Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-XL as a candidate apoptotic binding partner for GRASP65

Abstract S4-2: Targeted intraoperative radiotherapy for early breast cancer: TARGIT-A trial- updated analysis of local recurrence and first analysis of survival

Abstract Background TARGIT-A, an international phase 3 randomised trial (Lancet 2010;376:91–102) compared outcomes in patients undergoing breast conserving surgery followed by either whole breast external beam radiotherapy (EBRT) over several weeks, or a risk-adaptive approach using single dose targeted intra-operative radiotherapy (TARGIT). Risk-adaptive approach meant that if the final pathology report demonstrated unpredicted pre-specified adverse features, then EBRT was to be added to TARGIT. Method 3451 women aged 45 years or older with invasive ductal carcinoma were enrolled from 33 centres in 10 countries between 2000 and 2012. Randomisation to TARGIT or EBRT arm was done either before lumpectomy (pre-pathology) or after lumpectomy (postpathology). If allocated to TARGIT, patients in the pre-pathology group received it immediately after surgical excision under the same anaesthesia; patients in the post-pathology group received it as a subsequent procedure. We pre-specified that analysis would be performed overall as the primary analysis and for these groups separately as a secondary analysis. The primary outcome was ipsilateral within breast recurrence (IBR) with an absolute non-inferiority margin of 2.5% at 5 years and secondary outcome was survival. We performed exploratory analyses for loco-regional recurrence, ‘all recurrence’ (ipsilateral or contralateral breast, axilla or distant), distant recurrence, and causes of death. Results 1721 patients were randomly allocated to receive TARGIT and 1730 to EBRT. 1010 patients have a minimum 4 years follow up and 611 patients have minimum 5 years follow up. Primary events have increased from 13 to 34 since 2010. For the primary outcome of ipsilateral breast recurrence, the absolute difference at 5-years was 2.0%, which was higher with TARGIT and reached the conventional levels of statistical significance (p = 0.042), but was within the pre-specified non-inferiority margin; in prepathology the absolute difference in 5-year IBR was 1%; in postpathology it was 3.7%. For the secondary outcome, there was a non-significant trend for improved overall survival with TARGIT (HR = 0.70(0.46–1.07)) due to fewer non-breast cancer deaths (17 vs. 35, HR 0.47 (0.26–0.84)). Cardiovascular deaths were 1 vs. 10 and deaths from cancers other than breast were 7 vs.16. Conclusion The risk-adapted approach using single dose TARGIT has a slightly higher local recurrence rate than EBRT for the primary endpoint of IBR, but was within the preset non-inferiority boundary, with the prepathology apparently performing better than the postpathology stratum. In addition there was a trend for improved overall survival in the TARGIT arm due to fewer non-breast cancer deaths. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S4-2.</jats:p