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Some studies suggest that the inactivation of the Ras-MAPK pathway in trophoblast cells can lead to recurrent abortion, but the molecular mechanism underlying the inactivation of this pathway in trophoblast cells is still unclear. This study aimed to explore the relationship between the mechanism of abnormal activation of RASA1, a regulatory protein of the Ras-MAPK pathway, and unexplained recurrent spontaneous abortion. RT-qPCR was used to detect the transcription levels of RASA1 gene. Immunohistochemistry and Western blot were used to detect the expression levels of the RASA1, Raf and MEK proteins. CCK-8, TUNEL and Transwell assays were used to detect the proliferative, apoptotic, and invasive capacities of HTR-8/SVneo cells. ChIP assays were used to detect the enrichment of H3K27me3 in RASA1 gene promoter. Abortion villi experiments showed that the enrichment of H3K27me3 in the RASA1 gene promoter was reduced, and that both RASA1 gene transcription and RASA1 protein expression were increased. Cell experiments confirmed that RASA1 could decrease the phosphorylated Raf and MEK proteins, inhibit the proliferation and invasion ability, and promote the apoptosis ability of HTR-8/SVneo cells. It was also found that the proliferation and invasion ability as well as the Ras-MAPK pathway activity of HTR-8/SVneo cells were inhibited when treated with histone methyltransferase inhibitor DZNep. RASA1 gene was abnormally activated in unexplained recurrent spontaneous abortion villi due to the decreased enrichment of H3K27me3 in the gene promoter. High expression of RASA1 could inhibit the activity of the Ras-MAPK pathway, and thus inhibit the proliferation and invasion ability of trophoblast cells.

Capillary malformation–arteriovenous malformation (CM–AVM) is a newly recognized clinical entity caused by mutation of the RASA1 gene, which encodes p120-RasGAP. Here we describe, for the first time, a patient with CM–AVM presenting during the late stages of pregnancy with pulmonary “capillary level” microvascular shunt, worsening cutaneous capillary malformations, and gross fluid overload. Sequencing revealed a novel mutation of the RASA1 gene involving a frameshift mutation in the RASGAP domain of RASA1. This report extends our current genetic and clinical understanding of CM–AVM. © 2013 Wiley Periodicals, Inc.

Hereditary capillary malformations are known to be caused by mutations in the RASA1 gene. The associated phenotype is still subject of debate. The purpose of this study was to conduct a RASA1 mutation analysis in the family that led to the initial discovery of the 5q locus, and to delineate the associated phenotype. A novel truncating mutation was identified in all clinically affected individuals and in none of the unaffected members. The associated phenotype was widely variable; all individuals had multifocal CM with at least one area of high flow. Various additional features were observed, some previously reported and others novel, including limb overgrowth, varicosities, possible lymphatic malformations, localized hyperhidrosis and exercise induced redness. The cause of this wide intramutational phenotypic variability remains to be elucidated. (C) 2012 Published by Elsevier Masson SAS

p120 Ras GTPase-activating protein (RasGAP) encoded by the rasa1 gene in mice is a prototypical member of the RasGAP family of proteins involved in negative-regulation of the p21 Ras proto-oncogene. RasGAP has been implicated in signal transduction through a number of cell surface receptors. In humans, inactivating mutations in the coding region of the RASA1 gene cause capillary malformation arteriovenous malformation. In mice, generalized disruption of the rasa1 gene results in early embryonic lethality associated with defective vasculogenesis and increased apoptosis of neuronal cells. The early lethality in this mouse model precludes its use to further study the importance of RasGAP as a regulator of cell function. Therefore, to circumvent this problem, we have generated a conditional rasa1 knockout mouse. In this mouse, an exon that encodes a part of the RasGAP protein essential for catalytic activity has been flanked by loxP recognition sites. With the use of different constitutive and inducible Cre transgenic mouse lines, we show that deletion of this exon from the rasa1 locus results in effective loss of expression of catalytically-active RasGAP from a variety of adult tissues. The conditional rasa1 mouse will be useful for the analysis of the role of RasGAP in mature cell types.

Melanoma shows frequent Ras-Raf-MAPK pathway activation via frequent mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, NF1 (neurofibromatosis type 1), one of the Ras GTPase activating proteins (RasGAPs), has been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs has not been addressed in melanoma. To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). Our whole genome analyses also identified two novel somatic missense mutations (p.Tyr472His and p.Leu481Phe) in or around the PH domain in RASA1 gene. RASA1 (RAS p21 protein activator (GTPase activating protein) 1, also called as p120RasGAP) is a GTPase activation protein that negatively regulates Ras by catalyzing the hydrolysis of active Ras-GTP to inactive Ras-GDP. In this study, we addressed possible roles of RASA1 in melanoma tumorigenesis. To determine RASA1 level in melanocytic lesions, immunohistochemical analysis of RASA1 on human melanoma tissue microarray (TMA) containing nevi (n=34), primary melanomas (n=63), lymph node metastasis (n=35), and distal metastasis (n=29) was performed. This analysis has shown the inverse correlation of RASA1 expression to melanoma progression. Functionally, ectopic expression of RASA1 mutant increased soft-agar colony formation and tumor growth while that of wild-type RASA1 decreased. shRNA-mediated knock down of RASA1 promoted soft-agar colony formation and invasion of melanocytes and melanoma cells with BRAF mutation as a consequence of the elevated Ras activity. Here, we established the functional significance of novel somatic missense mutations in RASA1 identified in our WGS study. Collectively, these data implicated that RASA1 inactivation by loss of expression and/or mutation may promote melanoma tumorigenesis. Citation Format: Hyeran Sung, Li Ding, Krishna L. Kanchi, Jane L. Messina, Vernon K. Sondak, Mulé J. James, Richard K. Wilson, Jeffrey S. Weber, Minjung Kim. RASA1 alteration promotes melanoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2014-442