Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

[Background] The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded.[Methods] In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes.[Results] In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction.[Conclusions] These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.This work was supported by Junta de Andalucía, project of Excellence from Junta de Andalucía P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520- R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO. EB1s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundación Domingo Martínez (call 2019).Peer reviewe

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

This work was supported by Junta de Andalucia, project of Excellence from Junta de Andalucia P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011C02-01, SAF2015-70520-R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cancer ISCIII CB16/12/00421 to FJO. EB1s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundacion Domingo Martinez (call 2019).We would like to acknowledge Laura L´opez for technical assistance; Eduardo Andr´es and Laura Terr´on (Bioinformatic core IPBLN, CSIC) and Pan Hui (Bioinformatic Core, Joslin Diabetes center, Harvard Medical School).Background: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/ activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. Methods: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. Results: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. Conclusions: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α overactivation.Junta de Andalucia P10-CTS-0662 P12-CTS-38Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01 SAF2015-70520- R RTI2018-098968-B-I00 RTICC RD12/0036/0026CIBER Cancer ISCIII CB16/12/00421Basque Department of Industry, Tourism and Trade (Etortek)MINECO CB16/12/00421Fundacion Domingo Martine

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

Background: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/ activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. Methods: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. Results: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. Conclusions: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α overactivation.Junta de AndalucíaMinisterio de Economía. Españ

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

Rodríguez-Vargas, José Manuel et al.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.ES was supported by Newcastle University's Institute of Neuroscience and MS performed this work as part of her degree in Newcastle University's MRes Programme in Medical and Molecular Biosciences.Peer Reviewe

Interaction between PARP-1 and HIF-2 in the hypoxic response

et al.Hypoxia-inducible factors (HIFs) mediate the transcriptional adaptation of hypoxic cells. The extensive transcriptional programm regulated by HIFs involves the induction of genes controlling angiogenesis, cellular metabolism, cell growth, metastasis, apoptosis, extracellular matrix remodeling and others. HIF is a heterodimer of HIF- and HIF-β subunits. In addition to HIF-1, HIF-2 has evolved as an isoform that contributes differently to the hypoxic adaptation by performing non-redundant functions. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein involved in the control of DNA repair and gene transcription by modulating chromatin structure and acting as part of gene-specific enhancer/promoter-binding complexes. Previous results have shown that PARP-1 regulates HIF-1 activity. In this study, we focused on the cross-talk between HIF-2 and PARP-1. By using different approaches to suppress PARP-1, we show that HIF-2 mRNA expression, protein levels and HIF-2-dependent gene expression, such as ANGPTL4 and erythropoietin (EPO), are regulated by PARP-1. This regulation occurs at both the transcriptional and post-trancriptional level. We also show a complex formation between HIF-2 with PARP-1. This complex is sensitive to PARP inhibition and seems to protect against the von Hippel-Lindau-dependent HIF-2 degradation. Finally, we show that parp-1-/-mice display a significant reduction in the circulating hypoxia-induced EPO levels, number of red cells and hemoglobin concentration. Altogether, these results reveal a complex functional interaction between PARP-1 and the HIF system and suggest that PARP-1 is involved in the fine tuning of the HIF-mediated hypoxic response in vivo.This work was supported by Ministerio de Ciencia e Innovación (SAF2006-01094 and SAF2009-13281-C02-01), Fundación La Caixa (BM06-219-0) and Junta de Andalucía (P07-CTS-0239) to FJO; Ministerio de Educación y Ciencia (SAF2007-64597 and SAF-2010-20067) and the BIZKAIA XEDE Program from the Bizkaia County to EB.Peer Reviewe

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

Background: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expres sion/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. Methods: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. Results: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. Conclusions: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over activationThis work was supported by Junta de Andalucía, project of Excel lence from Junta de Andalucía P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011- C02-01, SAF2015-70520- R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO. EB1 s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundación Domingo Martínez (call 2019).Ye

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.This work was supported by Ministerio de Ciencia e Innovación SAF2006-01094, SAF2009-13281-C02-01, Fundación La Caixa BM06-219-0 and Junta de Andalucía P07-CTS-0239 and CTS-6602 to FJO, Ministerio de Educación y Ciencia SAF2007-64597; CICYT: SAF2009-13281-C02-02; Junta de Andalucía, P06-CTS-01385 to JMRdA and grants CEIC (P10-CTS5865) and FEDER-ISCIII (PI10/00883) to JCR-M. AGdH has been funded by grants from ‘‘Fundación Científica de la Asociación Española Contra el Cáncer’’, Ministerio de Ciencia y Tecnología SAF2010-16089, and ‘‘Fundación La Marató de TV3’’. JCR-M has been funded by Grants CEIC (P1=-CTS5865) and FEDER-ISCIII (PI10/00883). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptBaulida and García de Herreros' labs is supported by the Instituto de Salud Carlos III (PI12/ 00257 and D012/0036/0005, part of the Plan Nacional I+D+ I and cofounded by the ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional-FEDER), the Fundación Científica Asociación Española Contra el Cáncer (Ayudas a grupos estables de investigación, 2010-1) and the Ministerio de Economía y Competitividad (SAF2013-4889-C2-1R)

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