Cell Cycle Restriction Is More Important Than Apoptosis Induction for RASSF1A Protein Tumor Suppression

The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle. In part, cell cycle regulation appears to be dependent upon the ability of RASSF1A to complex with microtubules and regulate their dynamics. Which property of RASSF1A, apoptosis induction or microtubule regulation, is responsible for its tumor suppressor function is not known. We have identified a short conserved motif that is essential for the binding of RASSF family proteins with microtubule-associated proteins. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. Therefore, it appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects.     

The Tumor Suppressor Gene,RASSF1A,Is Essential for Protection against Inflammation -Induced Injury

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a^+/−, Rassf1a^−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a ^IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a^−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a^−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a^−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.     

Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity

The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity.     

The RASSF1A tumor suppressor activates Bax via MOAP-1

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. RASSF1A may serve as a node in the integration of signaling pathways controlling a range of critical cellular functions including cell cycle, genomic instability, and apoptosis. The mechanism of action of RASSF1A remains under investigation. We now identify a novel pathway connecting RASSF1A to Bax via the Bax binding protein MOAP-1. RASSF1A and MOAP-1 interact directly, and this interaction is enhanced by the presence of activated K-Ras. RASSF1A can activate Bax via MOAP-1. Moreover, activated K-Ras, RASSF1A, and MOAP-1 synergize to induce Bax activation and cell death. Analysis of a tumor-derived point mutant of RASSF1A showed that the mutant was defective for the MOAP-1 interaction and for Bax activation. Moreover, inhibition of RASSF1A by shRNA impaired the ability of K-Ras to activate Bax. Thus, we identify a novel pro-apoptotic pathway linking K-Ras, RASSF1A and Bax that is specifically impaired in some human tumors.     

Tumor Suppressor Ras Association Domain Family 5 (RASSF5/NORE1) Mediates Death Receptor Ligand-induced Apoptosis

Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.     

N-terminal RASSF family: RASSF7-RASSF10

Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children''s cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analyzed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7–RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs.Key words: N-terminal RASSF, RAS, cancer, epigenetic, tumor suppressor     

N-terminal RASSF family

Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children’s cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analysed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7-RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs.     

53BP1‐mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double‐strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.     

The tumour suppressor RASSF1A is a novel substrate of PKC

Ras association domain family 1A (RASSF1A) is a tumour suppressor that contains an amino-terminal cysteine-rich region, similar to the diacylglycerol (DAG)-binding domain (C1 domain) found in the protein kinase C (PKC) family of proteins, and a carboxy-terminal Ras-association (RA) domain. In the present study, RASSF1A was identified as a substrate for PKC. Using classical biochemical approaches, it was established that S197 and S203 within the RA domain of RASSF1A are phosphorylated by PKC in vitro and in vivo. Unlike the WT protein, the S197, 203D double mutant of RASSF1A failed to modulate microtubule organization and perinuclear vimentin collapse. By contrast, the equivalent AA mutant of RASSF1A phenocopied the WT protein. These findings indicate that PKC phosphorylation of RASSF1A regulates its ability to reorganize the microtubule network.     

The tumor suppressor RASSF1A in human carcinogenesis: an update

Loss of heterozygosity of the small arm of chromosome 3 is one of the most common alterations in human cancer. Most notably, a segment in 3p21.3 is frequently lost in lung cancer and several other carcinomas. We and others have identified a novel Ras effector at this segment, which was termed Ras Association Domain family 1 (RASSF1A) gene. RASSF1 consists of two main variants (RASSF1A and RASSF1C), which are transcribed from distinct CpG island promoters. Aberrant methylation of the RASSF1A promoter region is one of the most frequent epigenetic inactivation events detected in human cancer and leads to silencing of RASSF1A. Hypermethylation of RASSF1A was commonly observed in primary tumors including lung, breast, pancreas, kidney, liver, cervix, nasopharyngeal, prostate, thyroid and other cancers. Moreover, RASSF1A methylation was frequently detected in body fluids including blood, urine, nipple aspirates, sputum and bronchial alveolar lavages. Inactivation of RASSF1A was associated with an advanced tumor stage (e.g. bladder, brain, prostate, gastric tumors) and poor prognosis (e.g. lung, sarcoma and breast cancer). Detection of aberrant RASSF1A methylation may serve as a diagnostic and prognostic marker. The functional analyses of RASSF1A reveal an involvement in apoptotic signaling, microtubule stabilization and mitotic progression. The tumor suppressor RASSF1A may act as a negative Ras effector inhibiting cell growth and inducing cell death. Thus, RASSF1A may represent an epigenetically inactivated bona fide tumor suppressor in human carcinogenesis.     

RASSF tumor suppressor gene family: Biological functions and regulation

Genetic changes through allelic loss and nucleic acid or protein modifications are the main contributors to loss of function of tumor suppressor proteins. In particular, epigenetic silencing of genes by promoter hypermethylation is associated with increased tumor severity and poor survival. The RASSF (Ras association domain family) family of proteins consists of 10 members, many of which are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers such as leukemia, melanoma, breast, prostate, neck, lung, brain, colorectal and kidney cancers. In addition to their tumor suppressor function, RASSF proteins act as scaffolding agents in microtubule stability, regulate mitotic cell division, modulate apoptosis, control cell migration and cell adhesion, and modulate NFκB activity and the duration of inflammation. The ubiquitous functions of these proteins highlight their importance in numerous physiological pathways. In this review, we will focus on the biological roles of the RASSF family members and their regulation.     

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.     

RASSF1A suppresses melanoma development by modulating apoptosis and cell-cycle progression

The tumor suppressor candidate gene Ras association domain family 1, isoform A (RASSF1A) encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A-deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell-cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell-cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.     

RASSF1A在细胞自噬及凋亡中的功能

肿瘤抑制因子Ras相关结构域家族成员1A（Ras association domain family 1A，RASSF1A）是Ras超家族蛋白重要的下游效应因子，具有调控自噬及凋亡的作用。自噬及凋亡是影响机体生存发育的重要生命过程，其调节紊乱与肿瘤的发生发展密切相关。本文针对RASSF1A对自噬及凋亡的调节机制及其与肿瘤发生发展之间的关系展开综述，分析翻译后修饰对于RASSF1A调节自噬及凋亡过程中功能切换的作用，探讨自噬及凋亡在肿瘤发生中的调节作用，以期为RASSF1A启动子高甲基化型肿瘤的治疗提供新思路。     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor-suppressor network

The Ras Association Domain Family 1A (RASSF1A) gene is one of the most frequently silenced genes in human cancer. RASSF1A has been shown to interact with the proapoptotic kinase MST1. Recent work in Drosophila has led to the discovery of a new tumor-suppressor pathway involving the Drosophila MST1 and MST2 ortholog, Hippo, as well as the Lats/Warts serine/threonine kinase and a protein named Salvador (Sav). Little is known about this pathway in mammalian cells. We report that complexes consisting of RASSF1A, MST2, WW45 (the human ortholog of Sav), and LATS1 exist in human cells. MST2 enhances the RASSF1A-WW45 interaction, which requires the C-terminal SARAH domain of both proteins. Components of this complex are localized at centrosomes and spindle poles from interphase to telophase and at the midbody during cytokinesis. Both RASSF1A and WW45 activate MST2 by promoting MST2 autophosphorylation and LATS1 phosphorylation. Mitosis is delayed in Rassf1a(-/-) mouse embryo fibroblasts and frequently results in cytokinesis failure, similar to what has been observed for LATS1-deficient cells. RASSF1A, MST2, or WW45 can rescue this defect. The complex of RASSF1A, MST2, WW45, and LATS1 consists of several tumor suppressors, is conserved in mammalian cells, and appears to be involved in controlling mitotic exit.     

Control of APC-Cdc20 by the Tumor Suppressor RASSF1A

The tumor suppressor gene RASSF1A is frequently hypermethylated in various tumors. However, how RASSF1A functions in tumor suppression was unknown. Our study shows that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression by inhibiting APC-Cdc20. RASSF1A appears to work in early pro-metaphase, after Emi1 destruction and before activation of the Mad2-dependent spindle checkpoint. Loss of RASSF1A expression induces premature APC activation, thereby resulting in acceleration of mitotic cyclin degradation and mitotic progression as well as induction of mitotic abnormalities. Thus, RASSF1A plays a critical role in mitotic progression and tumor suppression by controlling APC-Cdc20 activity.     

RASSF1A: A potential novel therapeutic target against cardiac hypertrophy

Cardiac hypertrophy is a key risk factor for chronic heart failure. Current treatments predominantly focus on both reducing the peripheral vascular resistance and activating nerve-humoral system. However, these efforts can't reverse cardiac hypertrophy fundamentally. Ras association domain family 1 isoform A (RASSF1A) is a regulatory tumor suppressor whose inactivation by inappropriate promoter methylation has been implicated in the development of many human cancers. Recently, there have been a number of studies investigating the roles of RASSF1A in the pathophysiology of cardiac hypertrophy. In this review, we focus on the present progresses of cardiac RASSF1A under physiological and pathological conditions, trying to systematically elucidate how the RASSF1A-mediated signal pathways contribute to the maintenance of normal cardiac myocyte structure and function and lead to the regression of pathological cardiac hypertrophy. These pathways exert multiple functions such as regulating cardiac contractility, physiologically increasing stability of microtubule, preventing cardiac dysfunction, attenuating interstitial fibrosis and mediating cell apoptosis. These specific roles are highly relevant with cardiac hemodynamics and therapeutic strategies, indicating RASSF1A may have the potential to reverse pathological cardiac hypertrophy thus prevent heart failure fundamentally.     

Tumor suppressor RASSF1A promoter: p53 binding and methylation

Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ～300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment.