Suppressor Ras-association Domain Family Research Articles

A dRASSF-STRIPAK-Imd-JAK/STAT axis controls antiviral immune response in Drosophila.

Host antiviral immunity suffers strong pressure from rapidly evolving viruses. Identifying host antiviral immune mechanisms has profound implications for developing antiviral strategies. Here, we uncover an essential role for the tumor suppressor Ras-association domain family (RASSF) in Drosophila antiviral response. Loss of dRassf in fat body leads to increased vulnerability to viral infection and impaired Imd pathway activation accompanied by detrimental JAK/STAT signaling overactivation. Mechanistically, dRASSF protects TAK1, a key kinase of Imd pathway, from inhibition by the STRIPAK PP2A phosphatase complex. Activated Imd signaling then employs the effector Relish to interfere with the dimerization of JAK/STAT transmembrane receptor Domeless, therefore preventing excessive JAK/STAT signaling. Moreover, we find that RASSF and STRIPAK PP2A complex are also involved in antiviral response in human cell lines. Our study identifies an important role for RASSF in antiviral immunity and elucidates a dRASSF-STRIPAK-Imd-JAK/STAT signaling axis that ensures proper antiviral responses in Drosophila.

DNA Damage Triggers the Nuclear Accumulation of RASSF6 Tumor Suppressor Protein via CDK9 and BAF53 To Regulate p53 Target Gene Transcription.

RASSF6, a member of the tumor suppressor Ras-association domain family (RASSF) proteins, regulates cell cycle arrest and apoptosis via p53 and plays a tumor suppressor role. We previously reported that RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. In this study, we demonstrated that RASSF6 has nuclear localization and nuclear export signals and that DNA damage triggers the nuclear accumulation of RASSF6. We found that RASSF6 directly binds to BAF53, the component of SWI/SNF complex. DNA damage induces CDK9-mediated phosphorylation of BAF53, which enhances the interaction with RASSF6 and increases the amount of RASSF6 in the nucleus. Subsequently, RASSF6 augments the interaction between BAF53 and BAF60a, another component of the SWI/SNF complex, and further promotes the interaction of BAF53 and BAF60a with p53. BAF53 silencing or BAF60a silencing attenuates RASSF6-mediated p53 target gene transcription and apoptosis. Thus, RASSF6 is involved in the regulation of DNA damage-induced complex formation, including BAF53, BAF60a, and p53.

Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.

The tumor suppressor RASSF1A modulates inflammation and injury in the reperfused murine myocardium

Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type-specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury.

The RASSF6 Tumor Suppressor Protein Regulates Apoptosis and Cell Cycle Progression via Retinoblastoma Protein.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. RASSF6 is frequently suppressed in human cancers, and its low expression level is associated with poor prognosis. RASSF6 regulates cell cycle arrest and apoptosis and plays a tumor suppressor role. Mechanistically, RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. However, RASSF6 also induces cell cycle arrest and apoptosis in a p53-negative background, which implies that the tumor suppressor function of RASSF6 does not depend solely on p53. In this study, we revealed that RASSF6 mediates cell cycle arrest and apoptosis via pRb. RASSF6 enhances the interaction between pRb and protein phosphatase. RASSF6 also enhances P16INK4A and P14ARF expression by suppressing BMI1. In this way, RASSF6 increases unphosphorylated pRb and augments the interaction between pRb and E2F1. Moreover, RASSF6 induces TP73 target genes via pRb and E2F1 in a p53-negative background. Finally, we confirmed that RASSF6 depletion induces polyploid cells in p53-negative HCT116 cells. In conclusion, RASSF6 behaves as a tumor suppressor in cancers with loss of function of p53, and pRb is implicated in this function of RASSF6.

BCL-XL binds and antagonizes RASSF6 tumor suppressor to suppress p53 expression.

RASSF6, a member of the tumor suppressor Ras-association domain family proteins, induces apoptosis in the caspase-dependent and caspase-independent manners. RASSF6 interacts with MDM2 and stabilizes p53. BCL-XL is a prosurvival member of BCL-2 family proteins. BCL-XL directly inhibits proapoptotic BAX and BAK. BCL-XL also traps tBID, a proapoptotic activator BH3-only protein, and sequesters p53. In addition, BCL-XL regulates the mitochondrial membrane permeability via voltage-dependent anion channel. In these manners, BCL-XL plays an antiapoptotic role. We report the interaction of BCL-XL with RASSF6. BCL-XL inhibits the interaction between RASSF6 and MDM2 and suppresses p53 expression. Consequently, BCL-XL antagonizes RASSF6-mediated apoptosis. Thus, the inhibition of RASSF6-mediated apoptosis also underlies the prosurvival role of BCL-XL.

Domain analysis of Ras‐association domain family member 6 upon interaction with MDM2

The tumor suppressor Ras-association domain family member 6 (RASSF6) has Ras-association domain (RA) and Salvador/RASSF/Hippo domain (SARAH). RASSF6 antagonizes MDM2, stabilizes p53, and induces apoptosis and cell cycle arrest. We previously demonstrated the interaction between RASSF6 and MDM2, but did not determine how both proteins interact with each other. We have shown here that N-terminal, RA, and SARAH domains of RASSF6 interact with MDM2 at distinct regions. RA binds to the RING-finger region of MDM2 and stabilizes p53. SARAH binds RA and blocks the interaction between RA and MDM2. RA overexpression induces p53-dependent apoptosis and senescence. In the presence of active KRas, the interaction between RA and MDM2 is recovered. In this way, RA and SARAH play an important role in Ras-mediated regulation of p53.

The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes.

AimsTumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes.Methods and resultsWe used RASSF1A knockout (RASSF1A−/−) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-α (10 µg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A−/− mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-α receptor complex. In the absence of RASSF1A, signal transmission from the TNF-α receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca2+ channel and ryanodine receptors.ConclusionOur data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets.

Mitotic Arrest by Tumor Suppressor RASSF1A Is Regulated via CHK1 Phosphorylation

The tumor suppressor RAS-association domain family 1 isoform A (RASSF1A) is known to play an important role in cell-cycle regulation. However, the molecular details about RASSF1A protein regulation are unclear. In this report, checkpoint kinase 1 (CHK1) is identified as a novel RASSF1A kinase that phosphorylates RASSF1A in vitro and under cellular conditions. Using tandem mass spectrometry and biochemical analysis, it was determined that CHK1 phosphorylates RASSF1A on Serine 184, which has been shown to be mutated in a subset of human primary nasopharyngeal carcinomas. Furthermore, Serine 184 phosphorylation of RASSF1A was significantly diminished by a CHK1-specific kinase inhibitor. Similarly, a kinase-dead CHK1 mutant was unable to phosphorylate Serine 184 whereas constitutively active-CHK1 enhanced phosphorylation. Molecular substitution of Serine 184 with aspartic acid, mimicking phosphorylation, abolished the ability of RASSF1A to interact with microtubules and induce M-phase arrest. Combined, these data indicate that phosphorylation of RASSF1A by CHK1 is important for mitotic regulation and provide valuable new insight into the regulatory mechanisms of RASSF1A function. This study reveals that CHK1-mediated phosphorylation of RASSF1A, at Serine 184, plays an important role in cell-cycle regulation and highlights that mutation of this CHK1 phosphorylation site in nasopharyngeal carcinoma has disease relevance.

The tumor suppressor RAS-association domain family protein 1A (RASSF1A) regulates TNF-alpha signaling in cardiomyocytes

Purpose: Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Here we show a novel regulator of TNF-α signaling, the Ras-association domain family 1 isoform A (RASSF1A), which regulates cardiac contractility and intracellular calcium through interaction with TNFR1. Methods and results: We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10μg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/mL), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P<0.05) which is consistent with previously published data (Circulation 2004; 109:406-411). However, KO mice showed a blunted contractile response following acute TNF-a treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p<0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNFR1 in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Mechanistically, RASSF1A is essential in regulating calcium transients and contractility in cardiomyocytes downstream of TNF-α signaling through regulation of cytoplasmic phospholipase A2 (cPLA2) and phosphorylation of calcium handling molecules. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFκB, a downstream target of TNF-α. Conclusion: Our data indicates an essential role of RASSF1A in regulating TNF-α signaling in cardiomyocytes. Moreover, our present work contributes a novel effector pathway via RASSF1A which transmits the positive inotropic effect of TNF-α and should therefore be preserved or even stimulated in the treatment of heart failure. In addition, enhancement of RASSF1A function/expression might well benefit patients with heart failure.

Methylation of RASSF1A gene promoter is regulated by p53 and DAXX

Inactivation of the tumor suppressor Ras-association domain family 1 isoform A (RASSF1A) due to epigenetic silencing occurs in a variety of human cancers, and still largely unknown are the regulators and mechanisms underlying RASSF1A gene promoter methylation. Herein, we report that this methylation is regulated by p53 and death-associated protein 6 (DAXX) in acute lymphoblastic leukemia (ALL). We found that p53 bound to the RASSF1A promoter, recruiting DAXX as well as DNA methyltransferase 1 (DNMT1) for DNA methylation, which subsequently resulted in inactivation of RASSF1A in wild-type p53 ALL cells. Although the presence of p53 was required for the recruitment of DAXX and DNMT1 to the RASSF1A promoter, fluctuation in p53 protein levels did not affect the rates of RASSF1A methylation. Conversely, methylation of RASSF1A promoter was critically controlled by DAXX, as the enforced overexpression of DAXX led to enhanced RASSF1A promoter methylation, whereas inhibition of DAXX reduced RASSF1A methylation. Interestingly, we found that the p53/DAXX-mediated RASSF1A methylation regulated murine double minute 2 (MDM2) protein stability in ALL. Our results reveal a novel function for p53 in the methylation of RASSF1A promoter by its interaction with DAXX. Discovery of this mechanism provides new insight into the interactions among the tumor-related factors p53, RASSF1A, DAXX, and MDM2 in cancer pathogenesis.

The tumor suppressor RASSF1A is a novel effector of small G protein Rap1A

Rap1A is a small G protein implicated in a spectrum of biological processes such as cell proliferation, adhesion, differentiation, and embryogenesis. The downstream effectors through which Rap1A mediates its diverse effects are largely unknown. Here we show that Rap1A, but not the related small G proteins Rap2 or Ras, binds the tumor suppressor Ras association domain family 1A (RASSF1A) in a manner that is regulated by phosphorylation of RASSF1A. Interaction with Rap1A is shown to influence the effect of RASSF1A on microtubule behavior.

Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type-specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.

Frequent hypermethylation of RASSF1A tumour suppressor gene promoter and presence of Merkel cell polyomavirus in small cell lung cancer

In small cell lung cancer (SCLC), hypermethylation of the tumour suppressor Ras association domain family 1A ( RASSF1A) is frequent. It is associated with SV40 polyomaviral infection in other tumours. Merkel cell polyomavirus (MCPyV) infection has been reported in Merkel cell carcinoma (MCC), a neuroendocrine carcinoma with biological similarity to SCLC. In our study, we investigated polyomavirus infection (SV40 and MCPyV) and promoter hypermethylation of the tumour suppressors RASSF1A and p16 in 18 SCLCs (14 primaries and 4 regional lymph node metastases) and 18 blood control samples. MCPyV was found in 39% (7 of 18) of the tumour tissues but not observed in controls. SV40 was not observed in the tumour tissue. RASSF1A promoter hypermethylation (94%; 17 of 18) was more frequent compared to p16 methylation (56%, 10 of 18). We found no significant correlation between RASSF1A or p16 promoter hypermethylation and infection with the investigated polyoma viruses. Our results show a high frequency of hypermethylation of the RASSF1A promoter and occurrence of MCPyV infection in the tumour tissue of SCLC. These events may contribute to the pathogenesis of SCLC.

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