene have been detected in a small subset of human HCC, thus suggesting the use of PARP inhibitors at least in specific HCC cases [81,82]. In the absence of specific mutations that render liver tumors vulnerable to DNA repair inhibition, an alternative strategy could consist in the combinatorial administration of a DNA-damaging agent together with an inhibitor of the DNA repair system. We have tested the strength of this alternative therapeutic approach in in vitro growing HCC cell lines, treating them with the DNAdamaging agent doxorubicin, either alone or in combination with the DNA-PKcs inhibitor NU7441 [23]. We found that DNA-PKcs inactivation reduced proliferation and induced apoptosis in the six HCC cell lines used. Of note, the effects on growth restraint were significantly higher in HCC cell lines when compared with WRL-68 and Chang non-transformed liver cell lines [23]. Strikingly, when suppression of DNA-PKcs was associated with treatment with doxorubicin, an impressive growth restraint, elevated apoptosis, and massive DNA damage occurred in all HCC cell lines [23]. Once again, only a slight decline in proliferation and increase in apoptosis and DNA damage was detected in Chang and WRL-68 non-transformed subjected to the same treatment scheme [23]. Furthermore, the striking growth restraint effects on HCC cell lines were independent of p53 mutation status, indicating that DNAPKcs inhibition might be ubiquitously deleterious for HCC cells [23]. Equivalent results were obtained by Cornell et al. in a recent study [24]. The authors found indeed that NU7441 significantly potentiates ionizing radiation induced cytotoxicity by 3- to 40-fold and doxorubicin cytotoxicity by 2- to 50-fold in HCC cell lines, and resulted in a strong delay of liver tumor xenograft growth in vivo. In addition, the same study showed a synergistic antiproliferative effect in HCC cells when NU7441 was administered in association with the ATM inhibitor KU55933 [24]. In another investigation, a remarkable inhibition of HCC cell growth in vitro and in vivo was achieved with the recently-developed water-soluble DNA-PKcs inhibitor KU-0060648 [83]. However, the contribution of DNA-PKcs inhibition on KU-0060648 dependent growth restraint of HCC cells requires to be better defined, as the authors found that KU-0060648 also suppresses the AKT/mTOR pathway in the same cells [83]. Altogether, these data suggest a possible addiction of HCC cells to DNA-PKcs for proliferation and survival, especially when subjected to DNA damaging insults.
9. Concluding remarks Mounting evidence underlines a critical role for DNA-PKcs in HCC progression and patient’s prognosis. Previous studies conducted in other tumor types indicate that DNA-PKcs significantly contributes to many features of cancer cells, including uncontrolled growth, resistance to DNA damage-induced apoptosis, migration, invasion, and metastasis as well as metabolism and gene reprogramming. Further studies are required to determine the specific role, or roles of DNA-PKcs in HCC. Preliminary data from our and other groups indicate that treatment strategies aimed at
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suppressing DNA-PKcs are deleterious to HCC cells subjected to a DNA damaging agent in vitro and in vivo. Although promising, these results require to be further substantiated in pre-clinical animal models showing overexpression and/or activation of DNAPKcs. In these models, in particular, the effectiveness and side effects of the aforementioned treatment strategies should be determined. Furthermore, the identification of upstream inducers and/or downstream targets of DNA-PKcs in the liver might help in the development of additional targeted therapies against this deadly disease. Conflict of interest The authors declare no conflict of interest. Acknowledgement Supported by the Italian Association Against Cancer, Grant number IG 12139. References [1] J.M. Llovet, J. Bruix, Molecular targeted therapies in hepatocellular carcinoma, Hepatology 48 (2008) 1312–1327. [2] F. Feo, R.M. Pascale, M.M. Simile, M.R. De Miglio, M.R. Muroni, D.F. Calvisi, Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies, Crit. Rev. Oncog. 11 (2000) 19–62. [3] Y. Tanaka, K. Hanada, M. Mizokami, A.E. Yeo, J.W. Shih, T. Gojobori, H.J. Alter, A comparison of the molecular clock of hepatitis C virus in the United States and Japan predicts that hepatocellular carcinoma incidence in the United States will increase over the next two decades, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 15584–15589. [4] S.D. Taylor-Robinson, G.R. Foster, S. Arora, S. Hargreaves, H.C. Thomas, Increase in primary liver cancer in the UK, 1979–94, Lancet 350 (1997) 1142–1143. [5] D.F. Calvisi, M. Frau, M.L. Tomasi, F. Feo, R.M. Pascale, Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison, Biochim. Biophys. Acta 1826 (2012) 215–237. [6] J.A. Indulski, W. Lutz, Metabolic genotype in relation to individual susceptibility to environmental carcinogens, Int. Arch. Occup. Environ. Health 73 (2000) 71–85. [7] F. Feo, M.R. De Miglio, M.M. Simile, M.R. Muroni, D.F. Calvisi, M. Frau, R.M. Pascale, Hepatocellular carcinoma as a complex polygenic disease Interpretive analysis of recent developments on genetic predisposition, Biochim. Biophys. Acta 1765 (2006) 126–147. [8] T.A. Dragani, Risk of HCC: genetic heterogeneity and complex genetics, J. Hepatol. 52 (2010) 252–257. [9] F. Donato, U. Gelatti, R.M. Limina, G. Fattovich, Southern Europe as an example of interaction between various environmental factors: a systematic review of the epidemiologic evidence, Oncogene 25 (2006) 3756–3770. [10] M. Sherman, Modern approach to hepatocellular carcinoma, Curr. Gastroenterol. Rep. 1 (2011) 49–55. [11] D.F. Calvisi, R.M. Pascale, F. Feo, Dissection of signal transduction pathways as a tool for the development of targeted therapies of hepatocellular carcinoma, Rev. Recent Clin. Trials 2 (2007) 217–236. [12] A. Schipler, G. Iliakis, DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice, Nucleic Acids Res. 41 (2013) 7589–7605. [13] M. Frau, F. Biasi, F. Feo, R.M. Pascale, Prognostic markers and putative therapeutic targets for hepatocellular carcinoma, Mol. Aspects Med. 31 (2010) 179–193. [14] A. Villanueva, P. Newell, D.Y. Chiang, S.L. Friedman, J.M. Llovet, Genomics and signaling pathways in hepatocellular carcinoma, Semin. Liver Dis. 27 (2007) 55–76. [15] M.A. Kern, K. Breuhahn, P. Schirmacher, Molecular pathogenesis of human hepatocellular carcinoma, Adv. Cancer Res. 86 (2002) 67–112. [16] S. Imbeaud, Y. Ladeiro, J. Zucman-Rossi, Identification of novel oncogenes and tumour suppressors in hepatocellular carcinoma, Semin. Liver Dis. 30 (2010) 75–86. [17] D.A. Dungl, E.N. Maginn, E.A. Stronach, Preventing damage limitation: targeting DNA-PKcs and DNA double-Strand Break repair pathways for ovarian cancer therapy, Front. Oncol. 5 (2015) 240. [18] N. Albarakati, T.M. Abdel-Fatah, R. Doherty, R. Russell, D. Agarwal, P. Moseley, C. Perry, A. Arora, N. Alsubhi, C. Seedhouse, E.A. Rakha, A. Green, G. Ball, S. Chan, C. Caldas, I.O. Ellis, S. Madhusudan, Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy, Mol. Oncol. 9 (2015) 204–217.
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Review
DNA-PKcs: A promising therapeutic target in human hepatocellular carcinoma? Rosa M. Pascale a,1 , Christy Joseph b,1 , Gavinella Latte a , Matthias Evert c , Francesco Feo a , Diego F. Calvisi a,b,∗ a
Department of Clinical and Experimental Medicine, University of Sassari, 07100 Sassari, Italy Institute of Pathology, Universitätsmedizin Greifswald, 17489 Greifswald, Germany c Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany b
a r t i c l e
i n f o
Article history: Received 25 August 2016 Accepted 11 October 2016 Available online xxx Keywords: Hepatocellular carcinoma DNA repair Signaling pathways Targeted therapies
a b s t r a c t Hepatocellular carcinoma (HCC) is a frequent and deadly disease worldwide. The absence of effective therapies when the tumor is surgically unresectable leads to an extremely poor outcome of HCC patients. Thus, it is mandatory to elucidate the molecular pathogenesis of HCC in order to develop novel therapeutic strategies against this pernicious tumor. Mounting evidence indicates that suppression of the DNA damage response machinery might be deleterious for the survival and growth of the tumor cells. In particular, DNA dependent protein kinase catalytic subunit (DNA-PKcs), a major player in the non-homologous end-joining (NHEJ) repair process, seems to represent a valuable target for innovative anti-neoplastic therapies in cancer. DNA-PKcs levels are strongly upregulated and associated with a poor clinical outcome in various tumor types, including HCC. Importantly, DNA-PKcs not only protects tumor cells from harmful DNA insults coming either from the microenvironment or chemotherapeutic drug treatments, but also possesses additional properties, independent from its DNA repair activity, that provide growth advantages to cancer cells. These properties (metabolic and gene reprogramming, invasiveness and metastasis, resistance to apoptosis, etc.) have started to be elucidated. In the present review, we summarize the physiologic and oncogenic roles of DNA-PKcs, with a special emphasis on liver cancer. In particular, this work focuses on the molecular mechanism whereby DNA-PKcs exerts its pro-tumorigenic activity in cancer cells. In addition, the upstream regulator of DNA-PKcs activation as well as its downstream effectors thus far identified are illustrated. Furthermore, the potential therapeutic strategies aimed at inhibiting DNA-PKcs activity in HCC are discussed. © 2016 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The repair mechanims of double-strand breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA-PKCS: a key component of the NHEJ machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The final phase of NHEJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA repair-independent functions of DNA-PKCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mechanistic and prognostic role of DNA-PKCS in human hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Molecular mechanisms responsible for DNA-PKCS activation in hepatocellular carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 DNA-PKCS inhibitors for the treatment of liver cancer: preliminary evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Abbreviations: CHIP, chromatin immunoprecipitation; DNA-PKcs, DNA dependent protein kinase catalytic subunit; DSBs, double strand breaks; GI, genomic instability; HCC, hepatocellular carcinoma; NHEJ, non-homologous end-joining. ∗ Corresponding author at: Department of Clinical and Experimental Medicine, University o Sassari, via P. Manzella 4, 07100 Sassari, Italy. E-mail address: [email protected] (D.F. Calvisi). 1 Pascale RM and Joseph C equally contributed to this work. http://dx.doi.org/10.1016/j.dnarep.2016.10.004 1568-7864/© 2016 Elsevier B.V. All rights reserved.
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Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction
2. The repair mechanims of double-strand breaks
Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer, with 0.25-1 million new cases per year and a life expectancy of about 6 months from the time of the diagnosis [1,2]. HCC incidence changes with age, sex, ethnic group, and geographic region and is rapidly rising in Western countries [3,4]. Major risk factors associated with HCC development include chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, alcoholic and nonalcoholic steatohepatitis, food contamination by Aflatoxin B1, and some inherited metabolic disorders [1,5]. In addition, HCC incidence shows differences within the human population in response to risk factors, thus suggesting the implication of a polygenic control in HCC incidence [6–8]. The interaction of genetic and environmental risk factors creates a wide genotypic and phenotypic heterogeneity within human HCC [7–9]. Partial liver resection, radiofrequency ablation, and liver transplantation potentially curative options for HCC [10]. However, only a small subset of HCC cases is amenable to these treatments due the late diagnosis of the disease [1,10,11]. Therapies with pharmacological agents, trans-arterial chemo-embolization or administration of yttrium-90 microspheres, and percutaneous ethanol injection do not improve substantially the prognosis of patients with advanced disease [1,10,11]. Thus, the study of the mechanisms involved in HCC progression could provide opportunities for more efficacious targeted therapies. Different etiologic stimuli may induce hepatocarcinogenesis, which is preceded in most cases by the development of hepatitis and cirrhosis in humans and/or in animals (Fig. 1). Hepatocarcinogenesis is a multiphasic process: the interaction of DNA with carcinogens, oxidative damage and reactive radicals, produced during carcinogen metabolism and/or inflammation, and compensatory hepatocyte regeneration in early phases of the process, induce lesions in the DNA, including the highly toxic double strand breaks (DSBs). This extensive DNA damage leads to chromosomal aberrations, resulting in loss of heterozygosity and genomic instability (GI) [12]. This process is followed by the development of foci of altered hepatocytes (Fig. 1), which partly evolve to low-grade and high-grade dysplastic nodules, and then to HCC with increasingly lower levels of differentiation (Fig. 1). During these events, GI progressively increases in liver lesional cells, with consequent chromosome aberrations as well as oncogene and oncosuppressor mutations and progressive deregulation of multiple signaling pathways, reaching their maximum in poorly differentiated, highly metastatic HCC [13–16]. Recent studies focused on strategies to directly interfere with DNA repair mechanisms in cancer, such as non-homologous end joining (NHEJ), a DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and, according to recent findings, a potential chemo-sensitization target in numerous cancer types including ovarian cancer [17], breast cancer [18], non-small cell lung cancer [19], colon cancer [20], glioblastoma [21]. Recent evidence implicating a critical role of DNA-PKcs in HCC [22–24] suggest that this protein could be a potential candidate target for new personalized therapies against liver cancer. The present review is principally devoted to an interpretive analysis of recent finding on DNA-PKcs in HCC as a potential target of new therapeutic approaches.
Living organisms have developed refined systems of genome stability maintenance to prevent cancer [25], by recognition of broken DNA sites and repair of the DNA lesions [26]. In mammalian cells, DSBs may be repaired by two main processes: homologous recombination and non-homologous end-joining (NHEJ). In the homologous recombination process, the repair of the damaged DNA strand is achieved by using a homologous stretch of DNA from the sister chromatid. NHEJ consists instead in the re-ligation of the broken DNA molecule [26–28]. NHEJ is a multiphasic process (Fig. 2), which includes: (a) the recognition of DSB by the Ku70/80 ring-shaped protein heterodimer; (b) the recruitment, assembly, and stabilization of the NHEJ complex at the site of damage; (c) the binding to the ends of the broken chains and their stabilization; (d) the activation of the catalytic subunit od DNA-PKcs and the ligation of broken ends [28]. NHEJ is initiated by the recognition and binding of the Ku70/80 dimer to the sugar backbone at the site of DNA damage. Bound Ku70/80 recruits the NHEJ apparatus to the DNA lesion by directly linking the canonical NHEJ proteins: DNA-PKcs (DNA dependent protein kinase catalytic subunit), XRCCA (X-ray cross complementing protein 4), XLF (XRCC4-like factor), and DNA Ligase IV. This protein complex ties the DNA ends and activates DNA-PKcs. The latter process is then followed by the ligation of the broken ends, which is mediated by DNA Ligase IV, and the disintegration of the NHEJ complex [26–30]. The active NHEJ complex may require other components, such as DCLRE1C (DNA cross-link repair protein 1C or Artemis), DNA polymerase and , TDT (terminal dinucletidyltrasferase), PNKP (polynucleotide kinase-phosphatase), APTX (aprataxin), and APLF (aprataxin-PNKP-like factor) [31]. 3. DNA-PKCS: a key component of the NHEJ machinery DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3) kinase-like kinase family (PIKK), which includes other two proteins responsive to DNA damage: ATM (ataxia-telengectasia mutated) and ATR (Rad3-related protein) [32,33]. The N-terminal region of DNA-PKcs is composed of HEAT (Huntington-elongation-factor 3, regulatory subunit A of PP2A, TOR1) repeats and other ␣-helical regions, constituting the 74% of DNA-PKcs. The C region of the protein contains the PI3 kinase domain, which is involved in phosphorylation of other proteins as well as auto-phosphorylation, and is flanked N-terminally by the FAT (FRAP, ATM, TRRAP) domain and C-terminally by the FATC (FAT C-terminal) domain [34–36] (Fig. 3). Structural studies have shown that the N-terminal portion of DNA-PKcs has a pincer-shaped structure forming a central channel that probably binds electrostatically to double strand-DNA (DSDNA) and the C-terminal domains, containing the kinase domain, forms an head/crown placed on top of the pincer-shaped structure [37,38] (Fig. 3). However, according to recent studies, DS-DNA seems to interact closer to the base of DNA-PKcs [39,40]. Following the recognition of the DSB ends by the Ku (Ku70/80) complex, during initiation of NHEJ, the binding of DNA-PKcs to KuDNA complex, involving the final 13 aminoacidic residues of Ku80, induces a conformational change in the FAT and FATC domains, surrounding the PIK3 kinase domain, followed by the activation of the kinase activity [41]. Further, the N-terminus region of DNAPKcs also plays a role in modulating its enzymatic activity [42]. However, the mechanisms whereby the interaction of DNA-PKcs
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interaction with PP6c is required for DNA-PKcs activation after DNA damage [48], and this interaction occurs primarily through PP6R1 [49]. 4. The final phase of NHEJ
Fig. 1. Schematic representation of liver carcinogenesis. Hepatitis/cirrhosis induced in humans and/or animals by different carcinogenic stimuli, are followed by the development of hepatocellular carcinoma. The clonal expansion of cells initiated by carcinogens leads to the development of foci of altered hepatocytes (FAH), which may evolve to dysplastic nodules (DN). This is followed by the development of well-differentiated hepatocellular carcinomas (WDH) that further progress to moderately-differentiated (MDH) and poorl-differentiated (PDH) carcinomas. During this process, progressively increase of genomic instability (GI) occurs.
with Ku and DNA leads to DNA-PKcs activation are not yet completely understood. Available evidence indicates a role of the kinase domain [43]. Surprisingly, only a few phosphorylation events are required for NHEJ in vivo, most of which consist of auto-phosphorylation, although DNA-PKcs may be also phosphorylated by different kinases, such as PIKKs, ATM, and ATR (36). Proteomics studies have also revealed that DNA-PKcs is modified by acetylation [44] and ubiquitination in vivo [45], but it is not known how these posttranslational modifications affect its function. Interestingly, various proteins have been shown to interact with DNA-PKcs. Among these proteins is phosphatase 6 (PP6), composed of catalytic (PP6c) and regulatory subunits (PP6R1, PP6R2 and PP6R3) [46,47]. In particular, it has been reported that DNA-PKcs
The mechanism whereby Ku complex is removed from the DNA ends during the repair process remains poorly defined. Ku heterodimer ubiquitination has been proposed as a mechanism to remove the Ku complex from DNA ends [50]. To complete NHEJ, DNA synthesis fills-in the gaps, and end joining is carried out by DNA Ligase IV, which is stabilized and activated by XRCC4 that promotes its adenylation [51]. DNA Ligase IV capacity to ligate non-cohesive ends of DNA is enhanced by XLF [52,53]. An important role is also played by APLF, which stimulates the ligation by XRCC4-DNA Ligase IV, facilitates the assembly of the NHEJ complex, and promotes the retention of XRCC4 in association with PARP3 and APLF [54,55]. In conclusion, the basic steps of NHEJ include: (1) DSB detection by the Ku heterodimer with subsequent formation of the DNAPKcs-Ku-DNA complex, (2) lesion processing, (3) DNA end ligation by DNA Ligase IV in complex with XRCC4 and XLF. The results of structural studies suggest that the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular helixes suitable to store/release conformational energy to regulate NHEJ complexes, and DNA end protection, processing, and ligation [56]. 5. DNA repair-independent functions of DNA-PKCS Recent experimental findings convincingly indicate that DNAPKcs possesses additional functions that are not dependent on its DNA repair activity. In particular, a mounting body of data points to a crucial role of DNA-PKcs during mitosis. This hypothesis was generated following the observation that siRNA-induced depletion of DNA-PKcs or suppression of DNA-PKcs kinase activity by the chemical inhibitor NU7441, results in mitotic defects [57]. The molecular mechanisms responsible for the activation of DNA-PKcs in mitosis are not completely understood. Present evidence suggests that they differ from the Ku-dependent activation of DNA-PKcs after
Fig. 2. Schematic representation of non-homologous end-joining (NHEJ) and protein concerned with the different steps. Abbreviations: APLF, aprataxin-PNKP-like factor; XLF, XRCC4-like factor; XRCCA, X-ray cross complementing protein 4. Other details are in the text.
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Fig. 3. Molecular structure of DNA-PKcs. Details are in the text.
DNA damage, and that PLK1 (polo-like kinase 1) and PP6 regulate DNA-PKcs phosphorylation during mitosis [58]. Other important functions of DNA-PKcs are the protection of telomeres [59–61] and the regulation of inflammatory and immune responses [62,63]. DNA-PKcs promotes the phosphorylation of RNA polymerase II C-terminal domain, thus controlling transcriptional elongation [64,65]. It is also involved in directing transcriptional factors like AIRE (Autoimmune Regulator) to the promoter regions of Tolllike receptor (TLR) genes [66]. In hormone-dependent cancers, a circuit of reciprocal regulation between the Androgen Receptor (AR) and Estrogen Receptor (ER) with DNA-PKcs has been identified [67–69]. Furthermore, several studies indicate that DNA-PKcs is a major, positive regulator of lipogenic genes. Indeed, it has been found that DNA-PKcs catalyzes the phosphorylation of the upstream transcription factor 1 (USF-1) gene, which is responsible for coordinating changes in lipogenesis and gluconeogenesis during the fasting–feeding cycle [70,71] The functions of DNA-PKcs independent of its DNA repair activity are schematically illustrated in Fig. 4. Together with aberrant fatty acid biosynthesis, considered a hallmark of malignancy [71], DNA-PKcs is involved also in the upregulation of a specific set of genes, including PREX1, ROCK2, Integrin b4, and VAV3, that are primarily responsible for other cancer traits, namely invasion and metastasis [72,73]. The importance of DNA-PKcs in mediating the metastatic properties of cancer cells was underscored by in vivo experiments on mouse models as well as by the finding that levels of DNA-PKcs predict clinical disease recurrence and metastasis development in prostate cancer [72,73]. Besides its activity in the nucleus, DNA-PKcs is also involved in the regulation of cytoplasmic organelles, such as the Golgi apparatus [74]. Specifically, following DNA damage, DNA-PKcs phosphorylates GOLPH3 (Golgi phosphoprotein 3) and increases its binding to Myosin XVIIIA (MYO18A), which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival [74].
6. Mechanistic and prognostic role of DNA-PKCS in human hepatocellular carcinoma Cumulating evidence points to an important role played by DNA-PKcs along HCC development and progression. Among different histotypes of liver tumors, hepatocellular carcinoma was found to be the one with the highest expression of DNA-PKcs, followed by cholangiocarcinoma and biliary cystadenocarcinoma [22]. In addition, it has been described that the expression and activity of DNA-PKcs are concurrently increased with hypoxia-inducible factor 1-alpha (HIF-1alpha) levels under hypoxic conditions in the HepG2 human hepatoma cell line [76]. In these conditions, DNAPKcs can both directly interact with and phosphorylate HIF-1alpha, thus suggesting that DNA-PKcs promotes HIF-1alpha stabilization [76]. These important findings envisage the possibility that the DNA-PKcs/HIF-1alpha complex could significantly contribute to therapy resistance in hypoxic tumor cells. Using the same hepatoma cell line, An et al. found that DNA-PKcs contributes to the stabilization of c-Myc oncoprotein via the Akt/GSK-3beta signaling cascade [77]. In HepG2 cells, similar to that described in the HeLa cell line, DNA-PKcs inhibited the proteolysis of c-Myc by suppressing its proteasomal degradation [77]. The importance of DNA-PKcs as a main modulator of c-Myc oncoprotein was further confirmed by the same authors in the LO2 normal liver cell line. In these cells, indeed, overexpression of DNA-PKcs led to increased protein levels of c-Myc as well as upregulation of AKT [77]. Taken together, these data indicate that DNA-PKcs induction is associated with pro-oncogenic functions in the liver, via its ability to positively regulate important oncogenes, such as AKT and c-Myc, and to stimulate cell survival under hypoxic conditions via its interaction with HIF-1alpha. Following these pioneering and promising studies, our group has further investigated the importance of DNA-PKcs in human HCC. In a recent study using a large collection of human liver tumor specimens, we showed that DNA-PKcs levels and activity are
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Fig. 4. Functions of DNA-PKcs independent of DNA-repair activity. (A) Induction by DNA damage of DNA-PKcs interaction with polo-like kinase 1 (PLK1), followed by activation of phosphorylation of checkpoint kinase 2 (Chk2) in mitosis and phosphorylation of BRCA1, which facilitates DNA repair and regulates mitotic spindle formation. (B) Maintenance of telomere endcapping by telomerase action and homologous recombination favored by DNA-PKcs. (C) Role of DNA-PKcs in activation of immune system and inflammatory response upon recognition of foreign DNA. (D) Control of the transcriptional initiation and elongation through phosphorylation of RNA polymerase II by DNA-PKcs. (E) Circuit of activation of androgen receptors (ARs). ARs activated by dihydrotestosterone (DHT) promote DNA-PKcs expression, which coactivates ARs. (F) The succession fasting/feeding stimulated phosphorylation of USF-1 (upstream transcription factor 1) by DNA-PKcs, leading to fatty acid synthetase (FAS) up-regulation and lipogenesis.
progressively induced from non-neoplastic surrounding liver tissues to HCC. In particular, the highest levels of DNA-PKcs were detected in the biologically most aggressive liver tumors [23]. Subsequently, when investigating the possible relationship(s) between DNA-PKcs and the clinic-pathological features of the patients, we found that DNA-PKcs activity directly correlates with genomic instability, proliferation and angiogenic (microvessel density) indices, and inversely with apoptosis and patients’ survival length [23]. These findings suggest a role of DNA-PKcs in the acquisition of malignant properties of liver cancer cells. In accordance with our data, the prognostic role of DNA-PKcs in human HCC has been subsequently confirmed and substantiated in an independent
investigation by Cornell et al. [24]. In the latter study, DNA-PKcs activity was identified as a marker of poor histological tumor grade and an independent indicator of short survival on HCC patients. In addition, DNA-PKcs levels were found to be positively and strongly associated with resistance to palliative doxorubicin treatment, as detected by the time to radiological progression of the tumor [24].
7. Molecular mechanisms responsible for DNA-PKCS activation in hepatocellular carcinoma The upstream effectors responsible for activation of DNA-PKcs in HCC have recently started to be elucidated. At the molecular level,
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Fig. 5. Mechanisms of DNA-PKcs upregulation. Transcriptional activation of DNA-PKcs gene by (A) amplification and (B) HSF1 stimulation of RAS/ERK signaling leading to AP-1 activation and DNA-PCcs overexpression. Post-transcriptional regulation of DNA-PKcs by (C) stabilization (hyper-folding) of DNA-PKcs by Reptin/RUVBL2 protein, and (D) physical interaction of TNKS1BP1 protein with DNA-PKcs and PARP1, followed by DNA-PKcs auto-phosphorylation and activation.
we found that upregulation of DNA-PKcs mRNA and protein levels is induced by the heat shock factor 1 (HSF1) transcription factor in HCC cell lines [23]. In these cell lines, however, a direct upregulation of DNA-PKcs by HSF1 could not be demonstrated. Indeed, although a putative binding site for HSF1 was identified by transcription binding site prediction tools, the subsequent Chromatin ImmunoPrecipitation (ChIP) experiment conducted in human hepatoblast WRL-68 cells as well as in HuH6 and HLE HCC cell lines did not confirm the interaction between HSF1 and DNA-PKcs [23]. On the other hand, several putative binding sites for AP-1, a pivotal effector of HSF1 downstream of MAPK and JNK pathways, were detected in the DNA-PKcs promoter. Importantly, when HSF1 forced overexpression in the SNU-423 HCC cell line was coupled to treatment with specific MAPK and JNK soluble inhibitors, HSF1-induced upregulation of DNA-PKcs mRNA and protein was abolished. In addition, ChIP analysis showed a functional interaction between c-Jun and c-Fos (members of the AP-1 complex) and the DNA-PKcs promoter [23]. Altogether, these data strongly suggest that HSF1 induces DNA-PKcs in HCC cell lines indirectly, via activation of members of the AP-1 complex [78]. Additional mechanisms responsible for DNA-PKcs upregulation in HCC have been subsequently identified. In particular, DNA-PKcs induction in this tumor type might be the consequence of genomic aberrations, as amplification of the DNA-PKcs gene locus (8q11.21) was detected in 55% of a large collection of HCC specimens, where it correlated significantly with DNA-PKcs gene expression [24]. Furthermore, recent evidence indicates that DNA-PKcs is also regulated at the post-transcriptional level in HCC. Indeed, it has been described that Reptin/RUVBL2, a member of the ATPase protein
family and a presumed liver oncogene, concurs to stabilize the levels of the DNA-PKcs protein in HCC cells via direct interaction [77]. Importantly, in the same study, the authors showed that Reptin/RUVBL2 promotes the stabilization of ATM kinase as well, thus implying a crucial role of Reptin/RUVBL2 in DSB repair in HCC cells. Finally, in another investigation, tankyrase 1 binding protein 1 (TNKS1BP1), which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily, was found to physically interact with DNAPKcs and PARP-1 as well as to promote the association between DNA-PKcs and PARP-1 in various cell lines, including HepG2 [78]. In particular, it was shown that overexpression of TNKS1BP1 induces the auto-phosphorylation of DNA-PKcs at Ser2056 in a PARP-1 dependent manner, which contributes to an increased activity of the DNA DSB repair system [78]. Altogether, the present data indicate that multiple molecular events might contribute to unrestrained induction of DNA-PKcs in liver cancer. The main molecular mechanisms responsible for DNAPKcs activation thus far identified in HCC are summarized in Fig. 5.
8. DNA-PKCS inhibitors for the treatment of liver cancer: preliminary evidence It is well established that DNA repair pathways are crucial for normal cell survival, and that defects in the execution and control of these mechanisms have been linked with the development of many cancer types [79]. However, although an effective DNA repair system possesses beneficial effects for normal cells, it is plausible to hypothesize that the same system plays a role in carcinogenesis by both protecting tumor cells from stress-induced
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death and promoting proliferation in the presence of a potentially lethal microenvironment [79]. Noticeably, only some DNA repair modules are retained by cancer cells, thus rendering the latter cells dependent on a few pathways for survival [79]. Therefore, the use of inhibitors against essential components of the retained DNA repair system might represent a promising path to eradicate specifically cancer cells. In support of the latter hypothesis, it has been demonstrated that breast cancer cells from Breast Cancer 2 Early Onset (BRCA2)-deficient tumors display a strong dependency on the DNA stress protein PARP-1, as PARP-1 inhibition impairs the DNA-damage response to the point of lethality [80]. Interestingly, mutations in the BRCA2 gene have been detected in a small subset of human HCC, thus suggesting the use of PARP inhibitors at least in specific HCC cases [81,82]. In the absence of specific mutations that render liver tumors vulnerable to DNA repair inhibition, an alternative strategy could consist in the combinatorial administration of a DNA-damaging agent together with an inhibitor of the DNA repair system. We have tested the strength of this alternative therapeutic approach in in vitro growing HCC cell lines, treating them with the DNAdamaging agent doxorubicin, either alone or in combination with the DNA-PKcs inhibitor NU7441 [23]. We found that DNA-PKcs inactivation reduced proliferation and induced apoptosis in the six HCC cell lines used. Of note, the effects on growth restraint were significantly higher in HCC cell lines when compared with WRL-68 and Chang non-transformed liver cell lines [23]. Strikingly, when suppression of DNA-PKcs was associated with treatment with doxorubicin, an impressive growth restraint, elevated apoptosis, and massive DNA damage occurred in all HCC cell lines [23]. Once again, only a slight decline in proliferation and increase in apoptosis and DNA damage was detected in Chang and WRL-68 non-transformed subjected to the same treatment scheme [23]. Furthermore, the striking growth restraint effects on HCC cell lines were independent of p53 mutation status, indicating that DNAPKcs inhibition might be ubiquitously deleterious for HCC cells [23]. Equivalent results were obtained by Cornell et al. in a recent study [24]. The authors found indeed that NU7441 significantly potentiates ionizing radiation induced cytotoxicity by 3- to 40-fold and doxorubicin cytotoxicity by 2- to 50-fold in HCC cell lines, and resulted in a strong delay of liver tumor xenograft growth in vivo. In addition, the same study showed a synergistic antiproliferative effect in HCC cells when NU7441 was administered in association with the ATM inhibitor KU55933 [24]. In another investigation, a remarkable inhibition of HCC cell growth in vitro and in vivo was achieved with the recently-developed water-soluble DNA-PKcs inhibitor KU-0060648 [83]. However, the contribution of DNA-PKcs inhibition on KU-0060648 dependent growth restraint of HCC cells requires to be better defined, as the authors found that KU-0060648 also suppresses the AKT/mTOR pathway in the same cells [83]. Altogether, these data suggest a possible addiction of HCC cells to DNA-PKcs for proliferation and survival, especially when subjected to DNA damaging insults.
9. Concluding remarks Mounting evidence underlines a critical role for DNA-PKcs in HCC progression and patient’s prognosis. Previous studies conducted in other tumor types indicate that DNA-PKcs significantly contributes to many features of cancer cells, including uncontrolled growth, resistance to DNA damage-induced apoptosis, migration, invasion, and metastasis as well as metabolism and gene reprogramming. Further studies are required to determine the specific role, or roles of DNA-PKcs in HCC. Preliminary data from our and other groups indicate that treatment strategies aimed at
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suppressing DNA-PKcs are deleterious to HCC cells subjected to a DNA damaging agent in vitro and in vivo. Although promising, these results require to be further substantiated in pre-clinical animal models showing overexpression and/or activation of DNAPKcs. In these models, in particular, the effectiveness and side effects of the aforementioned treatment strategies should be determined. Furthermore, the identification of upstream inducers and/or downstream targets of DNA-PKcs in the liver might help in the development of additional targeted therapies against this deadly disease. Conflict of interest The authors declare no conflict of interest. Acknowledgement Supported by the Italian Association Against Cancer, Grant number IG 12139. References [1] J.M. Llovet, J. Bruix, Molecular targeted therapies in hepatocellular carcinoma, Hepatology 48 (2008) 1312–1327. [2] F. Feo, R.M. Pascale, M.M. Simile, M.R. De Miglio, M.R. Muroni, D.F. Calvisi, Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies, Crit. Rev. Oncog. 11 (2000) 19–62. [3] Y. Tanaka, K. Hanada, M. Mizokami, A.E. Yeo, J.W. Shih, T. Gojobori, H.J. Alter, A comparison of the molecular clock of hepatitis C virus in the United States and Japan predicts that hepatocellular carcinoma incidence in the United States will increase over the next two decades, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 15584–15589. [4] S.D. Taylor-Robinson, G.R. Foster, S. Arora, S. Hargreaves, H.C. Thomas, Increase in primary liver cancer in the UK, 1979–94, Lancet 350 (1997) 1142–1143. [5] D.F. Calvisi, M. Frau, M.L. Tomasi, F. Feo, R.M. Pascale, Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison, Biochim. Biophys. Acta 1826 (2012) 215–237. [6] J.A. Indulski, W. Lutz, Metabolic genotype in relation to individual susceptibility to environmental carcinogens, Int. Arch. Occup. Environ. Health 73 (2000) 71–85. [7] F. Feo, M.R. De Miglio, M.M. Simile, M.R. Muroni, D.F. Calvisi, M. Frau, R.M. Pascale, Hepatocellular carcinoma as a complex polygenic disease Interpretive analysis of recent developments on genetic predisposition, Biochim. Biophys. Acta 1765 (2006) 126–147. [8] T.A. Dragani, Risk of HCC: genetic heterogeneity and complex genetics, J. Hepatol. 52 (2010) 252–257. [9] F. Donato, U. Gelatti, R.M. Limina, G. Fattovich, Southern Europe as an example of interaction between various environmental factors: a systematic review of the epidemiologic evidence, Oncogene 25 (2006) 3756–3770. [10] M. Sherman, Modern approach to hepatocellular carcinoma, Curr. Gastroenterol. Rep. 1 (2011) 49–55. [11] D.F. Calvisi, R.M. Pascale, F. Feo, Dissection of signal transduction pathways as a tool for the development of targeted therapies of hepatocellular carcinoma, Rev. Recent Clin. Trials 2 (2007) 217–236. [12] A. Schipler, G. Iliakis, DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice, Nucleic Acids Res. 41 (2013) 7589–7605. [13] M. Frau, F. Biasi, F. Feo, R.M. Pascale, Prognostic markers and putative therapeutic targets for hepatocellular carcinoma, Mol. Aspects Med. 31 (2010) 179–193. [14] A. Villanueva, P. Newell, D.Y. Chiang, S.L. Friedman, J.M. Llovet, Genomics and signaling pathways in hepatocellular carcinoma, Semin. Liver Dis. 27 (2007) 55–76. [15] M.A. Kern, K. Breuhahn, P. Schirmacher, Molecular pathogenesis of human hepatocellular carcinoma, Adv. Cancer Res. 86 (2002) 67–112. [16] S. Imbeaud, Y. Ladeiro, J. Zucman-Rossi, Identification of novel oncogenes and tumour suppressors in hepatocellular carcinoma, Semin. Liver Dis. 30 (2010) 75–86. [17] D.A. Dungl, E.N. Maginn, E.A. Stronach, Preventing damage limitation: targeting DNA-PKcs and DNA double-Strand Break repair pathways for ovarian cancer therapy, Front. Oncol. 5 (2015) 240. [18] N. Albarakati, T.M. Abdel-Fatah, R. Doherty, R. Russell, D. Agarwal, P. Moseley, C. Perry, A. Arora, N. Alsubhi, C. Seedhouse, E.A. Rakha, A. Green, G. Ball, S. Chan, C. Caldas, I.O. Ellis, S. Madhusudan, Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy, Mol. Oncol. 9 (2015) 204–217.
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