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Poly (ADP-ribosylation), known as PARylation, is a post-translational modification catalyzed by poly (ADP-ribose) polymerases (PARP) and primarily removed by the enzyme poly (ADP-ribose) glycohydrolase (PARG). While the aberrant removal of post-translation modifications including phosphorylation and methylation has known tumorigenic effects, deregulation of PARylation has not been widely studied. Increased hydrolysis of PARylation chains facilitates cancer growth through enhancing estrogen receptor (ER)-driven proliferation, but oncogenic transformation has not been linked to increased PARG expression. In this study, we find that elevated PARG levels are associated with a poor prognosis in breast cancers, especially in HER2-positive and triple-negative subtypes. Using both in vitro and in vivo models, we demonstrate that heightened expression of catalytically active PARG facilitates cell transformation and invasion of normal mammary epithelial cells. Catalytically inactive PARG mutants did not recapitulate these phenotypes. Consistent with clinical data showing elevated PARG predicts poor outcomes in HER2+ patients, we observed that PARG acts in synergy with HER2 to promote neoplastic growth of immortalized mammary cells. In contrast, PARG depletion significantly impairs the growth and metastasis of triple-negative breast tumors. Mechanistically, we find that PARG interacts with SMAD2/3 and significantly decreases their PARylation in non-transformed cells, leading to enhanced expression of SMAD target genes. Further linking SMAD-mediated transcription to the oncogenicity of PARG, we show that PARG-mediated anchorage-independent growth and invasion are dependent, at least in part, on SMAD expression. Overall, our study underscores the oncogenic impact of aberrant protein PARylation and highlights the therapeutic potential of PARG inhibition in breast cancer.

Poly(ADP-ribose) polymerase (Parp) is an enzyme responsible for catalyzing post-translational modifications through the addition of poly(ADP-ribose) chains (known as PARylation). Modification by PARylation modulates numerous cellular processes including transcription, chromatin remodeling, apoptosis, and DNA damage repair. In particular, the role of Parp activation in response to DNA damage has been intensely studied. Tumors bearing mutations of the breast cancer susceptibility genes, Brca1/2, are prone to DNA breakages whose restoration into functional double-strand DNA is Parp dependent. This concept has been exploited therapeutically in Brca mutated breast and ovarian tumors, where acute sensitivity to Parp inhibitors is observed. Based on in vitro and clinical studies it remains unclear to what extent Parp inhibitors can be utilized beyond treating Brca mutated tumors. This review will focus on the often overlooked roles of PARylation in chromatin remodeling, epigenetics, and transcription to explain why some cancers may be unresponsive to Parp inhibition. We predict that understanding the impact of PARylation on gene expression will lead to alternative approaches to manipulate the Parp pathway for therapeutic benefit.

Triple-negative breast cancers are associated with poor clinical outcomes and new therapeutic strategies are clearly needed. Gallotannin (Gltn) has been previously demonstrated to have potent anti-tumor properties against cholangiocarcinoma in mice, but little is known regarding its capacity to suppress tumor outgrowth in breast cancer models. We tested Gltn for potential growth inhibitory properties against a variety of breast cancer cell lines in vitro. In particular, triple-negative breast cancer cells display higher levels of sensitivity to Gltn. The loss of proliferative capacity in Gltn exposed cells is associated with slowed cell cycle progression and S phase arrest, dependent on Chk2 phosphorylation and further characterized by changes to proliferation related genes, such as cyclin D1 (CcnD1) as determined by Nanostring technology. Importantly, Gltn administered orally or via intraperitoneal (IP) injections greatly reduced tumor outgrowth of triple-negative breast cells from mammary fat pads without signs of toxicity. In conclusion, these data strongly suggest that Gltn represents a novel approach to treat triple-negative breast carcinomas.

Breast cancer patients whose tumors fall into the triple negative category have the poorest clinical outcomes. These patients are at high risk for metastatic recurrence and have poor overall survival. Clearly, new therapeutic strategies are needed to combat triple negative tumors both at the time of onset and if necessary, at recurrence. Toward this goal, we initially tested the anti-proliferative properties of Gallotannin (GLTN) against a variety of breast cancer cell lines in vitro. Gallotannin was chosen due to its potent chemotherapeutic effects against cholangiocarcinoma in mice, and the lack of data concerning its potential use as an anti-cancer agent in breast cancer models. We observed potent anti-proliferative properties of GLTN in the low micromolar range in most breast cancer cell lines tested, but not in corresponding non-transformed mammary epithelial cells. Notably, triple negative breast cancer cells displayed the highest level of sensitivity to GLTN. The loss of proliferative capacity in these cells is due to cell cycle arrest in S phase. This corresponded with increased Chk1 phosphorylation, decreased Cyclin D1 protein levels, and changes to growth related genes as determined by Nanostring technology. GLTN may have several cellular targets that could potentially mediate growth arrest. The most thoroughly described target of GLTN is PARG, which is responsible for removing poly(ADP)ribose moieties from target proteins. GLTN potently inhibits PARG activity both in vitro and in vivo. Surprisingly, we found that PARG knockdown results in a dramatic growth inhibition of breast cancer cells, similar to what is observed in cells exposed to GLTN. To our knowledge, this is the first report indicating PARG inhibition may be exploited therapeutically to impair breast cancer growth. Importantly, PARG knockdown cells showed less sensitivity to GLTN, indicating PARG is a primary effector of GLTN-mediated growth inhibition. To test the capacity of GLTN to diminish tumor outgrowth in vivo, we utilized a mouse xenograft model where MDA-MB-468 triple negative breast cancer cells were injected into the mammary fat pad and allowed to grow to a palpable size before drug treatment. GLTN was administered ad libitum orally at 0.05% in drinking water or via intraperitoneal (IP) injections dosed at 10mg/kg daily. Both orally and IP administered GLTN greatly reduced tumor outgrowth by approximately 3 fold within a 25 day period without significant signs of weight loss, morbidity or liver toxicity. Beyond 25 days, orally ingested GLTN continued to prevent tumor growth by 3-fold, whereas tumors exposed to GLTN given by IP exhibited decreased sensitivity. In conclusion, these data strongly suggest that orally administered GLTN represents a novel approach to treat triple negative breast carcinomas and that PARG may represent a new therapeutic target for anti-cancer compounds. Citation Format: Tiejun Zhao, Qiang Sun, Amanda Lovato, Sonia del Rincon, Michael Witcher. In vivo efficacy of the PARG inhibitor Gallotannin against triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2013-5515

Poly(ADP-ribose)polymerase (PARP) is an enzyme responsible for catalyzing the addition of poly(ADP-ribose) chains onto target proteins in a process known as PARylation. This post-translational modification has been implicated in numerous cellular processes including gene expression, chromatin remodeling, apoptosis and DNA damage repair. In particular, the role of PARP activation in response to DNA damage has been intensely studied. BRCA mutated tumors are highly sensitive to DNA breaks and are therefore critically dependent on alternative repair mechanisms involving PARP. This forms the basis for a synthetic lethal therapeutic approach that has been met with much success in the clinic. BRCA mutated breast and ovarian tumors, in particular, are acutely sensitive to PARP inhibitors. Currently, it is unclear from in vitro and clinical studies to what extent PARP inhibitors can be used as chemotherapeutics to treat tumors beyond those harboring BRCA mutations. Due to the insensitivity of most solid tumors to PARP inhibitors, we have begun to study the role of the dePARylating enzyme poly(ADP-ribose)glycohydrolase (PARG), as a possible target in cancer therapy. PARG, like PARP, maintains a critical role in DNA repair and can therefore be a target in cells with defective repair mechanisms (e.g. BRCA mutant cells). We further hypothesize that targeting this factor may activate the expression of tumor suppressor genes, thus limiting cell proliferation. Thus far, we have novel data indicating that targeting PARG may indeed have therapeutic benefit. We show that PARG knockdowns of the T47D and MDA-MB-468 breast cancer cell lines have dramatically reduced rates of cellular proliferation. Gene expression analyses of the knockdown cells indicated that cell cycle related pathways may be affected. As such, we examined cell cycle profiles by BrdU and PI staining and found that PARG knockdown cells were arrested in S phase. These results are particularly striking given that these cells show resistance to PARP inhibition. Subsequent studies using the PARG inhibitor gallotannin successfully reconstituted the results obtained with the PARG knockdowns. Overall, our data illustrates that PARG has great potential as a new anti-cancer target. Citation Format: Amanda Lovato, Tiejun Zhao, Qiang Sun, Michael Witcher. Poly(ADP-ribose)glycohydrolase (PARG) is a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2013-4378