RAS P21 Protein Activator Research Articles

RNA-binding protein QKI suppresses breast cancer via RASA1/MAPK signaling pathway.

BackgroundRNA-binding protein Quaking (QKI) has been linked with the pathogenesis and development of various human malignancies. Herein, we explored the particular role of QKI in breast cancer (BC) progression.MethodsThe methods employed in the study included public dataset analysis, western blot, quantitative real-time PCR (qRT-PCR), cell count kit-8 (CCK8) assay, colony formation assay, flow cytometric analysis, RNA immunoprecipitation (RIP), messenger RNA (mRNA) stability assay, QKI overexpression and knockdown, and Ras p21 protein activator 1 (RASA1) knockdown.ResultsAberrant expression levels of QKI and RASA1 were detected in BC and compared with those in noncancerous tissues. A moderately positive correlation between QKI and RASA1 was verified within BC tissues. Low expression of QKI was associated with positive estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, non-triple-negative breast cancer (TNBC), non-basal-like BC, and poor clinical outcomes in BC patients. QKI overexpression suppressed BC cell proliferation and colony formation, and arrested cell cycle at G1 phase. RIP assay and mRNA stability assay confirmed that QKI directly bound to RASA1 transcript and increased its stability, thus inactivating the MAPK pathway and inhibiting BC progression. RASA1 knockdown could partly attenuate the inhibitory effect of QKI on BC cell proliferation via activating the mitogen-activated protein kinase (MAPK) pathway.ConclusionsQKI, which was frequently downregulated in BC, could significantly inhibit cell proliferation and arrest cell cycle at G1 phase by binding and enhancing RASA1 mRNA expression. Low expression of QKI was prominently associated with unfavorable clinical outcomes in BC patients, indicating the prognostic value of QKI in BC.

Role of RASA1 in cancer: A review and update (Review).

Ras p21 protein activator 1 (RASA1) is a regulator of Ras GDP and GTP and is involved in numerous physiological processes such as angiogenesis, cell proliferation, and apoptosis. As a result, RASA1 also contributes to pathological processes in vascular diseases and tumour formation. This review focuses on the role of RASA1 in multiple tumours types in the lung, intestines, liver, and breast. Furthermore, we discuss the potential mechanisms of RASA1 and its downstream effects through Ras/RAF/MEK/ERK or Ras/PI3K/AKT signalling. Moreover, miRNAs are capable of regulating RASA1 and could be a novel targeted treatment strategy for tumours.

RASA1 inhibits the progression of renal cell carcinoma by decreasing the expression of miR-223-3p and promoting the expression of FBXW7.

RAS p21 protein activator 1 (RASA1), also known as p120-RasGAP, is a RasGAP protein that functions as a signaling scaffold protein, regulating pivotal signal cascades. However, its biological mechanism in renal cell carcinoma (RCC) remains unknown. In the present study, RASA1, F-box/WD repeat-containing protein 7 (FBXW7), and miR-223-3p expression were assessed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Then, the targeted correlations of miR-223-3p with FBXW7 and RASA1 were verified via a dual-luciferase reporter gene assay. CCK-8, flow cytometry, and Transwell assays were implemented independently to explore the impact of RASA1 on cell proliferation, apoptosis, migration, and cell cycle progression. Finally, the influence of RASA1 on tumor formation in RCC was assessed in vivo through the analysis of tumor growth in nude mice. Results showed that FBXW7 and RASA1 expression were decreased in RCC tissues and cell lines, while miR-223-3p was expressed at a higher level. Additionally, FBXW7 and RASA1 inhibited cell proliferation but facilitated the population of RCC cells in the G0/G1 phase. Altogether, RASA1 may play a key role in the progression of RCC by decreasing miR-223-3p and subsequently increasing FBXW7 expression.

MicroRNA-144 relieves chronic constriction injury-induced neuropathic pain via targeting RASA1.

MicroRNAs (miRNAs) have been shown to participate in development of neuropathic pain. However, the role of microRNA-144 (miR-144) in neuropathic pain remains unclear. In the present study, we established a neuropathic pain mouse model via chronic constriction injury (CCI)-induction. The successful establishment of this model was confirmed via evaluation of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). By using this model, we found that miR-144 was significantly downregulated in CCI-induced neuropathic pain mice. In addition, intrathecal injection of miR-144 agomiR alleviated mechanical and thermal hyperalgesia in neuropathic pain mice as shown by the increased of PWT and PWL. Moreover, miR-144 negatively regulated neuroinflammation by decreasing the expression of proinflammatory mediators, including TNF-α (tumor necrosis factor-α), IL (interleukin)-1β, and IL-6, thus facilitating the inhibition of neuropathic pain development. Mechanistically, RASA1 (RAS P21 Protein Activator 1) was downregulated following the injection of agomiR-144, and was verified to be a target of miR-144. Furthermore, overexpression of RASA1 reversed the inhibitory effect of miR-144 on neuropathic pain. Therefore, the present study suggested that miR-144 has the potential to be explored as therapeutic target for treatment of neuropathic pain.

RAS P21 Protein Activator 3 (RASA3) specifically promotes pathogenic T helper 17 cell generation by repressing T helper 2-biased programs

Abstract Th17 cells that produce the signature cytokine interleukin 17 (IL-17) can be both pathogenic and non-pathogenic. Pathogenic-Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17-subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3), a GTPase activating protein, was highly expressed by IL-1β/IL-6/IL-23 induced-pTh17 cells relative to TGFβ1/IL-6 induced-non-pTh17 cells and was required specifically for pTh17 generation in vitro and in vivo. Mice conditionally deficient for Rasa3 in T cells showed less pathology during experimental autoimmune encephalomyelitis. By RNA-seq analysis, Rasa3-deficient T cells were found to acquire a Th2-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2-bias of Rasa3-deficient T cells was due to aberrantly elevated transcription factor IRF4 expression. Furthermore, RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 programs. These findings indicate that a distinct molecular program directs pTh17 generation and reveals targets for treating pTh17-related pathology and diseases.

MicroRNA‑4530 suppresses cell proliferation and induces apoptosis by targeting RASA1 in human umbilical vein endothelial cells.

MicroRNAs (miRNAs/miRs) are a class of endogenous and non-coding RNAs that are present in eukaryotes. In previous studies, miRNAs have been revealed to have an important role in cell growth and apoptosis. In the present study, the function of a novel and rarely studied miRNA, miR-4530, was investigated in human umbilical vein endothelial cells (HUVECs). The expression level of miR-4530 in HUVECs was investigated using reverse transcription-quantitative polymerase chain reaction following transfection with miR-4530 precursor plasmids, anti-miR-4530 plasmids and empty vector plasmids. Following this, it was revealed that overexpression of miR-4530 can suppress cell proliferation and enhance cell apoptosis. TargetScan analysis suggested that Ras p21 protein activator 1 (RASA1) is a target gene of miR-4530. The results of a dual-luciferase reporter assay also suggested that miR-4530 targets RASA1. Furthermore, the results of dual-luciferase reporter assay suggested that miR-4530 enhanced luciferase activity of the wild-type reporter, but not the mutant RASA1 reporter activity, thus suggesting that miR-4530 enhances the expression of RASA1. In addition, western blot analysis demonstrated that the protein expression level of RASA1 was enhanced following upregulation of miR-4530. The exact mechanism underlying this process has not yet been determined and requires further investigation. In addition, a RASA1 overexpression plasmid vector was transfected into HUVECs. The results suggest that overexpression of RASA1 suppresses cell growth and promotes apoptosis, which was in agreement with the results regarding the overexpression of miR-4530. To investigate how miRNA-4530 affects cellular function, numerous proteins associated with the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase pathways were investigated via western blot analysis. The results suggested that miRNA-4530 suppresses cell proliferation and enhances apoptosis by targeting RASA1 via the ERK/MAPK and PI3K/AKT signaling pathways.

MicroRNA‑21 and its impact on signaling pathways in cervical cancer (Review)

Oncogenic microRNA-21 (miR-21/miRNA-21) is a stable inhibitor of gene expression that is often upregulated in cervical cancer, a disease that affects the health of women and tends to transform and spread. Previous studies investigating miR-21 in biopsies and cells from cervical cancer patients have identified that miR-21 binds target mRNAs in signaling pathways or long non-coding RNAs (lncRNA). Furthermore, studies have elucidated the molecular mechanisms of two tumor necrosis factor α (TNF-α) signaling pathways that promote cell proliferation and inhibit cell apoptosis. miR-21 inhibits the TNF receptor 1 (TNFR1) signaling pathway and activates the TNFR2 signaling pathway. Moreover, miR-21 enhances cervical cancer cell proliferation by influencing the protein kinase B/mammalian target of rapamycin and RAS p21 protein activator 1 signaling pathways. The present review discusses the evidence that miR-21 may impact cervical cancer through inhibiting apoptosis and enhancing proliferation, and may therefore be a target for clinical intervention.

Long non-coding RNA LOC554202 promotes acquired gefitinib resistance in non-small cell lung cancer through upregulating miR-31 expression.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation inevitably have a relapse due to the occurrence of acquired resistance, resulting in treatment failure. However, little is known about the mechanisms of acquired resistance of NSCLC patients. Here, we elucidated the expression pattern of LOC554202 and miR-31, and their biological functions and mechanisms in NSCLC with acquired EGFR TKI resistance to gefitinib. In the present study, we observed that LOC554202 and miR-31 promoted proliferation and clonogenic growth of gefitinib-resistant NSCLC cells in vitro. LOC554202 upregulated miR-31 expression and they both reduced sensitivity of NSCLC cells to gefitinib. In a xenograft mice model, we found that knockdown of miR-31 significantly repressed gefitinib-resistant NSCLC cells growth in vivo. Furthermore, both LOC554202 and miR-31 levels were significantly increased in NSCLC patients acquiring resistance to gefitinib, and the expression of LOC554202 was positively correlated with the expression of miR-31. By luciferase reporter assays, we identified RAS P21 Protein Activator 1 (RASA1) and Hypoxia Inducible Factor 1 Subunit Alpha Inhibitor (FIH-1) as direct targets of miR-31 in NSCLC cells. Mechanistically, miR-31 directly repressed RASA1 and FIH-1 expression, and thus, at least partially activated the RAF-MEK-ERK and PI3K-AKT signaling pathways in NSCLC with acquired resistance to gefitinib. In conclusion, these data will help us develop potential therapeutic targets for the diagnosis and treatment of acquired EGFR TKI resistance in EGFR-mutant NSCLC.

MicroRNA‑21 regulates the biological behavior of esophageal squamous cell carcinoma by targeting RASA1.

MicroRNA-21 (miR-21) has been revealed to play a crucial role in regulating the biological behavior, including proliferation, migration, invasion and metastasis in certain cancers. However, its role in esophageal squamous cell carcinoma (ESCC) has yet to be elucidated. Based on the data of GSE13937 downloaded from Gene Expression Omnibus (GEO) database, miR-21 was revealed to be one of the top 20 differentially expressed (DE) miRNAs screened using the Morpheus online tool. RAS p21 protein activator 1 (RASA1) was predicted as the target gene of miR-21 using the predicting software and was combined with miR-21 using the luciferase reporter assay. Its relative expression was significantly decreased, however, miR-21 was increased in the tumor tissues compared to the normal adjacent tissues in patients with ESCC as determined by quantitative polymerase chain reaction (q-PCR). Furthermore, overexpression of miR-21 (mimic) could significantly decrease the gene level of RASA1. Conversely, downregulation of miR-21 (inhibitor) significantly increased the gene level of RASA1, while downregulation of RASA1 (siRASA1) markedly increased the gene expression of miR-21. Notably, the expression of Snail and vimentin were significantly increased by upregulation of miR-21 and downregulation of RASA1. Transwell results revealed that miR-21 and RASA1 regulated proliferation, migration and invasion in ESCC cells. In an in vivo model, miR-21 inhibitor (antagomir) could inhibit tumor growth. In conclusion, miR-21 regulated cell proliferation, migration, invasion and tumor growth of ESCC by directly targeting RASA1, which may have been achieved via regulation of Snail and vimentin. Anti-miR-21 revealed an antitumor effect. Thus, it may be considered as a possible target for ESCC therapy.

RAS P21 Protein Activator 3 (RASA3) Specifically Promotes Pathogenic T Helper 17 Cell Generation by Repressing T-Helper-2-Cell-Biased Programs

Pathogenic Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17 cell subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3) was highly expressed by pTh17 cells relative to non-pTh17 cells and was required specifically for pTh17 generation invitro and invivo. Mice conditionally deficient for Rasa3 in Tcells showed less pathology during experimental autoimmune encephalomyelitis. Rasa3-deficientTcells acquired a Th2 cell-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2 cell bias ofRasa3-deficient Tcells was due to aberrantly elevated transcription factor IRF4 expression. RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 cell programs. These findings indicate that a distinct molecular program directs pTh17 cell generation and reveals targets for treating pTh17 cell-related pathology and diseases.

Abstract 4364: R-Ras activation cooperates with BRAF mutation in melanoma tumorigenesis

Abstract The Ras family of small GTP binding proteins are tightly regulated through a complex network of proteins. In melanoma the Ras signaling pathway is frequently activated by mutations in NRAS (20%), KRAS (2%) and HRAS (1%); alternatively, Ras can also be activated by the inactivation of Ras GTPase activating proteins (RasGAPs) such as NF1, RASA1, and RASA2. Recently, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines and human melanoma specimens. In addition, RNAi-mediated knockdown of R-Ras suppressed anchorage-independent colony growth and tumor growth. Of the 3 major RAS effector pathways, specifically MEK/ERK, AKT and Ral-A, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a Rasa1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of Rasa1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in Rasa1L/L BRAFCA/CA mice compared to Rasa1+/+ BRAFCA/CA littermates; as well as, the development of cutaneous melanoma in Rasa1 deficient mice. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen S. Hill, Evan R. Roberts, Xue Wang, Youngchul Kim, Jane Messina, Minjung Kim. R-Ras activation cooperates with BRAF mutation in melanoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4364.

Abstract B12: The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis

Abstract The Ras family of small GTP-binding proteins is frequently activated by mutations, including NRAS (20%), KRAS (2%), and HRAS (1%), in melanoma. In addition to mutations, Ras isoforms can also be activated by the inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that the inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. We hypothesized that, although not mutated, R-Ras is activated in melanoma through the inactivation of RasGAPs and that RasGAP/R-Ras pathway activation cooperates with BRAF activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less-appreciated member of the Ras family, in melanoma tumorigenesis and investigated the molecular mechanisms underlying R-Ras signaling in BRAF mutant melanoma. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the three major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by RASA1 inactivation and subsequent R-Ras activation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacologic (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1 L/L; BRAF CA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in the expression of constitutively activated mutant BRAF and the deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1 L/L BRAF CA/CA mice compared to RASA1 +/+ BRAF CA/CA littermates and the development of melanoma in RASA1 mutant mice. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen S. Hill, Xue Wang, Evan R. Roberts, Youngchul Kim, Jane Messina, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B12.

MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1

Background/Aims: Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. Methods: Cultured cardiac fibroblasts (CFs) were activated by TGF-β1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson’s trichrome staining. Results: miR-223 was increased in CFs compared to cardiomypcytes, and TGF-β1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-β1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3’ untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. Conclusions: miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

Search for RASA1 Variants in Capillary Malformations of the Legs in 113 Children: Results from the French National Paediatric Cohort CONAPE.

Patients with an inherited autosomal-dominant disorder, capillary malformation-arteriovenous malformation (CM-AVM), frequently have mutations in Ras P21 protein activator 1 (RASA1). The aims of this study were to determine the prevalence of germline RASA1 variants in a French multicentre national cohort of children, age range 2-12 years, with sporadic occurrence of capillary malformation (CM) of the legs, whatever the associated abnormalities, and to identify genotype-phenotype correlates. DNA was extracted from leukocytes in blood samples, purified and amplified, and all exons of the RASA1 gene were analysed. Among 113 children analysed, 7 had heterozygous variants (6.1%). Four different variants were identified; 2 were new. In children with RASA1 variants, CMs were more frequently bilateral and multifocal. In conclusion, RASA1 variants are rarely found in children with sporadic CM of lower limbs without CM-AVM syndrome. CMs in this study were heterogeneous, and no disease-causing relationship could be proven.

Interactions between RASA2, CADM1, HIF1AN gene polymorphisms and body fatness with breast cancer: a population-based case-control study in China.

Genome-wide association studies (GWAS) have indicated that gene polymorphisms in alleles of RAS p21 protein activator 2 (RASA2), cell adhesion molecule 1 (CADM1) and hypoxia inducible factor 1 alpha subunit inhibitor (HIF1AN) are associated with the risk of obesity. In this study, we explored the interactions between candidate SNPs of RASA2 (rs16851483), CADM1 (rs12286929) and HIF1AN (rs17094222) and body fatness for breast cancer risk. Unconditional logistic regression models were applied to measure the associations of related factors with breast cancer by odds ratios (ORs) and 95% confidence intervals (CIs). It was observed that cases had a statistically higher body mass index (BMI ≥ 28 kg/m2, OR = 1.77), waist circumference (WC ≥ 90cm, OR = 2.89) and waist-to-hip ratio (WHR ≥ 0.9, OR = 3.41) as compared to controls. Significant differences were also found in the genotype distributions of RASA2 rs16851483 T/T homozygote and CADM1 rs12286929 G/A heterozygote between cases and controls, with an OR of 1.68 (95% CI: 1.10–2.56) and 0.80 (95% CI: 0.64–0.99), respectively. Furthermore, significant interactions were observed between polymorphisms of three genes and body fatness for the risk of breast cancer based on both additive and multiplicative scales. These results of our study suggest that body fatness possibly plays an important role in the development of breast cancer and this risk might be modified by specific genotypes of some potential genes, especially for obese women in China.

Co-expression and significance of Dok2 and Ras p21 protein activator 1 in breast cancer.

Docking protein 2 (Dok2) and Ras p21 protein activator 1 (RASA1) are tumor suppressors which have been identified in numerous solid tumors; however, the association between their expression in breast cancer and patient prognosis remains unclear. A total of 285 consecutive patients diagnosed histopathologically with breast cancer who underwent surgery at Jingzhou Central Hospital were selected for the present study. Dok2 and RASA1 protein were explored using histopathology and western blotting techniques, and the association of patient prognosis with clinicopathological parameters was investigated using univariate and multivariate analyses. Weak expression of Dok2/RASA1 was associated with poorly differentiated breast adenocarcinomas; negatively expressed Dok2 and RASA1 were associated with increased tumor size, a higher proportion of axillary lymph node metastasis and later clinical staging. Additionally, Dok2 and RASA1 expression were associated with disease-free survival of patients with breast cancer. As indicated by Cox's regression analysis, Dok2 and RASA1 expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for the recurrence of breast cancer. The results of the present study suggested that combined Dok2 and RASA1 negative expression may serve as an independent prognostic factor for patients following breast cancer surgery.

MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing

MicroRNA (miR)-132 has been identified as a top up-regulated miRNA during skin wound healing and its inhibition impairs wound repair. In a human in vivo surgical wound model, we showed that miR-132 was induced in epidermal as well as in dermal wound–edge compartments during healing. Moreover, in a panel of cells isolated from human skin wounds, miR-132 was found highly expressed in human dermal fibroblasts (HDFs). In HDFs, miR-132 expression was upregulated by TGF-β1. By overexpression or inhibition of miR-132, we showed that miR-132 promoted HDF migration. Mechanistically, global transcriptome analysis revealed that RAS signaling pathway was regulated by miR-132 in HDFs. We found that RAS p21 protein activator 1 (RASA1), a known target of miR-132, was downregulated in HDFs upon miR-132 overexpression. Silencing of RASA1 phenocopied the pro-migratory effect of miR-132. Collectively, our study reveals an important role for miR-132 in HDFs during wound healing and indicates a therapeutic potential of miR-132 in hard-to-heal skin wounds.

Abstract 1352: The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis

Abstract The Ras family of small GTP binding proteins are frequently activated by mutations in melanoma, as shown for NRAS (20%), KRAS (2%) and HRAS (1%). Ras isoforms can also be activated by inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the 3 major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1L/L BRAFCA/CA mice compared to RASA1+/+ BRAFCA/CA littermates. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen Suzanne Hill, Xue Wang, Youngchul Kim, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2017-1352

A Transposon-based Analysis Reveals RASA1 Is Involved in Triple-Negative Breast Cancer.

RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/- background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357-68. ©2017 AACR.

GNAQ mutation R183Q as a potential cause of familial Sturge-Weber syndrome: A case report.

Sturge-Weber syndrome (SWS) is a rare neurocutaneous disorder whose etiology remains unclear. To investigate the genetic contribution underlying this disease, the genetic variants of a 4-generation family with a history of SWS was analyzed in the present study. SWS was diagnosed in 3 of the family members (II-1, III-11 and IV-6). Sanger sequencing was performed to identify mutations in G protein subunit αq (GNAQ) and RAS p21 protein activator 1 exons in the 3 patients with SWS and other unaffected family members. Notably, a non-synonymous single-nucleotide variant at codon 183 on exon 4 of the GNAQ gene was identified as the only pathogenic site. This variant generated a substitution of arginine (R) with glutamine and resulted in a change of function of the encoded protein. Evolutionary conservation analysis revealed that the mutated residue 183 (R) of GNAQ is highly conserved across several vertebrate species. Furthermore, an immunofluorescence staining assay demonstrated that the substitution of arginine with glutamine resulted in a change in the sub-cellular localization of the GNAQ recombinant protein in vitro. These findings may aid in the development of novel diagnostic markers and/or therapeutic targets for the treatment of patients with familial SWS.