Poly (ADP-ribose) polymerase (PARP) catalyzes poly ADP-ribosylation reaction using NAD as a substrate. PARG is the main enzyme for poly (ADP-ribose) degradation. Strong PARP inhibitors have been developed and are in clinical trials as single agents or in combination with other chemotherapeutic drugs or radiation therapy. Synthetic lethality of BRCA1/2 mutation in cancer cells with PARP inhibitors led to a new strategy for individualized cancer therapy. Functional studies of PARG revealed that its inhibition leads to poly (ADP-ribose) accumulation, block of DNA repair, epigenetic changes and cell death, suggesting that it could serve as another potential cancer target. The differences and similarities of PARP and PARG as a cancer target will be discussed.

•    PARP
•    PARG
•    Cancer therapy
•    NAD
•    PAR

Fujimori H, Hirai T, Inoue K, Koizumi F, Masutani M (2014) The Distinctive Properties of Parp and Parg as a Cancer Therapeutic Target. JSM Clin Oncol Res 2(5): 1033.

PARP: Poly(ADP-ribose) Polymerase; PARG: Poly(ADP-ribose) Glycohydrrolase; NAD: Nicotinamide Adenosine Dinucleotide; PAR: Poly(ADP-ribose); HR: Homologous Recombination

PolyADP-ribosylation reactions, one of the post-translational modification of proteins, are catalyzed by poly(ADP-ribose) polymerase (PARP) using NAD as a substrate [1]. Among these PARP family proteins, PARP-1, PARP-2 and PARP-3 are activated by DNA strand breaks and are involved in DNA repair [2]. Poly(ADP-ribose) (PAR) was catabolized mainly by poly(ADP-ribose) glycohydrolase (PARG). PARP and PARG inhibitors have been focused for cancer therapy. These two enzymmes have apparently opposite roles in poly (ADP-ribose) metabolism and the consequences of inhibition of their function are distinctive, although partly overlap. Here we discuss how PARP and PARG inhibition leads to cell death through different action mechanisms.

PAR Signaling in Cancer

PARP inhibitors suppress DNA repair induced by spontaneous and exogenously introduced DNA damage [2]. Certain PARP inhibitors also produce bulky DNA lesions by stalling PARP-1 molecules at the sites of DNA breakages [3]. Poly(ADP-ribose) (PAR) signaling by three PAR binding proteins; ALC1, a PAR-dependent helicase, APLF [4], which has PAR-binding zinc finger and a ubiquitin-ligase, CTFR [5], facilitates DNA damage response and are involved in induction of cell cycle arrest.

Synthetic lethality of BRCA1/2 mutation in cancer cells with PARP inhibitors [6,7] or PARP-1 knockdown led to a new strategy for cancer therapy (Table 1); a single treatment with a PARP inhibitor is able to target BRCA1/2 mutated cancers [8]. In this strategy, normal cells, which retain at least one BRCA1/2 functional allele will not be affected by PARP inhibitors.

PARP inhibitors for cancer therapy

Several PARP inhibitors were demonstrated to be effective in clinical trials including BRCA-mutated breast cancers, and prostate cancers, triple-negative breast cancers [8,9] and ovarian cancers [10]. However, there are cases in which PARP inhibitors are not effective (Table1). The loss of 53BP1 is frequently observed in breast cancer and such cancers showed resistance to PARP inhibitors even when the BRCA1 gene is mutated [11]. Secondary mutations in the BRCA2 gene, which enable to recover partial activities of the mutated the BRCA2 gene, are reported to cause resistance to treatment with PARP inhibitors [12]. Some cancers may also become resistant to PARP inhibitors through MDR gene overexpression through high levels of drug excretion.

Action mechanism of PARG inhibitors

PAR degradation involves PARG [13], ARH3 [14], and TARG [15]. Among them, PARG is the main PAR degradation enzyme and its disruption in the cells leads to accumulation of PAR (Figure 1) [16,17]. PARG functions through interaction with PARP in base excision repair [18]. PARG inhibition thus leads to inhibition of DNA repair directly by blocking the base-excision repair process and indirectly by stalling PAR signaling.

By functional inhibition of PARG, PAR catabolism could be severely disturbed and causes extensive accumulation of PAR, which leads to cell death. In cancer cells and neuronal cells, apoptosis-inducing factor (AIF) dependent parthanatos is induced by PAR accumulation [19,20] (Figure 1).

Figure 1 PARP and PARG inhibition both lead to cell death through different action mechanisms. PARP inhibition causes decrease of PAR levels and PARP stalling at DNA ends that severely block DNA repair. On the other hand, PARG inhibition will induce PAR accumulation and may block DNA repair. Epigenetic and transcriptional dysregulation by PARG inhibitors may also contribute to inhibiting cancer cell growth.

Other types of cell death such as apoptosis and necrotic cell death are also enhanced when PARG deficient cells are treated with DNA damaging agents [21] or ionizing radiation [22-24]. PARG inhibition also disturbs growth signaling pathway and micro RNA regulation [25].

PARP-1 levels in cancer cells are frequently upregulated, and PARG levels in cancer cells are also shown to be divergent [26,27] and these PARP and PARG levels may affect the endogenous PAR levels. However, the relationship between the levels of PARP or PAR and the efficiency of cell death induction by PARG inhibition has not been studied yet. Because PARG inhibition also leads to indirect inhibition of PARP-1 by enhancing auto-PARylation of PARP-1, PARG inhibition is also expected to cause PARP-1 inhibition.

Factors affecting therapeutic effects of PARG inhibitors

A strong synthetic lethal combination has not been reported with PARG functional inhibition, however, a mild synthetic lethal action has been reported (Table 1)

with BRCA2 mutation [28]. There is a lack of clinically useful strong inhibitor of PARG, and finding of synthetic lethal target molecules or a pathway for PARG has not been progressed. Because of the recent elucidation of the structure of PARG molecules, development of effective PARG inhibitors are expected to progress in the near future. The approach with synthetic lethality could be effective and useful, therefore further studies are expected to be carried out.

Combination of PARG inhibitors with chemotherapy and radiation therapy

Combinations of PARG inhibition with chemotherapy or radiation therapy have been also suggested to be useful. As shown (Table 2),

PARG inhibitors show some overlapping spectra of sensitization in combination with chemotherapeutic agents. Of note, PARG inhibition attenuates the cytotoxicity of a topoisomerase inhibitor, camptothecin [29]. This is a contrasting phenomenon from the PARP inhibitor combination [30]. Another difference between PARP and PARG inhibition is the combined effect with cisplatin. PARP1 functional inhibition did not enhance cytotoxicity of cisplatin but PARG functional inhibition augmented cisplatin cytotoxicity [29] indicating the action mechanism of PARG inhibition should be different from that of PARP inhibitor. PARG inhibition also showed radiosensitization to particular cancer cells [23]. PARG inhibition was also shown to be effective for radiosensitization of particle-ion radiotherapy by accompanying augmented PAR accumulation [22].

PAR pathway addiction

PARylation signaling is upregulated in many cancer cells [31]. The dependence levels of cancer cell growth on PAR signaling, namely the addiction level of cancer cells to PAR signaling, may determine the effectiveness of PAR pathway blocking drugs. The more tumor cells acquire the high levels of genomic instability, the more the addiction level to PAR signaling may increase.

As described here, action mechanisms of PARP and PARG inhibitors show differences. This suggests that different cancers should be targeted by PARP and PARG inhibitors. PARG inhibitors also indirectly inhibit PARP. The NAD level of cancer cells should be divergent and certain cancers show dysfunction of nicotinate phosphoribosyltransferase, NAPRT [32]. PARG inhibitors may be more effective if higher levels of NAD are present because augmented levels of PAR accumulation could be expected to be induced. In contrast, lower NAD levels may be effective for treatment with PARP inhibitors. Further studies of PARP and PARG function and development of their inhibitors will enable the clinical application of the cancer drugs targeting the PAR pathway.

This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan (11104962), the Third Term Comprehensive 10-Year Strategy for Cancer Control (10103833) from the Ministry of Health, Labor and Welfare of Japan, and from the MEXT of Japan (22300343).

1. Sugimura T. Poly (adenosine diphosphate ribose). Prog Nucleic Acid Res Mol Biol. 1973; 13: 127-151.

2. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 2006; 7: 517-528.

3. Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res. 2012; 72: 5588-5599.

4. Rulten SL, Fisher AE, Robert I, Zuma MC, Rouleau M, Ju L, et al. PARP-3 and APLF function together to accelerate nonhomologous end-joining. Mol Cell. 2011; 41: 33-45.

5. Yu X, Minter-Dykhouse K, Malureanu L, Zhao WM, Zhang D, Merkle CJ, Ward IM. Chfr is required for tumor suppression and Aurora A regulation. Nat Genet. 2005; 37: 401-406.

6. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 2005; 434: 913-917.

7. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005; 434: 917-921.

8. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med. 2009; 361: 123-134.

9. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN, et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proofof-concept trial. Lancet. 2010; 376: 235-244.

10. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, BellMcGuinn KM, et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial. Lancet. 2010; 376: 245-251.

11. Bouwman P, Aly A, Escandell JM, Pieterse M, Bartkova J, van der Gulden H, et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol. 2010; 17: 688-695.

12. Sakai W, Swisher EM, Karlan BY, Agarwal MK, Higgins J, Friedman C, et al. Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature. 2008; 451: 1116-1120.

13. Miwa M, Sugimura T. Splitting of the ribose-ribose linkage of poly(adenosine diphosphate-robose) by a calf thymus extract. J Biol Chem. 1971; 246: 6362-6364.

14. Oka S, Kato J, Moss J. Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase. J Biol Chem. 2006; 281: 705-713.

15. Sharifi R, Morra R, Appel CD, Tallis M, Chioza B, Jankevicius G, et al. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/ C6orf130 in neurodegenerative disease. EMBO J. 2013; 32: 1225- 1237.

16. Cortes U, Tong W M, Coyle DL, Meyer-Ficca ML, Meyer RG, Petrilli V, et al. Depletion of the 110-kilodalton isoform of poly(ADP-ribose) glycohydrolase increases sensitivity to genotoxic and endotoxic stress in mice. Molecular and cellular biology 2004; 24: 7163-7178.

17. Koh DW, Lawler AM, Poitras MF, Sasaki M, Wattler S, Nehls MC, et al. Failure to degrade poly(ADP-ribose) causes increased sensitivity to cytotoxicity and early embryonic lethality. Proc Natl Acad Sci U S A. 2004; 101: 17699-17704.

18. Keil C, Gröbe T, Oei SL. MNNG-induced cell death is controlled by interactions between PARP-, poly(ADP-ribose) glycohydrolase, and XRCC1. J Biol Chem. 2006; 281: 34394-34405.

19. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, et al. Poly(ADP-ribose) (PAR) polymer is a death signal. Proc Natl Acad Sci U S A. 2006; 103: 18308-18313.

20. Koh DW, Dawson TM, Dawson VL. Mediation of cell death by poly(ADPribose) polymerase-1. Pharmacol Res. 2005; 52: 5-14.

21. Shirai H, Poetsch AR, Gunji A, Maeda D, Fujimori H, Fujihara H, et al. PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways. Cell Death Dis. 2013; 4: e656.

22. Shirai H, Fujimori H, Gunji A, Maeda D, Hirai T, Poetsch AR, et al. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation. Biochemical and biophysical research communications. 2013; 435: 100-106.

23. Nakadate Y, Kodera Y, Kitamura Y, Tachibana T, Tamura T, Koizumi F. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint. Biochemical and biophysical research communications. 2013; 44: 793-798.

24. Amé JC, Fouquerel E, Gauthier LR, Biard D, Boussin FD, Dantzer F, et al. Radiation-induced mitotic catastrophe in PARG-deficient cells. J Cell Sci. 2009; 122: 1990-2002.

25. Leung AK, Vyas S, Rood JE, Bhutkar A, Sharp PA, Chang P. Poly(ADPribose) regulates stress responses and microRNA activity in the cytoplasm. Mol Cell. 2011; 42: 489-499.

26. Ogino H, Nakayama R, Sakamoto H, Yoshida T, Sugimura T, Masutani M. Analysis of poly(ADP-ribose) polymerase-1 (PARP1) gene alteration in human germ cell tumor cell lines. Cancer Genet Cytogenet. 2010; 197: 8-15.

27. Bieche I, Pennaneach V, Driouch K, Vacher S, Zaremba T, Susini A. Variations in the mRNA expression of poly(ADP-ribose) polymerases, poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome. International journal of cancer. Journal international du cancer. 2013; 133: 2791-2800.

28. Fathers C, Drayton RM, Solovieva S, Bryant HE. Inhibition of poly(ADP-ribose) glycohydrolase (PARG) specifically kills BRCA2-deficient tumor cells. Cell Cycle. 2012; 11: 990-997.

29. Fujihara H, Ogino H, Maeda D, Shirai H, Nozaki T, Kamada N, et al. Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents. Curr Cancer Drug Targets. 2009; 9: 953-962.

30. Zhang YW, Regairaz M, Seiler JA, Agama KK, Doroshow JH, Pommier Y. Poly(ADP-ribose) polymerase and XPF-ERCC1 participate in distinct pathways for the repair of topoisomerase I-induced DNA damage in mammalian cells. Nucleic Acids Res. 2011; 39: 3607-3620.

31. Gottipati P, Vischioni B, Schultz N, Solomons J, Bryant HE, Djureinovic T, Issaeva N. Poly(ADP-ribose) polymerase is hyperactivated in homologous recombination-defective cells. Cancer Res. 2010; 70: 5389-5398.

32. Shames DS, Elkins K, Walter K, Holcomb T, Du P, Mohl D, et al. Loss of NAPRT1 expression by tumor-specific promoter methylation provides a novel predictive biomarker for NAMPT inhibitors. Clin Cancer Res. 2013; 19: 6912-6923.