Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Characterization of PARP-3 in genomic stability and epithelial plasticity. – PARP-3

Role of PARP-3 in genome integrity and epithelial plasticity

Our objectives are aimed to decipher the role of PARP-3 in genome integrity and determine the molecular mechanisms involved.

Is PARP-3 a new target in cancer therapy ?

Poly(ADP-ribosyl)ation is a post-translational modification of proteins with key functions in the monitoring and maintenance of genome integrity. This enzymatic reaction was initially discovered by Pierre Chambon in Strasbourg, in the lab of Paul Mandel almost 50 years ago. Very rapidly, the therapeutic value of inhibiting this activity appeared as promising to potentiate the cytotoxic or antiproliferative effects of chemotherapeutic drugs or radiation therapy. Poly (ADP-ribose) polymerases (PARPs) catalyse poly (ADP-ribose) synthesis onto various acceptor proteins and form a family of 17 members, many of which have been identified in the laboratory. So far, only PARP-1 and PARP-2 were shown to be directly involved in the maintenance of genome integrity and DNA repair. By combining biochemical and structural approaches with the development and characterization of loss of function mutant mice, our laboratory has largely contributed to determine the function of PARP-1 and PARP-2 in these processes and have promoted the development of PARP chemical inhibitors for therapeutic purposes. Many laboratories and pharmaceutical companies are now engaged in the development of these molecules to increase chemo-or radiotherapy but also to target various cancers (breast, ovarian, prostate) with a BRCA1 or BRCA2 mutation (clinical trials Phase I-III). Our laboratory now focuses his research on the characterization of the other PARP family members including the poorly characterized PARP-3. Our goals are to dissect the contribution of this protein in genome integrity, cell proliferation and cell plasticity.

To explore the biochemical properties of the protein, we express the protein in large amounts in insect cells and purify it. Its enzymatic properties (synthesis of poly (ADP-ribose)) are studied in vitro. Protein interactions are identified by immunoprecipitation of the protein using a specific antibody and determination of the protein partners by mass spectrometry. To evaluate the functional properties of PARP-3, we generated a loss of function mouse model in which the protein is not expressed, and we analyse the phenotype. We also use human cell lines in which PARP-3 expression is depleted using siRNA aproches and we explore the consequences of this depletion on a set of cellular processes.

Within the questions already developed, we have identified PARP-3 as a new player in cell response to double strand breaks and mitotic progression. PARP-3 is recruited to sites of DNA damage. Its absence sensitizes human cells to ionizing radiation and causes a significant delay in mitotic division. Our recent results define PARP-3 as a new actor in double-strand break repair by nonhomologous end-joining.
We have also demonstrated an important role of this protein in the repair of physiological strand breaks induced during lymphocyte maturation. Finally, initial data suggest a role of this protein in epithelial plasticity. Together, our results introduce PARP-3 as an important player in tumorigenesis and suggest that its inhibition might be of therapeutic benefit.

We continue this work by dissecting the role of PARP-3 in non-homologous end-joining. We study the consequences of its absence in lymphocyte maturation and especially V(D)J recombination. We explore the impact of its expression in epithelial plasticity. Finally, we study the consequences of its depletion in human tumor cell lines.

Our current results defining PARP-3 as a new player in cell response to double-strand breaks and cell division (mitosis) have been published in PNAS (2011), Cell Cycle (2011) and as a brief «Live from the Labs« at the CNRS.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins mediated by Poly(ADP-ribose) polymerases (PARPs), which catalyse the transfer and polymerisation of ADP-ribose units from NAD+ to form a ramified poly(ADP-ribose) polymer (PAR), covalently linked to acceptor proteins. DNA breaks are considered to be the major trigger of PAR synthesis through the activation of the founding member PARP-1, favoring DNA repair. PARP-1 inhibitors appeared to have promising pharmacological applications, to potentialize the antitumor effect of chimio- and radiotherapies (phase 1 to 3 clinical trials in progress). The recent discovery of a PARP family (17 members) and of inducers of PAR synthesis other than DNA strand breaks underline the potential of this molecule to control an increasing number of various biological functions including the cellular response to DNA damage, transcriptional regulation, cell cycle progression, cell differentiation, cell death, intracellular transport, lipid metabolism and tumorigenesis [1, 2]. In addition, recent studies revealed that whereas some PARP family members catalyze poly(ADP-ribosyl)ation, others function as mono(ADP-ribosyl)transferase [3]. Together, these findings support the necessity to biochemically and physiologically define the properties of individual PARPs.
By combining biochemical, structural and genetic approaches, our laboratory has extensively contributed to describe PARP-1 and PARP-2 as active players of the base excision repair/single strand break repair (BER/SSBR) process and have discovered both cooperative and more specific functions of both enzymes in heterochromatin integrity.
Recently, we and others have identified PARP-3 as another DNA-dependent PARP but its enzymatic and functional characterization is still poorly documented [4, 5]. The initial studies identified a centrosomal PARP-3 involved in cell cycle progression and a nuclear isoform involved in transcriptional silencing and in the cellular response to DNA damage [4, 6]. Our preliminary cellular evidences indicate a contribution of PARP-3 in the maintenance of genome integrity and identify a significant increased expression of PARP-3 during the processes of epithelial to mesenchymal transition (EMT), two hallmarks of cancer biology. In this proposal, we combine three distinct expertises to determine the biochemical specifics and functional properties of PARP-3 in cellular respose to DNA damage and in epithelial plasticity. By in vitro approaches, we will identify the DNA structures recognized by and activating PARP-3, define its enzymatic properties and identify its partners. We will generate and characterize PARP-3-deficient cellular and mouse models to explore its role in cellular response to DNA damage and in cell division. We have engineered a PARP-3 depleted breast cancer cell line to examine its contribution in epithelial to mesenchymal transition and in cancer invasiveness. Altogether, we believe that our results will define whether PARP-3, like PARP-1, can constitute a novel possible therapeutic target for cancer treatment.

[1] A. Hakme et al. EMBO reports 9 (2008) 1094-1100.
[2] V. Schreiber et al. Nat Rev Mol Cell Biol 7 (2006) 517-528.
[3] H. Kleine et al. Mol Cell 32 (2008) 57-69.
[4] A. Augustin et al. J Cell Sci 116 (2003) 1551-1562.
[5] M. Johansson et al. Genomics 57 (1999) 442-445.
[6] M. Rouleau et al. J Cell Biochem 100 (2007) 385-401.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 458,240 euros
Beginning and duration of the scientific project: - 36 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter