RAS Protein Activator Like 1 Research Articles

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K-AKT pathway.

The significance of the prominent tumor suppressor gene for RAS protein activator-like 1 (RASAL1) could be better understood by combined genetic, clinical, and functional studies. Here, we investigated the oncogenic and clinical impacts of genetic alterations of RASAL1, particularly when coexisting with genetic alterations of the gene for phosphatase and tensin homolog (PTEN), in 9924 cancers of 33 types in the TCGA database. We found common concurrent genetic alterations of the two genes, which were cooperatively associated with activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, with cancer progression and mortality rates being 46.36% and 31.72% with concurrent gene alterations, versus 29.80% and 16.93% with neither gene alteration (HR 1.64, 95% CI 1.46-1.84 and 1.77, 95% CI 1.53-2.05), respectively. This was enhanced by additional tumor protein p53 (TP53) gene alterations, with cancer progression and mortality rates being 47.65% and 34.46% with coexisting RASAL1, PTEN, and TP53 alterations versus 25.30% and 13.11% with no alteration (HR 2.21, 95% CI 1.92-2.56 and 2.76, 95% CI 2.31-3.30), respectively. In the case of breast cancer, this genetic trio was associated with a triple-negative risk of 68.75% versus 3.83% with no genetic alteration (RR 17.94, 95% CI 9.60-33.51), consistent with the aggressive nature of triple-negative breast cancer. Mice with double knockouts of Rasal1 and Pten displayed robust Pi3k pathway activation, with the development of metastasizing malignancies, while single gene knockout resulted in only benign neoplasma. These results suggest that RASAL1, like PTEN, is a critical player in negatively regulating the PI3K-AKT pathway; defect in RASAL1 causes RAS activation, thus initiating the PI3K-AKT pathway signaling, which cannot terminate with concurrent PTEN defects. Thus, the unique concurrent RASAL1 and PTEN defects drive oncogenesis and cancer aggressiveness by cooperatively activating the PI3K-AKT pathway. This represents a robust genetic mechanism to promote human cancer.

Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.

Background/Aim Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. Methods A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. Results DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. Conclusion Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.

RASAL1 and ROS1 Gene Variants in Hereditary Breast Cancer.

Simple SummaryBreast cancer is the second leading cause of death in women. Identifying novel genetic factors conferring BC predisposition is crucial to predict who is at increased risk of developing the disease, allowing for early detection and therapy, and optimized patient management. We identified germline pathogenic variants in familial breast cancer patients in ROS1 and RASAL1 genes. Further analysis in independent patient group will help understanding the role of these novel genes in breast cancer predisposition.Breast cancer (BC) is the second leading cause of death in women. BC patients with family history or clinical features suggestive of inherited predisposition are candidate to genetic testing to determine whether a hereditary cancer syndrome is present. We aimed to identify new predisposing variants in familial BC patients using next-generation sequencing approaches. We performed whole exome sequencing (WES) in first-degree cousin pairs affected by hereditary BC negative at the BRCA1/2 (BReast CAncer gene 1/2) testing. Targeted analysis, for the genes resulting mutated via WES, was performed in additional 131 independent patients with a suspected hereditary predisposition (negative at the BRCA1/2 testing). We retrieved sequencing data for the mutated genes from WES of 197 Italian unrelated controls to perform a case-controls collapsing analysis. We found damaging variants in NPL (N-Acetylneuraminate Pyruvate Lyase), POLN (DNA Polymerase Nu), RASAL1 (RAS Protein Activator Like 1) and ROS1 (ROS Proto-Oncogene 1, Receptor Tyrosine Kinase), shared by the corresponding cousin pairs. We demonstrated that the splice site alterations identified in NPL and ROS1 (in two different pairs, respectively) impaired the formation of the correct transcripts. Target analysis in additional patients identified novel and rare damaging variants in RASAL1 and ROS1, with a significant allele frequency increase in cases. Moreover, ROS1 achieved a significantly higher proportion of variants among cases in comparison to our internal control database of Italian subjects (p = 0.0401). Our findings indicate that germline variants in ROS1 and RASAL1 might confer susceptibility to BC.

Overexpression of RASAL1 Indicates Poor Prognosis and Promotes Invasion of Ovarian Cancer.

RAS protein activator like-1 (RASAL1) exists in numerous human tissues and has been commonly demonstrated to act as a tumor suppressor in several cancers. This study aimed to identify the functional characteristics of RASAL1 in ovarian adenocarcinoma and a potential mechanism of action. We analyzed RASAL1 gene expression in ovarian adenocarcinoma samples and normal samples gained from the GEO and Oncomine databases respectively. Then the relationship between RASAL1 expression and overall survival (OS) was assessed using the Kaplan-Meier method. Furthermore, the biological effect of RASAL1 in ovarian adenocarcinoma cell lines was assessed by Quantitative real time-PCR (qRT-PCR), Cell Counting Kit-8 (CCK-8), western blot, wound healing and transwell assay. The statistical analysis showed patients with higher RASAL1 expression correlated with worse OS. The in vitro assays suggested knockdown of RASAL1 could inhibit cell proliferation, cell invasion and migration of ovarian adenocarcinoma. Moreover, the key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway were also decreased in ovarian adenocarcinoma cells with RASAL1 silencing. These findings provide promising evidence that RASAL1 may be not only a powerful biomarker but also an effective therapeutic target of ovarian adenocarcinoma.

LncRNAPCAT29 inhibits pulmonary fibrosis via the TGF‑β1‑regulated RASAL1/ERK1/2 signal pathway.

Pulmonary fibrosis is a severe respiratory disease characterized by the aggregation of extracellular matrix components and inflammation‑associated injury. Studies have suggested that long non‑coding RNAs (lncRNA) may serve a role in the pathophysiological processes of pulmonary fibrosis. However, the potential molecular mechanisms involving the lncRNA, prostate cancer‑associated transcript29 (lncRNAPCAT29) in the progression of pulmonary fibrosis are yet to be determined. In the present study, the role of lncRNAPCAT29 and the potential signaling mechanism in pulmonary fibrosis progression was investigated. Reverse transcription‑quantitative polymerase chain reaction and immunohistochemistry revealed that the expression levels of lncRNAPCAT29 were downregulated within interstitial lung cells from mice with silica‑induced pulmonary fibrosis. Transfection with lncRNAPCAT29 was associated with upregulated expression of microRNA (miRNA)‑221 and downregulated expression of transforming growth factor‑β1 (TGF‑β1); reduced inflammation and fibrotic progression was also associated with lncRNAPCAT29 transfection. TGF‑β1 expression levels were inhibited within pulmonary fibroblasts due to lncRNAPCAT29 expression; NEDD4 binding protein2 and Plexin‑A4 expression levels were also suppressed. Analysis of the potential mechanism underlying silica‑induced pulmonary fibrosis revealed that the expression levels of RAS protein activator like 1 (RASAL1) and extracellular signal‑regulated kinases 1/2 (ERK1/2) were suppressed due to lncRNAPCAT29 expression. The results of the present study demonstrated that lncRNAPCAT29 induced miRNA‑221 upregulation and TGF‑β1 downregulation. These observations were associated with reduced inflammation and progression of silica‑induced pulmonary fibrosis via the TGF‑β1‑regulated RASAL1/ERK1/2 signaling pathway, which may serve as a potential target for the treatment of pulmonary fibrosis.

MiR-130b-5p promotes proliferation, migration and invasion of gastric cancer cells via targeting RASAL1.

The aim of the present study was to investigate the targeted interaction between microRNA (miR)-130b-5p and RAS protein activator like 1 (RASAL1) gene and elucidate the function of miR-130b-5p in cell proliferation, migration and invasion in gastric cancer. Expression of miR-130b-5p and RASAL1 in seven gastric cell lines was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). MGC803 cells were selected for further study since they exhibited a marked increase in expression of miR-130b-5p accompanied by decreased expression of RASAL1. MGC803 cells were transfected with miR-130b-5p mimics and miR-130b-5p inhibitor using Lipofectamine 2000 for over- and underexpression, respectively, with cells transfected with negative control (NC) sequence as the control. In addition, a luciferase reporter gene assay was performed to evaluate the targeted interaction between miR-130b-5p and RASAL1. Then, alterations in RASAL1 expression were detected by RT-qPCR and western blot analysis following transfection with miR-130b-5p mimics and miR-130b-5p inhibitor. Cell proliferation, colony formation, and migration and invasion ability were detected by MTT, colony formation and Transwell assays, respectively. RASAL1 was demonstrated to be a target gene of miR-130b-5p by luciferase reporter gene assay. In addition, the expression of RASAL1 was significantly lower in MGC803 cells that were transfected with miR-130b-5p mimics and significantly higher in cells transfected with miR-130b-5p inhibitor in comparison with cells transfected with NC (P<0.05). Furthermore, the experimental group transfected with miR-130b-5p mimics manifested significantly higher cell proliferation, increased colony formation and increased migratory and invasive capacities (P<0.05). By contrast, cells transfected with miR-130b-5p inhibitor exhibited significantly lower cell proliferation, decreased colony formation and decreased migratory and invasive capacities, compared with cells transfected with NC (P<0.05). In conclusion, RASAL1 was demonstrated to be a target gene of miR-130b-5p. Overexpression of miR-130b-5p results in promoted proliferation, colony formation and migration and invasion abilities through targeted modulation of RASAL1.

RASAL1 inhibits HepG2 cell growth via HIF-2α mediated gluconeogenesis.

RAS protein activator like 1 (RASAL1) is a member of the RAS GTPase-activating protein (GAP) family, and has been identified as a tumor suppressor in various types of cancer. In the present study, it was determined that decreased levels of RASAL1 were accompanied by a higher pathological stage and larger tumor size in human liver cancer. Therefore, it was hypothesized that RASAL1 may serve an inhibitory role in liver cancer. In the present study, the following was demonstrated: i) Exogenous expression of RASAL1 may inhibit the proliferation and invasion ability of HepG2 cells; ii) overexpression of RASAL1 may downregulate HIF-2α transcription activity and HIF-2α-mediated gluconeogenesis through extracellular signal-related kinase 1/2 activation; iii) RASAL1 may reduce the xenograft tumor size in nude mice by inhibiting the expression of hypoxia-inducible factor (HIF)-2α and gluconeogenesis enzymes. These data suggest that the RASAL1/HIF-2α axis may serve an essential role in the growth of HepG2 cells, and that this signaling cascade may be a novel therapeutic target for the treatment of liver cancer.

RAS protein activator-like 1 is functionally involved in hypoxia resistance in breast cancer cells by targeting hypoxia inducible factor-1α.

RAS protein activator-like 1 (RASAL1) is a member of the RAS GTPase-activating protein family, and previous studies indicate that RASAL1 is involved in the progression of hypoxia resistance in breast cancer cells. In the present study, increased levels of hypoxia inducible factor-1α (HIF-1α) were observed to be accompanied with increased expression of RASAL1 in the breast cancer cell lines MCF-7 and MDA-MB-231 cells under hypoxia. Based on this, it was postulated that RASAL1 may serve a functional role in the development of hypoxia resistant in breast cancer cells. In the present study it was demonstrated that: i) Exogenous expression of RASAL1 in MCF-7 and MDA-MB-231 sensitized its reaction to the treatment of hypoxia, which is associated with its ability to directly reduce HIF-1α expression, inhibit migration activity and decrease the accumulation of reactive oxygen species (ROS); ii) knockdown of RASAL1 reversed its reaction to treatment with hypoxia; iii) RASAL1 directly regulated the expression of HIF-1α through the ROS-mediated, extracellular signal-regulated kinase and Akt pathway. These findings provide direct evidence that the RASAL1/HIF-1α axis may serve an essential role in the hypoxia resistance of breast cancer cells, suggesting that this signaling cohort may serve as a novel therapeutic target for the treatment of breast cancer.

RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis.

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.

Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

Hypoxia-Induced Changes in DNA Methylation Alter RASAL1 and TGFβ1 Expression in Human Trabecular Meshwork Cells.

PurposeFibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) β1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFβ1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions.MethodsGlobal DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFβ1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O2, 5%CO2 and 37°C environment. NTM and GTM cells were treated with TGFβ1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5μM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression.ResultsWe found increased DNA methylation, increased TGFβ1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFβ1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFβ1 and COL1A1 expression, and increased RASAL1 expression in GTM cells.ConclusionsTGFβ1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFβ1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.

A new subtype of high-grade mandibular osteosarcoma with RASAL1/MDM2 amplification

In contrast to long bone osteosarcoma, mandibular osteosarcoma is highly heterogeneous and morphologically overlaps with benign tumors, obscuring diagnosis and treatment selection. Molecular characterization is difficult due to the paucity of available specimens of this rare disease. We aimed to characterize the spectrum of mandibular osteosarcoma using immunohistochemistry and molecular techniques (quantitative polymerase chain reaction and sequencing) and compare them with benign fibro-osseous lesions. Forty-nine paraffin-embedded mandible osteosarcoma tissue samples were collected retrospectively and compared with 10 fibrous dysplasia and 15 ossifying fibroma cases. These were analyzed for molecular markers thought to differ between the different diseases and subtypes: MDM2 (murine double-minute type 2) overexpression, GNAS (guanine nucleotide-binding protein/α subunit) mutations, and amplification of MDM2 and/or RASAL1 (RAS protein activator like 1). Five fibroblastic high-grade osteosarcoma subtypes showed MDM2 amplification, including 2 with a microscopic appearance of high-grade osteosarcoma with part low-grade osteosarcoma (differentiated/dedifferentiated osteosarcoma) and MDM2 overexpression. The other 3 contained a coamplification of MDM2 and RASAL1, a signature also described for juvenile ossifying fibroma, with no overexpression of MDM2. These were of the giant cell-rich high-grade osteosarcoma, with areas mimicking juvenile ossifying fibroma (ossifying fibroma-like osteosarcoma). Our results show that some diagnosed high-grade osteosarcomas are differentiated/dedifferentiated osteosarcomas and harbor an overexpression and amplification of MDM2. In addition, juvenile ossifying fibromas can potentially evolve into giant cell-rich high-grade osteosarcomas and are characterized by a RASAL1 amplification (osteosarcoma with juvenile ossifying fibroma-like genotype). Thus, the presence of a RASAL1 amplification in ossifying fibroma may indicate a requirement for closer follow-up and more aggressive management.

Mycophenolic acid reverses TGF beta-induced cell motility, collagen matrix contraction and cell morphology in vitro.

The aim of this study was to elucidate functional and molecular effects of mycophenolic acid (MPA) on non-lymphatic, kidney epithelial cells treated with transforming growth factor (TGF). MPA effects were studied using HK2 cells incubated with EGF and TGF. The reversibility of these effects was verified using guanosine and 8-aminoguanosine. The following assays were applied: cell proliferation, viability, collagen matrix contraction, scratch wound closure, spindle index, FACS with anti-CD29 and anti-CD326, promoter demethylation of RAS protein activator like 1 (RASAL1), as well as gene expression of RASAL1, integrin 1ß (ITGB1) (CD29) and epithelial cell adhesion molecule (EpCam) (CD326). Cell proliferation was inhibited by increasing concentrations of MPA, whereas neither apoptosis nor cytotoxicity was detected. Stimulation with EGF and/or TGF led to a significant collagen matrix contraction that was successfully inhibited by MPA. In addition, scratch wound closure was inhibited by incubation with TGF alone or with EGF. Under the same conditions, cell morphology (spindle shape) and molecular phenotype (ITGB1(High)EpCam(Low)/ITGB1(Low)EpCam(High)) were both significantly changed, suggesting an epithelial to mesenchymal transformation. Cell morphology and motility, as well as molecular phenotype, were reversible after MPA treatment with TGF transformation in both presence/absence of EGF, thereby suggesting a correlation with the previously described antifibrotic effects of MPA. Dysregulation of TGF signal transduction appears to be related to progression of fibrosis. A TGF-transformed kidney epithelial cell line derived from human proximal tubules was used to study whether the immunosuppressive drug: MPA possesses any functional or molecular antifibrotic effects. Functional and morphological in vitro changes induced by both the TGF and epithelial-growth-factor were reversible by treatment with MPA. An inhibitory effect of MPA on the TGF pathway appears to be responsible for the previously described antifibrotic effects of the MPA in the COL4A3-deficient mouse model of renal fibrosis.

RASAL1 attenuates gastric carcinogenesis in nude mice by blocking RAS/ERK signaling.

Recent studies have suggested that the RAS protein activator like-1 (RASAL1) functions as a tumor suppressor in vitro and may play an important role in the development of gastric cancer. However, whether or not RASAL1 suppresses tumor growth in vivo remains to be determined. In the present study, we investigated the role of RASAL1 in gastric carcinogenesis using an in vivo xenograft model. A lentiviral RASAL1 expression vector was constructed and utilized to transfect the human poorly differentiated gastric adenocarcinoma cell line, BGC-823. RASAL1 expression levels were verified by quantitative real-time RT-PCR and Western blotting analysis. Then, we established the nude mice xenograft model using BGC-823 cells either over-expressing RASAL1 or normal. After three weeks, the results showed that the over-expression of RASAL1 led to a significant reduction in both tumor volume and weight compared with the other two control groups. Furthermore, in xenograft tissues the increased expression of RASAL1 in BGC-823 cells caused decreased expression of p-ERK1/2, a downstream moleculein the RAS/RAF/MEK/ERK signal pathway. These findings demonstrated that the over-expression of RASAL1 could inhibit the growth of gastric cancer by inactivation of the RAS/RAF/MEK/ERK pathway in vivo. This study indicates that RASAL1 may attenuate gastric carcinogenesis.

Hypermethylation and Clinicopathological Significance of RASAL1 Gene in Gastric Cancer

Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer. Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting. The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR. Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.

Enforced expression of RASAL1 suppresses cell proliferation and the transformation ability of gastric cancer cells

RAS protein activator like 1 (RASAL1) is a member of the RAS GTPase-activating protein (GAP) family, and it is an important molecule in the regulation of RAS activation. In the present study, we investigated the role of RASAL1 in gastric carcinogenesis. Decreased expression pattern of RASAL1 in gastric cancer tissues and cell lines was found in protein and RNA levels, although there was no statistically significant relationship between RASAL1 and clinicopathological features. Restored expression of RASAL1 induced by DNA methylation inhibitor 5-aza-2'-deoxycytidine (5'-AZA) and HDAC inhibitor trichostatin A (TSA) implied that RASAL1 expression is regulated by epigenetic mechanisms. The biological role of RASAL1 in gastric carcinogenesis was determined by invitro tumorigenicity assays. Overexpression of RASAL1 showed suppression of cell proliferation due to cell apoptosis. Subsequently, enforced expression of RASAL1 repressed significantly the gastric cancer cell transformation ability. These findings demonstrated that decreased RASAL1 expression is a characteristic of gastric cancer and regulated by epigenetic mechanisms. RASAL1 may be a functional tumor suppressor involved in gastric cancer. This study provides novel insights into the biological role of RASAL1 in gastric carcinogenesis.

In vivo and in vitro expression of the RASAL1 gene in human gastric adenocarcinoma and its clinicopathological significance

Recent studies have suggested that the RAS protein activator like-1 (RASAL1) is a potential tumor suppressor, which is found to be reduced in certain human cancers. Its downregulation is involved in the progression of malignancies. However, whether or not RASAL1 plays a role in the development of gastric cancer remains to be determined. Our study aimed to clarify the role of RASAL1 in the progression of gastric adenocarcinoma. The expression of RASAL1 in primary gastric adenocarcinoma tissue specimens was determined by immunohistochemistry. The expression of RASAL1 mRNA and protein was detected by RT-PCR and western blotting in gastric adenocarcinoma cell lines with varying differentiation statuses, including well-differentiated MKN-28, moderately differentiated SGC-7901 and poorly differentiated BGC-823, respectively. A normal gastric epithelial cell line, GES-l, was used as the control line. The immunohistochemical results revealed that the expression of the RASAL1 protein was mainly observed in the cytoplasm. Among 50 cases of gastric adenocarcinoma tissues, 12 cases were identified as (-), 23 cases (+), 13 cases (++) and 2 cases (+++). Among 50 cases of normal gastric tissues, 16 cases were (++) and 34 cases (+++). The expression of the RASAL1 protein was found to be decreased in the gastric adenocarcinoma tissue compared with normal gastric tissue (p<0.01). Moreover, in the gastric carcinoma tissues, the expression of RASAL1 was correlated with carcinoma diameter, differentiation grades, invasive depth, lymph node metastasis and TNM. Additionally, the RASAL1 mRNA and proteins were decreased in the three gastric adenocarcinoma cell lines compared with the normal gastric epithelial cell line GES-l. In addition, the downregulation of RASAL1 correlated with the differentiation status of cancer cell lines. Based on the above investigation, we conclude that expression of the RASAL1 gene is decreased in gastric carcinoma tissues and cell lines. The results indicate that RASAL1 may be important in the tumorigenesis and development of gastric carcinoma.

Reduced expression of RAS protein activator like‐1 in gastric cancer

RAS signaling is frequently deregulated in human neoplasms. However, RAS mutations have been found in only a small proportion of human gastric cancers, implicating other mechanisms in the activation of RAS signaling in gastric tumorigenesis. We have previously reported that decreased expression of RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, contributes to colon tumor progression. In our study, we explored the involvement of decreased RASAL1 expression in gastric tumorigenesis. RASAL1 expression was reduced in 6 of 10 gastric cancer cell lines examined by immunoblotting. Knockdown of RASAL1 increased mitogen-activated protein kinase signaling in response to growth factor stimulation, and the forced expression of RASAL1 reduced proliferation of gastric cancer cells. Immunohistochemical analyses in primary gastric tumors showed that RASAL1 expression was reduced in 23 of 48 (48%) of the gastric cancers but in none of the adenomas (0/10). Methylation of the RASAL1 promoter region and loss of heterozygosity (LOH) at the RASAL1 locus were examined to investigate the causes of RASAL1 silencing. All cell lines with reduced RASAL1 had RASAL1 methylation, and two had LOH. In primary gastric cancers, methylation or LOH was detected in 50% (6/12) of those with reduced RASAL1. Furthermore, RASAL1 expression was restored in some cell lines by histone deacetylase inhibitor treatment. Our findings demonstrate that reduced RASAL1 expression, partly due to genetic and epigenetic changes, contributes to gastric carcinogenesis, and also re-emphasize the importance of RAS signaling in gastric cancer development.

DNA methyltransferase 1 (DMNT1); RAS protein activator like 1 (RASAL1)

DNA methyltransferase 1 (DMNT1); RAS protein activator like 1 (RASAL1)