Cell-free DNA in plasma and ascites as a biomarker of bevacizumab response- a translational research sub-study of the REZOLVE (ANZGOG-1101) clinical trial.

OBJECTIVE: To investigate cell-free DNA (cfDNA) in plasma and ascites and its association with clinical outcomes (paracentesis-free interval, overall survival) and CA125 level in participants with advanced ovarian cancer, treated with palliative intraperitoneal bevacizumab to delay re-accumulation of ascites. METHODS: cfDNA was extracted from 0.3 to 1 mL samples from 20/24 participants of the REZOLVE trial. Standard and methylation-specific PCRs were performed to measure 3 biomarkers: total cfDNA (Alu), tumour-derived cfDNA (ctDNA, methylated IFFO1 promoter) and endothelium-derived cfDNA (ec-cfDNA, unmethylated CDH5 promoter). Values were correlated to clinical outcomes. RESULTS: cfDNA was detected in all samples, with higher yield in ascites (mean 669 ng/mL) than plasma (mean 75 ng/mL, p < 0.0001). Ascites had a higher ctDNA proportion than plasma (74 % vs. 20 %, p < 0.0001) and plasma had a higher ec-cfDNA proportion than ascites (24 % vs. 16 %, p < 0.002). High ctDNA proportion (>75 %) in ascites was associated with a 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) and ctDNA presence in plasma 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 (p = 0.012). No significant difference in total cfDNA, ctDNA nor ec-cfDNA was observed between participants who were responders versus non-responders. CONCLUSION: Sufficient cfDNA was detected in both plasma and ascites to study three biomarkers. These samples can provide useful information and should be considered in the design of future ovarian cancer trials.

The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells.

RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.

ARHGAP18: an endogenous inhibitor of angiogenesis, limiting tip formation and stabilizing junctions.

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-associated stresses induce an anti-inflammatory senescent phenotype in endothelial cells.

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 ofSENEX (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.