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pARylation-mediated regulation of cancer pathways

Final Report Summary - PARYLOME (pARylation-mediated regulation of cancer pathways)

Final Report

Cancer is one of the leading causes of death worldwide. One approach to addressing this issue is to identify novel targets that selectively inhibit the growth and/or behaviour of tumour cells. One of the more promising advances in this field was the discovery in 2005 that small molecule inhibitors of the DNA repair proteins PARP1 and 2 (Poly (ADP-ribose) Polymerase 1 and 2) selectively targeted tumour cells with BRCA1 or BRCA2 gene defects. This observation not only highlighted the clinical potential of PARP inhibitors but also suggested that other enzymes with a similar function could be used as therapeutic targets in cancer.

PARP1 and 2 are members of the Poly (ADP-ribose) Polymerase superfamily of enzymes that utilise β NAD+ as a substrate to polymerise the synthesis of Poly (ADP-ribose) (PAR) chains on target proteins (PARylation). This post translational modification can, depending upon the specific target in question, result in alterations in protein stability, localisation and protein/protein interactions. The majority of small molecule PARP1/2 inhibitors act as β NAD+ mimics, preventing the formation of PAR chains. In addition to inhibitors of PARP1/2, small molecule inhibitors of another PARP superfamily member, PARP5 (also known as Tankyrase) have also been proposed as cancer therapeutics.

Despite the rapid progression of PARP1/2 inhibitor into the clinic, the range of protein substrates that are targeted for PARylation is poorly defined. It was our contention, that if additional targets of PARP superfamily enzymes could be identified, this could enhance our understanding of PARP biology, in both normal and cancerous conditions, and potentially could inform the more refined use of PARP1/2 and tankyrase inhibitors.

In order to identify novel PARylated proteins, (i.e. targets of PARP superfamily enzymes), we carried out immunoaffinity purification of ADP-ribose (ADPr) bound proteins, and identified these proteins using quantitative mass spectrometry. Specifically, we purified proteins from tumour cell lysates using anti-mono- and poly-ADPr specific antibodies and used SILAC (Stable Isotope Labeling by Amino acids in cell Culture) based LC-MS/MS to identify and quantify ADPr bound proteins.

Using this approach, we have generated proteomic maps of PARylation targets (the PARylome). We have complimented this approach by carrying out SILAC/MS on cell lysates exposed to either PARP1/2 or tankyrase inhibitors. One interesting observation has been that many proteins, once PARylated, become targets from proteosomal degredation. The exposure of cells to PARP1/2 or tankyrase inhibitors thus causes an increase in the levels of a number of PARP1/2 or tankyrase substrates as they are no longer PARylated and degraded. By integrating the PARylome data with the list of proteins whose expression is enhanced by PARP1/2 or tankyrase inhibitor exposure, we have been able to highlight those PARylation targets that are likely substrates for proteosomal degredation.

One of the novel observations from these studies has been the identification of Sjogren Syndrome/Scleroderma Autoantigen 1 (SSSCA1), as a PARylated protein. SSSCA1 has been proposed as a candidate autoimmune antigen, but in general its function is poorly understood. At present we are investigating the cellular consequences of SSSCA1 PARylation as well as investigating which PARP superfamily member is responsible for SSSCA1 PARylation. The integration of the PARylome data with SILAC profiles from tankyrase inhibitor treated cells has also identified a number of proteins involved in cellular focal adhesion. Again, we are following this observation, and investigating how tankyrase modulates the interaction between cells, and how this might be exploited in the treatment of cancer and diseases such as fibrosis.