The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.
Age is the greatest risk factor for cardiovascular disease. In addition, inflammation and age (senescence) have been linked at both the clinical and molecular levels. In general, senescent cells have been described as pro-inflammatory based on their senescence associated secretory phenotype (SASP). However, we have previously shown that senescence induced by overexpression of SENEX (or ARHGAP18), in endothelial cells results in an anti-inflammatory phenotype. We have investigated, at the individual cellular level, the senescent phenotype of endothelial cells following three of the chief signals associated with ageing; oxidative stress, disturbed flow and hypoxia. All three stimuli induce senescence and, based on neutrophil adhesion and expression of the adhesion molecules E-selectin and VCAM-1, a population of senescent cells is seen that is resistant to inflammatory stimuli and thus we define as anti-inflammatory. The proportion of anti-inflammatory cells increases with time but remains stable at approximately 50% by eight days after induction of senescence, suggesting that these are stable phenotypes of endothelial cell senescence. Similar to other senescent cell types, p38MAPK blockade inhibits the development of the pro-inflammatory phenotype but unique to EC, there is a corresponding increase in the number of anti-inflammatory senescent cells. Thus stress-induced senescent endothelial cells display a mosaic of inflammatory phenotypes. The anti-inflammatory population suggests that senescent endothelial cells may have an unique protective role, to inhibit uncontrolled proliferation and to limit the local inflammatory response.
Localization of a regulator of RhoGTPases (ARHGAP18) is important for microtubule stability and endothelial cell function. The localization is demonstrated by advanced imaging and biochemical techniques.
5561 Background: The REZOLVE clinical trial investigated the effect of administering bevacizumab via the intraperitoneal route to reduce re-accumulation of ascites in patients with ovarian cancer who were not suitable for further chemotherapy. Plasma and ascites were collected throughout for translational research. Ascites contains cell-free DNA (cfDNA), a large proportion of which is tumour derived (ctDNA). There is increasing interest in cfDNA in plasma, yet little is known of it in ascites. Objectives: To compare cfDNA in ascites and plasma in terms of total concentration, tumour proportion and endothelial-cell derived (ec-cfDNA) proportion and investigate their association with clinical outcomes (paracentesis-free interval, overall survival) and CA125 level. Methods: Longitudinal plasma and ascites samples were collected from 20/24 participants and stored at -70°C for up to 8.5 years. cfDNA was extracted from 0.3-1 mL fluid using conventional protocols. Standard and methylation-specific PCR was used to measure total cfDNA, ctDNA and ec-cfDNA. Values were correlated with time to paracentesis pre- and post-bevacizumab treatment (the primary trial outcome) as well as overall survival, using log-rank tests. Relationships with clinical CA125 levels were tested by Pearson’s correlation coefficient. Comparisons between plasma and ascites used non-parametric analyses. Results: cfDNA was detected in all samples, with higher yield in ascites (average 669 ng/mL) than plasma (average 75 ng/mL, p<0.0001). ctDNA was detected in 30/32 (94%) ascites samples and 37/56 (68%) plasma samples. ctDNA was detected in plasma and/or ascites from each patient at at least 1 time point. ctDNA proportion was higher in ascites than plasma (p<0.0001) and ec-cfDNA proportion was higher in plasma than ascites (p=0.002). High ctDNA (>75%) in ascites at baseline was associated with significantly shorter paracentesis-free interval (median interval 47.5 versus 84 days, hazard ratio (HR) 2.21, 95% confidence interval (CI) 0.85 to 5.73, p=0.039). ctDNA presence in plasma at baseline was unfavourable for survival (median survival 56 versus 242 days, HR 3.21, 95% CI 1.15 to 9.00, p=0.008). A significant positive correlation was observed between ctDNA proportion in plasma and CA125 level measured within 7 days (p=0.0006). No difference in total cfDNA, ctDNA nor ec-cfDNA was observed between participants who were bevacizumab responders and non-responders in the trial. Conclusions: Sufficient cfDNA was obtained from plasma and ascites to perform qPCR for three biomarkers. Ascites was found to contain proportionately more ctDNA, while plasma contained more ec-cfDNA. The early evidence presented here supports the potential value of cfDNA biomarkers in plasma and ascites, however incorporation of their collection in future clinical trials will allow further investigation.
e15079 Background: Homologous recombination deficiency (HRD) caused by germline BRCA1/2 mutations (gBRCAm) sensitize high grade serous ovarian cancers (HGSOC) and TNBC to PARP inhibitors (PARPi) such as olaparib. Loss of function of BRCA1 or RAD51C, due to promoter methylation or mutation of non-BRCA HR genes may also confer sensitivity to PARPi. This investigator-initiated, proof-of-concept trial evaluated olaparib in platinum(Pt)-sensitive, relapsed HGSOC and metastatic TNBC, with HRD due to mechanisms other than gBRCAm. EMBRACE is the first trial evaluating PARPi in loss of function of BRCA1/ RAD51C by promoter methylation. Methods: Single-arm, phase 2 trial of olaparib 300 mg orally twice daily in adults with metastatic TNBC or Pt-sensitive relapsed HGSOC and HRD due to mechanisms other than gBRCAm. 1 line of prior Pt-chemotherapy was allowed (HGSOC); additional lines of non-Pt therapy were allowed (TNBC). Tumor BRCA1/RAD51C promoter methylation was determined by methylation-sensitive high-resolution melting PCR and next generation sequencing (NGS, Pillar Biosciences BRCA1 & RAD51C Methylation test, high methylation threshold 70%). Unmethylated cases underwent NGS HR gene mutation testing (Pillar Biosciences HRD test, paired tumor/normal). The primary outcome was objective tumor response rate (OTRR) at 6 months(m). Secondary objectives: progression-free survival (PFS), OTRR according to HR gene mutation type, safety. Tertiary: associations between biomarkers and clinical outcomes. Results: 22 participants (15 HGSOC, 7 TNBC) enrolled from 208 screened (Sep 2018 - Mar 2022). Methylation was detected in 8/15 HGSOC (all BRCA1) and 5/7 TNBC (4 BRCA1, 1 RAD51C). Both cohorts included mutations in gBRIP1 (1), gPALB2 (1), gRAD51C (3), sBRCA1 (2), with 1 COSMIC mutational signature 3, and 1 positive HRD score in HGSOC. OTRR at 6m was 40% in HGSOC (6/15: 5 partial, 1 complete), and 0% in TNBC (0/7). OTRR was 38% (3/8) in methylated tumors vs 43% (4/7) with other HRD. Stable disease at 6m was 47% (7) in HGSOC and 43% (3) in TNBC durations were 7m in HGSOC and 3m in TNBC. PFS was 53% at 6m and 25% at 12m in HGSOC, vs 17% at 6m and NE at 12m in TNBC. TNBC 2-3 prior lines of therapy. Conclusions: Olaparib demonstrated clinically relevant activity in HGSOC without gBRCAm. Objective responses were seen in HGSOC with methylated BRCA1. Olaparib had limited activity in heavily pre-treated TNBC. Further research is needed into the effects of non-Pt chemotherapy on methylation of BRCA1/RAD51C to improve selection of HGSOC and TNBC for PARPi. (ANZCTRN12617000855325) Acknowledgements: Funding from Cancer Australia and the NBCF(PS-15-048), provision of study drug plus an untied educational grant from AstraZeneca and its group of companies. EMBRACE was led by the NHMRC CTC, University of Sydney in collaboration with ANZGOG, BCT-ANZ, and GCCTI. Clinical trial information: ACTRN12617000855325 .
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