RhoGAP Research Articles

ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions, cell migration, and invasion.

The Rho family of GTPases are inactivated in a cell context–dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, one of the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 and overexpressing its two mutually independent upstream regulators, SEPT2 and SEPT9, all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion–associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the up-regulated Integrin Beta 1. These results provide novel insights into cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.

Novel pathogenic OCRL mutations and genotype\u2013phenotype analysis of Chinese children affected by oculocerebrorenal syndrome: two cases and a literature review

BackgroundOculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by congenital cataracts, mental retardation, and proximal tubulopathy. This condition is caused by a mutation of OCRL gene (located at chromosome Xq26.1), which encodes an inositol polyphosphate 5-phosphatase.Case presentationWe identified two novel OCRL mutations in two unrelated Chinese boys, each with a severe phenotype of Lowe syndrome. A novel de novo deletion (hemizygous c.659_662delAGGG, p.E220Vfs*29) was present in patient 1 and a novel splicing mutation (hemizygous c.2257-2A > T) that was maternally inherited was present in patient 2. A renal biopsy in patient 2 indicated mild mesangial proliferative glomerulonephritis, mild focal mononuclear cells infiltration, and interstitial focal fibrosis. Moreover, renal expression of OCRL-1 protein in patient 2 was significantly reduced compared to a control patient with thin basement membrane disease.ConclusionsThis study reports two novel OCRL variants associated with severe ocular and neurologic deficiency, despite only mild renal dysfunction. Based on our two patients and a literature review, the genotype–phenotype correlation of OCRL mutations with this severe phenotype of Lowe syndrome suggest a possible clustering of missense, deletion, and nonsense mutations in the 5-phosphatase domain and Rho-GAP domain in the Chinese population.

Genome-Wide Association Study (GWAS) of High-Risk Minimal Residual Disease and Relapse in B-Cell Acute Lymphoblastic Leukemia on Trial AIEOP-BFM ALL 2000

▪A significant proportion of patients with pediatric acute lymphoblastic leukemia (ALL) still suffers from relapse and therapy-related toxicities. Thus, the focus of further improvement of therapy for them is not only cure, but minimizing both short-term and long-term therapy-associated toxicity. This can be achieved by better personalized adjustment of therapy to the risk of relapse. Continuing research has identified numerous features with prognostic potential, including clinical and genetic variables as well as early response to chemotherapy being associated with treatment outcome. While most research activities on genetic prognostic factors have focused on somatic features, relatively little is known on hereditary contributions to treatment response and outcome in pediatric ALL. Therefore, we conducted a genome-wide association study (GWAS) of high-risk minimal residual disease (MRD) and relapse in B cell precursor (BCP) ALL patients treated on trial AIEOP-BFM ALL 2000.On this trial, patients were stratified into three risk groups, mainly by MRD analyses. MRD standard-risk (MRD-SR) patients were MRD-negative on treatment days 33 and 78, MRD high-risk (MRD-HR) patients had residual disease of ≥5x10-4 on treatment day 78. All the remaining MRD results were considered MRD intermediate-risk (MRD-IR). Besides MRD, established high-risk parameters were also retained for stratification: prednisone poor-response, induction failure, positivity for t(4;11)/ MLL-AF4 . Treatment used standard drugs and, in some of the patients, cranial irradiation and/or hematopoietic stem cell transplantation. DNA isolated from remission samples was genotyped using Illumina Human1M-Duo BeadChips containing 1.048.711 single SNV markers. For GWAS analyses gPLINK v2.050, PLINK v.1.09 and RStatistics v3.3.1 were used. The analysis on MRD was conducted in 69 MRD-HR patients (cases) and in 1034 patients of the MRD-SR and MRD-IR groups (controls). The analysis on relapse employed 145 relapsed (cases) and 1007 non-relapsed patients (controls). After quality control assessment, the final analyses included 749.262 SNPs among 62 MRD-HR patients compared to 927 controls and 129 relapsed patients compared to 925 controls.In the analysis on MRD-HR five SNVs reached genome-wide significance at P ≤5×10−8. An intronic SNV (rs1570765) in STARD13 demonstrated the strongest association (P =1.73x10-10; odds ratio, OR=5.06). Two additional SNVs with P ≤5×10−8 in STARD13 were in linkage disequilibrium with rs1570765. The two remaining SNVs with P ≤5×10−8 were located in FEZ2 and NRIP1. Five additional SNVs demonstrated significant levels of ≤1×10−6 and were associatedwith the following genes: STARD13 , VIT and FICD . STARD13 is located on chromosome 13, belongs to the family of RhoGap proteins and is frequently lost in hepatocellular and breast carcinomas. Its RhoGAP domain activates members of the Ras superfamily which leads to a suppression of cell growth and differentiation (Lin et al. Oncogene 2010). In the GWAS approach on relapse three SNVs reached significant levels of ≤1×10−6 and were associatedwith the genes ASB3 (rs1549749, P =3.51x10-7; rs12465316, P =4.26x10-7) on chromosome 2 and NDN (rs9972595, P =1.04x10-6) on chromosome 15. Twenty additional SNVs demonstrated significance levels of ≤1×10−5. ASB3 codes for a member of the ankyrin repeat and SOCS box-containing (ASB) family of proteins containing ankyrin repeat sequence and a SOCS box domain. The latter serves to couple suppressor of cytokine signalling (SOCS) proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. NDN , a p53 target gene, codes for Necdin which was previously shown to regulate the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells (Asai et al., Blood 2012). Further replication results, an analysis of additive effects and multivariate analyses will be presented.In conclusion, our results demonstrate that germline genetic variation is associated with high levels of MRD after induction/consolidation treatment for ALL and relapse on an AIEOP-BFM ALL protocol. In line with previous studies (Yang et al., JAMA 2009), our data suggest that genome-wide approaches may be useful to identify novel and clinically relevant SNVs for further refinement of risk-adapted treatment strategies in pediatric ALL. DisclosuresSchrappe:JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Medac: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.

Biocomputational Analysis of ARAP1, a novel GTPase‐Activating‐Protein Involved in Osteoclast‐Mediated Bone Digestion

RhoGTPases are important regulators of many cellular activities including cell proliferation, trafficking of proteins, DNA transcription, and apoptosis. RhoGTPases are also involved in bone remodeling, the continuous process of bone digestion, and formation that occurs during one's lifespan. Any abnormalities in these Rho Proteins can lead to physiological effects such as osteoporosis, characterized by porous bone, or osteopetrosis, characterized by highly-dense bone tissue. RhoA-GTP is responsible for disrupting sealing zone dynamics and promoting cell motility in osteoclasts. On the other hand, Cdc42-GTP and Rac1-GTP maintain osteoclast attachment to the bone surface, thereby allowing it to continue enzymatic degradation of bone. To employ RhoGTPases as potential drug targets, we need to further understand the complex physiology and control mechanisms for these G-proteins. ARAP1 has been shown to localize to the osteoclast sealing zones upon the release of phosphoinositide (3,4,5) trisphosphate, which binds to its first and third PH domains. ARAP1 belongs to the RhoGAP family of proteins and has a RhoGAP domain responsible for catalyzing the intrinsic GTPase activity of G proteins. However, it is still unknown which GTPase (i.e. RhoA, Cdc42, Rac1) preferentially binds to the ARAP1 catalytic domain. Delineating the substrate specificity for the RhoGAP domain is crucial to its characterization as a pro-migration or pro-digestion protein, allowing us to specifically target its function in managing osteoporosis or osteopetrosis-related illnesses. In this computational study, we have modeled the ARAP1 RhoGAP domain using homology modeling and ab initio techniques, characterized its biophysical features, and used docking analysis to scrutinize its interactions with potential substrates. The docking results suggest that ARAP1 shows a preferential substrate specificity for RhoA. This indicates that ARAP1-RhoGAP may behave in a similar manner as ARAP3-RhoGAP, which has been shown to prefer RhoA-targeting in an experimental setup. Lastly, we report an in silico mutagenesis analysis that induces RhoA-like mutations in Cdc42/Rac1 and provides structural data that supports our hypothesis of an ARAP1-RhoA preferential attraction. Overall, this study reveals new insights into the structure-function relations of the RhoGAP domain, specifically pertaining to its role in osteoclast-mediated bone digestion, using an integrated and comprehensive strategy of sequence and structure analyses. The novel findings from our study regarding the molecular mechanism of GTPases in the osteoclast lays the groundwork towards developing therapies that improve the quality of life for those suffering from chronic bone pathologies.

HNRNPL Circularizes ARHGAP35 to Produce an Oncogenic Protein.

Circular RNAs (circRNAs) are an intriguing class of widely prevalent endogenous RNAs, the vast majority of which have not been characterized functionally. Here, we identified a novel oncogenic circRNA originating from the back‐splicing of Exon2 and Exon3 of a tumor suppressor gene, ARHGAP35 (also known as P190‐A), termed as circARHGAP35. have observe that circARHGAP35 and linear ARHGAP35 have antithetical expression and functions. Interestingly, circARHGAP35 contains a 3867 nt long ORF with an m6A‐modified start codon and encodes a truncated protein comprising four FF domains and lacking the Rho GAP domain. Mechanistically, circARHGAP35 protein promotes cancer cell progression by interacting with TFII‐I protein in the nucleus. The RNA binding protein, HNRNPL, facilitates the formation of circARHGAP35. Clinically, circARHGAP35 is associated with poor survival in cancer patients. Our findings characterize an oncogenic circRNA and demonstrate a novel mechanism of oncogene activation in cancer by circRNA through the production of a truncated protein.

Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a podocytopathy leading to kidney failure, whose molecular cause frequently remains unresolved. Here, we describe a rare MYO9A loss of function nonsense heterozygous mutation (p.Arg701∗) as a possible contributor to disease in a sibling pair with familial FSGS/proteinuria. MYO9A variants of uncertain significance were identified by whole exome sequencing in a cohort of 94 biopsy proven patients with FSGS. MYO9A is an unconventional myosin with a Rho-GAP domain that controls epithelial cell junction assembly, crosslinks and bundles actin and deactivates the small GTPase protein encoded by the RHOA gene. RhoA activity is associated with cytoskeleton regulation of actin stress fiber formation and actomyosin contractility. Myo9A was detected in mouse and human podocytes invitro and invivo. Knockin mice carrying the p.Arg701∗MYO9A (Myo9AR701X) generated by gene editing developed proteinuria, podocyte effacement and FSGS. Kidneys and podocytes from Myo9AR701X/+ mutant mice revealed Myo9A haploinsufficiency, increased RhoA activity, decreased Myo9A-actin-calmodulin interaction, impaired podocyte attachment and migration. Our results indicate that Myo9A is a novel component of the podocyte cytoskeletal apparatus that regulates RhoA activity and podocyte function. Thus, Myo9AR701X/+ knock-in mice recapitulate the proband FSGS phenotype, demonstrate that p.R701X Myo9A is an FSGS-causing mutation in mice and suggest that heterozygous loss-of-function MYO9A mutations may cause a novel form of human autosomal dominant FSGS. Hence, identification of MYO9A pathogenic variants in additional individuals with familial or sporadic FSGS is needed to ascertain the gene contribution to disease.

Identifying Cancer-Relevant Mutations in the DLC START Domain Using Evolutionary and Structure-Function Analyses.

Rho GTPase signaling promotes proliferation, invasion, and metastasis in a broad spectrum of cancers. Rho GTPase activity is regulated by the deleted in liver cancer (DLC) family of bona fide tumor suppressors which directly inactivate Rho GTPases by stimulating GTP hydrolysis. In addition to a RhoGAP domain, DLC proteins contain a StAR-related lipid transfer (START) domain. START domains in other organisms bind hydrophobic small molecules and can regulate interacting partners or co-occurring domains through a variety of mechanisms. In the case of DLC proteins, their START domain appears to contribute to tumor suppressive activity. However, the nature of this START-directed mechanism, as well as the identities of relevant functional residues, remain virtually unknown. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset and evolutionary and structure-function analyses, we identify several conserved residues likely to be required for START-directed regulation of DLC-1 and DLC-2 tumor-suppressive capabilities. This pan-cancer analysis shows that conserved residues of both START domains are highly overrepresented in cancer cells from a wide range tissues. Interestingly, in DLC-1 and DLC-2, three of these residues form multiple interactions at the tertiary structural level. Furthermore, mutation of any of these residues is predicted to disrupt interactions and thus destabilize the START domain. As such, these mutations would not have emerged from traditional hotspot scans of COSMIC. We propose that evolutionary and structure-function analyses are an underutilized strategy which could be used to unmask cancer-relevant mutations within COSMIC. Our data also suggest DLC-1 and DLC-2 as high-priority candidates for development of novel therapeutics that target their START domain.

In-Silico analysis to identify the potent inhibitor of Rho GTPase activating protein for the usage of the Glaucoma

Considered as the one of the leading irreversible loss of vision causing optic neuropathy, Glaucoma is estimated to hit more than 80 million by the end of 2020 via population survey. Increased intraocular pressure is taken as one of the risk factors among others behind the root of causing the disease. The imbalance in the secretion and the excretion of the aqueous humour inside and out of the ocular results in the IOP to deviate from the normal value of 22 mm of Hg. The Rho GAP pathway playing a crucial role in the modulation of the contractile and relaxation property of the actin smooth muscle, has shown in negatively regulating the protein kinase and subsequently increased IOP. In-vitro and Ex-vitro inhibition of the Rho GTPase protein through inhibitors have resulted in the relaxation of the actin and hence the IOP. The aim of the study is to use the in-silico technique such as, Autodock4, to explore the ligand interaction and its ability to inhibit the activity of RhoGTPase. The molecules AZA1 and Azaindole, with the least binding energies, have proven to be a potent inhibitor of the protein, hence as a lead towards the treatment of the glaucoma by decreasing the IOP to an extent.

ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases.

As membrane-associated master regulators of cytoskeletal remodeling, Rho GTPases coordinate a wide range of biological processes such as cell adhesion, motility, and polarity. In the last years, Rho GTPases have also been recognized to control intracellular membrane sorting and trafficking steps directly; however, how Rho GTPase signaling is regulated at endomembranes is still poorly understood. In this review, we will specifically address the local Rho GTPase pools coordinating intracellular membrane trafficking with a focus on the endo- and exocytic pathways. We will further highlight the spatiotemporal molecular regulation of Rho signaling at endomembrane sites through Rho regulatory proteins, the GEFs and GAPs. Finally, we will discuss the contribution of dysregulated Rho signaling emanating from endomembranes to the development and progression of cancer.

808P - Investigation on gastric cancer susceptibility genes in Chinese early-onset diffuse gastric cancer

BackgroundHereditary diffuse gastric cancer (HDGC) is closely related to germline mutation of CDH1. In addition, potential pathogenic germline variants in other candidate genes were detected in FGC and HDGC families. However, comprehensive genetic profile of HDGC predisposing gene variants in Chinese Familial gastric cancer (FGC) and HDGC patients are yet to be elucidated. MethodsWe collected peripheral blood from 28 early-onset DGC patients and from a first degree relative DGC patient who was diagnosed at age of 50. All 29 patients fulfilled IGCLC 2015 criteria for HDGC clinical diagnosis and genetic testing. Genomic DNA was extracted from peripheral blood followed by Whole Exome Sequencing (WES) with average sequencing depth of at least 100X. Gastricancer predisposing SNV and Indel candidates were filtered based on population allele frequencies in public nucleotide polymorphism databases and corresponding functional impact was predicted by in silico tools. ResultsAmong previously reported gastric cancer susceptibility genes, CDH1, BRCA2, MAP3K6 expressed the most predisposing SNV and Indel candidates. In addition, a novel splice-site variant, c.2165-1G>A, in CDH1 was detected in a Chinese early-onset HDGC patient who developed ovarian metastasis. Moreover, BCR gene exhibited as the most prevalent gastric cancer predisposing gene with filtered variants primarily enriched in RhoGAP domain. Pathway enrichment analysis showed that the top three predisposing variant candidates were enriched in the following signalling pathway: ECM-receptor interaction (P-Value: 0.001), focal adhesion (P-Value: 0.02) and protein digestion and absorption (P-Value: 0.002). ConclusionsThis study suggested that CDH1, BRCA2, MAP3K6 may act as potential gastric cancer susceptibility genes in Chinese early-onset HDGC patients. Further study may be required to investigate the functional role of BCR abnormality, especially in its RhoGAP domain, in the tumorigenesis of gastric cancer. Moreover, to further investigate molecular mechanisms of early-onset gastric cancer, one may consider genes involved in three signalling pathways: ECM-receptor interaction, focal adhesion and protein digestion and absorption. Legal entity responsible for the studyPeking University Cancer Hospital. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 438: GRAF1 is a Novel Mediator of Parkin-dependent Mitophagy

Selective autophagic removal of damaged mitochondria by Parkin-mediated mitophagy is essential for metabolic homeostasis of long-lived cells and defects in this clearance pathway leads to the accumulation of toxic intermediates and accelerates the pathogenesis of numerous cardiometabolic diseases. This process is facilitated by the recruitment of the autophagosomal protein, LC3, by LC3-binding receptors that accumulate on damaged mitochondria. However, little is known about the precise LC3-receptors that mediate Parkin-dependent mitophagy in cardiomyocytes. Having previously identified a cardioprotective role for GRAF1, our current data indicate that GRAF1 plays critical role in promoting Parkin-dependent mitochondrial clearance. GRAF1 is expressed at high levels in the heart from E17 onwards and is poised to co-regulate actin- and lipid-dynamics by virtue of its multi-domain structure that includes a lipid binding/bending BAR domain, a phospholipid binding PH domain, a Rho-GAP domain, and a protein-interaction SH3 domain. Importantly, GRAF1 depletion in primary cardiomyocytes led to impaired mitochondrial OXPHOX-mediated ATP generation, mitochondrial membrane depolarization, increased mitochondrial-associated ROS, and increased ischemia/reperfusion-dependent myocyte death. These outcomes were accompanied by reduced LC3 mediated autophagic flux and accumulation of damaged mitochondria (and Parkin) in GRAF1 deficient cardiomyocytes and hearts. Mechanistically, we showed that GRAF1 facilitates Parkin-dependent mitophagy by serving as a novel LC3 receptor. GRAF1 is recruited to damaged mitochondria, is phosphorylated in a PINK1/Parkin-dependent fashion, and physically interacts with Parkin and LC3. While multiple LC3 receptors are recruited to depolarized mitochondria and act in a redundant function to promote mitochondrial engulfment, our studies reveal a unique and indispensable role for GRAF1 in this process. Our studies reveal that GRAF1 is distinctive because it facilitates mitochondrial clearance by further facilitating recruitment of Parkin and additional down-stream receptors to damaged mitochondria in a feed-forward fashion.

The tumor suppressive roles of ARHGAP25 in lung cancer cells.

AimSeveral Rho GTPase-activating proteins (Rho GAPs) have been proved to serve as tumor suppressors in diverse human cancers. Among them, ARHGAP25 has also been found to be associated with hematopoietic cells and regulate phagocytosis. Little is known about the role of ARHGAP25 in lung cancer cells.MethodsQuantitative real-time PCR and Western blot were used to measure the expression levels of ARHGAP25. The ability of cell growth and mobility were measured by cell proliferation and Transwell assays. Chromatin immunoprecipitation and luciferase assay were conducted to identify the transcriptional regulation.ResultsLung cancer tissues had much lower expression level of ARHGAP25 compared to non-cancerous specimens as well as for lung cancer cells. Cell growth and mobility were strongly reduced when ARHGAP25 was overexpressed. Further, significantly negative correlation between ARHGAP25 expression and Wnt signaling pathway was observed. Overexpression of ARHGAP25 reduced the expression of β-catenin and matrix metalloproteinase-7. ARHGAP25 knockdown effect of increased abilities of cell proliferation, migration and invasion could be reversed by adding XAV939 inhibitor. The promoter site of ARHGAP25 could be bound with HOXA4. HOXA4 could regulate the transcriptional activity of ARHGAP25.ConclusionsThis study suggests that ARHGAP25 may inhibit lung cancer cell growth, migration and invasion through Wnt/β-catenin signaling pathway and its transcriptional activity can be regulated by HOXA4.

Abstract 1680: RhoGAP and PAP2 domain-containing fusions are recurrent and prognostic in gastric cancer

Abstract We conducted an RNA sequencing study to identify novel in-frame gene fusions in 80 discovery dataset tumors collected from young Korean patients with diffuse gastric cancer. Twenty-five in-frame fusions were associated with diffuse gastric cancer, three of which (CLDN18-ARHGAP26, CTNND1-ARHGAP26, and ANXA2-MYO9A) were recurrent in 384 diffuse gastric cancers based on RT-PCR. All three fusions contained a RhoGAP domain in their 3’ partner genes. Patients with one of these three RhoGAP domain fusions had a significantly worse prognosis than those without such fusions. The median survival was 29.1 [95% CI, 5.7–not reached] and 94.6 [95% CI, 62.0–not reached] months, respectively (P=0.011, log-rank; HR, 2.8 [95% CI, 1.5–5.3]). Ectopic expression of CLDN18-ARHGAP26 promoted the migration and invasion capacities of diffuse gastric cancer cells. Parallel targeted RNA sequencing analysis additionally identified PAP2 domain-containing fusions as recurrent and poor prognostic in-frame fusions. Overall, in-frame gene fusions containing either RhoGAP or PAP2 domain clearly defined the aggressive subset (7.5%) of diffuse gastric cancer, and their prognostic impact (HR, 3.4 [95% CI, 2.0–5.7]) was greater than, and independent of, chromosomal instability and CDH1mutations. Our study may provide novel genomic insights guiding future strategies for managing diffuse gastric cancer. Citation Format: Hark K. Kim. RhoGAP and PAP2 domain-containing fusions are recurrent and prognostic in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1680.

Analysis of circulating lncRNA expression profiles in patients with diabetes mellitus and diabetic nephropathy: Differential expression profile of circulating lncRNA .

In order to elucidate the epigenetic mechanism and explore new biomarkers for diabetes and diabetic nephropathy, circulating lncRNA and mRNA expression profiles of normal control, diabetes mellitus, and diabetic nephropathy patients were analyzed. Serum samples from diabetic nephropathy patients (DN), diabetes mellitus patients without microalbuminuria (DM), and healthy controls (N) were collected. Arraystar Human LncRNA/mRNA V3.0 expression spectrum biochips were used for serum lncRNA and mRNA expression profile analysis. The urinary microalbumin/creatinine ratio and serum creatinine level were higher in diabetic nephropathy patients, and the estimated glomerular filtration rate (eGFR) was significantly decreased compared to that in diabetic patients and healthy controls (<0.05). Compared with healthy controls, 245 upregulated and 680 downregulated lncRNAs were identified in the serum of diabetic patients, and 45 and 813 lncRNAs were up- and downregulated in the serum of diabetic nephropathy patients compared with diabetic patients. Levels of lncRNA-ARAP1-AS2 gradually increased during the progression of diabetes and diabetic nephropathy (2.82 times in DM/N and 2.47 times in DN/DM), whereas those of lncRNA-ARAP1-AS1 gradually decreased (2.24 times in DM/N, 4.79 times in DN/DM). Additionally, mRNA levels of their target gene ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat, and PH domain 1) gradually increased (2.25 times in DM/N and 2.45 times in DN/DM). lncRNA-ARAP1-AS1 and ARAP1-AS2 enhanced ARAP1 mRNA expression and may be involved in the pathogenesis of diabetes and DN. Circulating lncRNA-ARAP1-AS1, ARAP1-AS2, and ARAP1 may serve as new biomarkers for diabetes and diabetic nephropathy.

Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1

Abstract The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain. We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1. We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1. Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.

The association of novel recurrent in-frame gene fusions with prognosis of diffuse gastric cancer.

34 Background: Among the two histologic subtype of gastric cancer (GC), diffuse gastric cancer (DGC) is increasingly being considered distinct from intestinal type gastric cancer (IGC). Despite the relative importance of DGC, few whole transcriptomic analyses have been performed for this histological subtype. We therefore conducted an RNA-sequencing study to search for novel driver fusions in DGC. Methods: We conducted a whole transcriptomic and targeted RNA sequencing study of 384 Korean DGCs to identify gene fusions that may be novel prognostic markers or therapeutic targets. Targeted DNA sequencing and SNP6.0 array analyses were conducted in parallel. Results: Whole transcriptomic analyses were conducted in 80 discovery dataset tumors collected from young patients with DGC who had not been treated with chemotherapy or radiation. Twenty-five in-frame fusions were associated with DGC, four of which were recurrent in 384 DGCs based on targeted RNA sequencing and RT-PCR sequencing analyses. Three of the four recurrent fusions contained a RhoGAP domain in their 3’ partner genes. Patients with one of these three fusions have a significantly worse prognosis than those without (HR, 2.8 [95% CI, 1.5‒5.3]). The fusion that harbored a PAP2 domain in the 3’ partner gene was also identified as recurrent and poor prognostic in-frame fusions. Overall, RhoGAP and PAP2 domain-containing fusions were present in 7.5% of DGCs, but not in adjacent normal tissue, and clearly defined the worst prognosis subgroup. Their prognostic impact (adjusted HR 4.1 [95% CI, 2.1‒7.9]) was higher than, and independent of, chromosomal instability (CIN) and CDH1 mutation, which we previously identified as the strongest adverse prognostic genomic abnormalities in DGCs (adjusted HRs, 2.5 (1.5‒4.4) and 2.4 (1.3‒4.4), respectively). Our comprehensive in-frame fusion screen also identified several clinically-actionable fusions amenable to ALK or FGFR inhibition, which had not been previously associated with gastric cancer. Conclusions: Our findings may provide novel genomic insights guiding future personalized strategies for managing DGCs, given the strong prognostic impact of RhoGAP and PAP2 domain-containing gene fusions.

Rho GTPase Activating Protein 24 (ARHGAP24) Regulates the Anti-Cancer Activity of Sorafenib Against Breast Cancer MDA-MB-231 Cells via the Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathway.

BackgroundSTAT3 has emerged as a novel potential target for sorafenib, a multikinase inhibitor, in the context of cancer therapy. ARHGAP24 is a Rac-specific Rho GTPase-activating protein (Rho GAP), which can convert Rho GTPases to an inactive state. It has been proved to be an oncosuppressor protein in renal cancer. In the present study, we investigated its anti-cancer effect in breast cancer (BC).Material/MethodsQuantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the expression of ARHGAP24 in clinical tissue samples. Then, BC MDA-MB-231 cells were virally transduced with ARHGAP24 silencing or overexpression lentiviral vectors in the absence or presence of sorafenib. Cell viability and metastatic ability were evaluated by using the Cell Counting Kit-8 (CCK-8) and Transwell assays. Proteins belonging to the STAT3 pathway were detected by Western blot.ResultsARHGAP24 decreased in BC tissues compared with the adjacent normal tissues. Forced expression of ARHGAP24 and sorafenib treatment significantly suppressed the viability, migration, and invasion of MDA-MB-231 cells. Conversely, elimination of the endogenous ARHGAP24 with shRNA promoted cell viability, migration, and invasion. The phosphorylation of STAT3 and the expression of MMP-2 and MMP-9 were attenuated by ARHGAP24 ectopic expression and sorafenib treatment. Furthermore, forced expression of ARHGAP24 significantly enhanced sorafenib-induced decrease of cell viability, migration, and invasion of MDA-MB-231 cells, while elimination of the endogenous ARHGAP24 with shRNA inhibited it.ConclusionsARHGAP24 can suppress the development of MDA-MB-231 cells via the STAT3 signaling pathway, and sorafenib inhibits cell viability, migration, invasion, and STAT3 activation in MDA-MB-231 cells through ARHGAP24.

BAR domain proteins—a linkage between cellular membranes, signaling pathways, and the actin cytoskeleton

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.

RhoGAP domain-containing fusions and PPAPDC1A fusions are recurrent and prognostic in diffuse gastric cancer

We conducted an RNA sequencing study to identify novel gene fusions in 80 discovery dataset tumors collected from young patients with diffuse gastric cancer (DGC). Twenty-five in-frame fusions are associated with DGC, three of which (CLDN18-ARHGAP26, CTNND1-ARHGAP26, and ANXA2-MYO9A) are recurrent in 384 DGCs based on RT-PCR. All three fusions contain a RhoGAP domain in their 3’ partner genes. Patients with one of these three fusions have a significantly worse prognosis than those without. Ectopic expression of CLDN18-ARHGAP26 promotes the migration and invasion capacities of DGC cells. Parallel targeted RNA sequencing analysis additionally identifies TACC2-PPAPDC1A as a recurrent and poor prognostic in-frame fusion. Overall, PPAPDC1A fusions and in-frame fusions containing a RhoGAP domain clearly define the aggressive subset (7.5%) of DGCs, and their prognostic impact is greater than, and independent of, chromosomal instability and CDH1 mutations. Our study may provide novel genomic insights guiding future strategies for managing DGCs.