José Manuel Rodríguez-Vargas

PARP-1 is involved in autophagy induced by DNA damage

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. PARP-1 has been implicated in different pathways leading to cell death and its inhibition potentiates chemotherapy-induced cell death. Whether PARP-1 participates in the cell’s decision to commit to autophagy following DNA damage is still not known. To address this issue PARP-1 wild type and deficient cells have been treated with a dose of doxorubicin that induces autophagy. Electron microscopy examination and GFP-LC3 transfection revealed autophagic vesicles and increased expression of genes involved in autophagy (bnip-3, cathepsin b and l and beclin-1) in wild type cells treated with doxo but not in parp-1-/- cells or cells treated with a PARP inhibitor. Mechanistically the lack of autophagic features in PARP-1 deficient/PARP inhibited cells is attributed to prevention of ATP and NAD+ depletion and to the activation of the key autophagy regulator mTOR. Pharmacological or genetical inhibition of autophagy results in increased cell death, suggesting a protective role of autophagy induced by doxorubicin. These results suggest that autophagy might be cytoprotective during the response to DNA damage and suggest that PARP-1 activation is involved in the cell’s decision to undergo autophagy.

PARP inhibitors: New partners in the therapy of cancer and inflammatory diseases

Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD(+) to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, which currently comprises 18 members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress. PARP is involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Inhibitors of PARP-1 have been shown to enhance the cytotoxic effects of ionizing radiation and DNA-damaging chemotherapy agents, such as the methylating agents and topoisomerase I inhibitors. There are currently at least five PARP inhibitors in clinical trial development. Recent in vitro and in vivo evidence suggests that PARP inhibitors could be used not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in the breast cancer-associated genes (BRCA1 and BRCA2). PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes and promotes an inflammatory response associated with multiple organ failure. Inhibition of PARP activity is protective in a wide range of inflammatory and ischemia-reperfusion-associated diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. The aim of this review is to overview the emerging data in the literature showing the role of PARP in the pathogenesis of cancer and inflammatory diseases and unravel the solid body of literature that supports the view that PARP is an important target for therapeutic intervention in critical illness.

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.

Modulation of Transcription by PARP-1: Consequences in Carcinogenesis and Inflammation

Post-translational modification of proteins by poly(ADP-ribosyl)ation is involved in the regulation of a number of biological functions. While an 18 member superfamily of poly(ADP-ribose) polymerases (PARP)s has been described PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells. PARP-1 act as a DNA nick sensor and is activated by DNA breaks to cleave NAD(+) into nicotinamide and ADP-ribose to synthesize long branching poly(ADP-ribose) polymers (PAR) covalently attached to nuclear acceptor proteins. Whereas activation of PARP-1 by mild genotoxic stimuli facilitate DNA repair and cell survival, severe DNA damage triggers different pathways of cell death including PARP-mediated cell death through the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. PAR and PARP-1 have also been described as having a function in transcriptional regulation through their ability to modify chromatin-associated proteins and as a cofactor of different transcription factors, most notably NF-kappaB and AP-1. Pharmacological inhibition or genetic ablation of PARP-1 not only provided remarkable protection from tissue injury in various oxidative stress-related disease models but it result in a clear benefit in the treatment of cancer by different mechanisms including selective killing of homologous recombination-deficient tumor cells, down regulation of tumor-related gene expression and decrease in the apoptotic threshold in the co-treatment with chemo and radiotherapy. We will summarize in this review the current findings and concepts for the role of PARP-1 and poly(ADP-ribosyl)ation in the regulation of transcription, oxidative stress and carcinogenesis.

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.

The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria‐mediated apoptosis and DNA damage in p53 mutant leukemic T cells

DNA methyltransferase (DNMT)‐inhibiting nucleoside analogs reactivate the expression of tumor suppressor genes and apoptosis‐related genes silenced by methylation, thus favoring the induction of apoptosis in tumor cells. Moreover, induction of DNA damage seems to contribute to their antitumor effect. However, the apoptotic signaling pathway induced by these demethylating drugs is not well understood. Here, we have investigated the induction of apoptosis by two nucleoside DNMT inhibitors, decitabine and zebularine, in leukemic T cells. Both inhibitors induced caspase‐dependent apoptosis in Jurkat, CEM‐6 and MOLT‐4 leukemia T cell lines, all with mutant p53, whereas resting and activated normal T lymphocytes were highly resistant to these demethylating agents. Although decitabine and zebularine showed different ability to induce apoptosis and cell cycle arrest among the three cell lines, they similarly activated the intrinsic apoptotic pathway by inducing mitochondrial alterations such as Bak activation, loss of transmembrane potential and generation of reactive oxygen species (ROS). Accordingly, Bcl‐2‐ and Bcl‐xL‐overexpressing Jurkat cells, as well as caspase‐9‐deficient Jurkat cells, were resistant to apoptosis induced by decitabine and zebularine. Interestingly, ROS production seemed to be necessary for the induction of apoptosis. Apoptotic events, such as Bak and caspase activation, started as soon as 20 hr after treatment with either decitabine or zebularine. In addition, progression of apoptosis triggered by both DNMT inhibitors was paralleled by the induction of DNA damage. Our results suggest that the mitochondrial apoptotic pathway activated by decitabine and zebularine in p53 mutant leukemic T cells depends mainly on the induction of DNA damage.

PARP3 controls TGFβ and ROS driven epithelial-to-mesenchymal transition and stemness by stimulating a TG2-Snail-E-cadherin axis

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFβ and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFβ-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFβ-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFβ-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFβ-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export

AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation.

Role of Poly(ADP-Ribose) in Catalyzing Starvation-Induced Autophagy

Poly(ADP-ribosyl)ation (PARylation) is a reversible posttranslational modification of proteins mediated by poly(ADP-ribose) polymerases (PARP proteins, 17 members). Most of the studies developed over the last decades have essentially focused on the biochemical, physiological, and pathological properties of the founding members of the PARP family, PARP-1, PARP-2, and PARP-3. PARylation is carried out by the balance of poly(ADP-ribose) (PAR) synthesis enzymes (PARPs) and PAR decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions (transcription regulation, replication, organization of chromatin domains, genome stability, cell cycle progression, differentiation, and metabolism and cell survival pathways) but uncontrolled PARP activation (overproduction of PAR) may also lead to cell death. PARylation has been implicated in apoptosis, necroptosis, and autophagic cell death (or type II programmed cell death). Macroautophagy (hereafter called autophagy) is an evolutionarily conserved pathway of lysosomes-mediated cellular self-digestion involving the formation of a double-membrane vesicle, the autophagosome, which engulfs cytoplasmic components and delivers them to the lysosomes for degradation. Starvation alarms eukaryotic cells to adjust metabolism to survive. PARP-1 activation is involved in amplifying autophagy by feeding back reactive species of oxygen production/DNA damage/NAD+ consumption axis, leading to AMPK activation/mTORC1 inhibition and subsequently opening autophagy. In the current review we focus on the contribution of PARP and PARylation, as a cellular process involved in cell’s energy homeostasis and posttranslational protein modification, as modulator of autophagy after nutrient deprivation.