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Supplementary Figure 2 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

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Supplementary Table 1 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

RASSF1A is a Ras effector that promotes the anti-proliferative properties of Ras. It acts as a scaffold protein that regulates several pro-apoptotic signaling pathways, thereby linking Ras to their regulation. However, accumulating evidence suggests that RASSF1A functions as a regulator of other additional biological processes, such as DNA repair and transcription, thereby implicating Ras in the modulation of these biological processes. The mechanisms by which RASSF1A modulates these processes is not fully understood but likely involves interacting with other effectors associated with these functions and coordinating their activity. Thus, to fully understand how RASSF1A manifests its activity, it is critical to identify RASSF1A interacting partners.Unfortunately, the reagents available for the detection of RASSF1A are of poor quality and also exhibit low sensitivity. Here we describe an immunoprecipitation protocol, taking into consideration the limitations of currently available reagents, that can reliably detect the endogenous interaction between RASSF1A and its binding partners.

RASSF1A is a Ras effector that promotes the anti-proliferative properties of Ras. It acts as a scaffold protein that regulates several pro-apoptotic signaling pathways, thereby linking Ras to their regulation. However, accumulating evidence suggests that RASSF1A functions as a regulator of other additional biological processes, such as DNA repair and transcription, thereby implicating Ras in the modulation of these biological processes. The mechanisms by which RASSF1A modulates these processes is not fully understood but likely involves interacting with other effectors associated with these functions and coordinating their activity. Thus, to fully understand how RASSF1A manifests its activity, it is critical to identify RASSF1A interacting partners.Unfortunately, the reagents available for the detection of RASSF1A are of poor quality and also exhibit low sensitivity. Here we describe an immunoprecipitation protocol, taking into consideration the limitations of currently available reagents, that can reliably detect the endogenous interaction between RASSF1A and its binding partners.

Simple Summary The RASSF1A tumor suppressor can serve as a pro-apoptotic effector of the K-RAS oncoprotein. It is frequently inactivated epigenetically in lung cancer, and genetic inactivation of RASSF1A in transgenic mice enhances the ability of mutant K-RAS to promote tumorigenesis. Here we show that RASSF1A complexes with and stabilizes the protein DAB2IP. DAB2IP is a tumor suppressor itself and acts, in part, as a negative regulator (GAP) for RAS. Thus, loss of RASSF1A results in the reduced expression of DAB2IP, which promotes the activation of wild type RAS. Therefore, RASSF1A negative cells are likely to show enhanced RAS activity. This may be the first example of a RAS effector being able to back-regulate RAS activity. Abstract Lung cancer is the leading cause of cancer-related death worldwide. Lung cancer is commonly driven by mutations in the RAS oncogenes, the most frequently activated oncogene family in human disease. RAS-induced tumorigenesis is inhibited by the tumor suppressor RASSF1A, which induces apoptosis in response to hyperactivation of RAS. RASSF1A expression is suppressed in cancer at high rates, primarily owing to promoter hypermethylation. Recent reports have shown that loss of RASSF1A expression uncouples RAS from apoptotic signaling in vivo, thereby enhancing tumor aggressiveness. Moreover, a concomitant upregulation of RAS mitogenic signaling upon RASSF1A loss has been observed, suggesting RASSF1A may directly regulate RAS activation. Here, we present the first mechanistic evidence for control of RAS activation by RASSF1A. We present a novel interaction between RASSF1A and the Ras GTPase Activating Protein (RasGAP) DAB2IP, an important negative regulator of RAS. Using shRNA-mediated knockdown and stable overexpression approaches, we demonstrate that RASSF1A upregulates DAB2IP protein levels in NSCLC cells. Suppression of RASSF1A and subsequent downregulation of DAB2IP enhances GTP loading onto RAS, thus increasing RAS mitogenic signaling in both mutant- and wildtype-RAS cells. Moreover, co-suppression of RASSF1A and DAB2IP significantly enhances in vitro and in vivo growth of wildtype-RAS cells. Tumors expressing wildtype RAS, therefore, may still suffer from hyperactive RAS signaling when RASSF1A is downregulated. This may render them susceptible to the targeted RAS inhibitors currently in development.

Mutations that activate the RAS oncoproteins are common in cancer. However, aberrant upregulation of RAS activity often occurs in the absence of activating mutations in the RAS genes due to defects in RAS regulators. It is now clear that loss of function of Ras GTPase-activating proteins (RasGAPs) is common in tumors, and germline mutations in certain RasGAP genes are responsible for some clinical syndromes. Although regulation of RAS is central to their activity, RasGAPs exhibit great diversity in their binding partners and therefore affect signaling by multiple mechanisms that are independent of RAS. The RASSF family of tumor suppressors are essential to RAS-induced apoptosis and senescence, and constitute a barrier to RAS-mediated transformation. Suppression of RASSF protein expression can also promote the development of excessive RAS signaling by uncoupling RAS from growth inhibitory pathways. Here, we will examine how these effectors of RAS contribute to tumor suppression, through both RAS-dependent and RAS-independent mechanisms.

Background Dysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa). Methods Serum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis. Results MiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and β-catenin levels were down-regulated in miR-186-5p inhibited PCa cells. Conclusions Our findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer. Electronic supplementary material The online version of this article (10.1186/s12885-018-4258-0) contains supplementary material, which is available to authorized users.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

Sir, the recent editorial of our article describing the first animal model for KRAS-driven lung tumors in a RASSF1A-deficient background (1) succinctly and accurately describes our work. I would like to take the opportunity to elaborate and to describe some additional pertinent experiments.

The TOR pathway is a vital component of cellular homeostasis that controls the synthesis of proteins, nucleic acids and lipids. Its core is the TOR kinase. Activation of the TOR pathway suppresses autophagy, which plays a vital but complex role in tumorigenesis. The TOR pathway is regulated by activation of the Ras-related protein Rheb, which can bind mTOR. The Hippo pathway is a major growth control module that regulates cell growth, differentiation and apoptosis. Its core consists of an MST/LATS kinase cascade that can be activated by the RASSF1A tumor suppressor. The TOR and Hippo pathways may be coordinately regulated to promote cellular homeostasis. However, the links between the pathways remain only partially understood. We now demonstrate that in addition to mTOR regulation, Rheb also impacts the Hippo pathway by forming a complex with RASSF1A. Using stable clones of two human lung tumor cell lines (NCI-H1792 and NCI-H1299) with shRNA-mediated silencing or ectopic overexpression of RASSF1A, we show that activated Rheb stimulates the Hippo pathway, but is suppressed in its ability to stimulate the TOR pathway. Moreover, by selectively labeling autophagic vacuoles we show that RASSF1A inhibits the ability of Rheb to suppress autophagy and enhance cell growth. Thus, we identify a new connection that impacts coordination of Hippo and TOR signaling. As RASSF1A expression is frequently lost in human tumors, the RASSF1A status of a tumor may impact not just its Hippo pathway status, but also its TOR pathway status.

The Ras oncoprotein may participate in driving half of human tumors and as such remains the single most significant therapeutic target in cancer. Attempts to develop targeted inhibitors of RAS have proved extremely challenging. We have developed a novel Ras binding molecule that blocks the ability of Ras to interact with its downstream effector components and thus down-regulates Ras signaling. We have also developed the first direct inhibitor of the RAS effector RALGDS. Both compounds suppresses the transforming activity of mutant forms of Ras as well as the wild type protein when it is over-active due to defects in the NF1 protein. The compounds have no detectable toxicity in animals and readily crosses the blood-brain barrier. They are potently active in xenograft models of Ras mediated transformation and can eradicate the ability of Ras addicted tumor cells to form tumors in animals. We propose the compounds may serve as the basis for the development of new treatments for oncogenic RAS driven tumors and for many Rasopathy symptoms. Citation Format: Geoffrey Justin Clark. Novel RAS inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6374.

The tumor microenvironment (TME) is composed of a heterogeneous biological ecosystem of cellular and non-cellular elements including transformed tumor cells, endothelial cells, immune cells, activated fibroblasts or myofibroblasts, stem and progenitor cells, as well as the cytokines and matrix that they produce. The constituents of the TME stroma are multiple and varied, however cancer associated fibroblasts (CAF) and their contribution to the TME are important in tumor progression. CAF are hypothesized to arise from multiple progenitor cell types, including mesenchymal stem cells. Currently, isolation of TME stroma from patients is complicated by issues such as limited availability of biopsy material and cell stress incurred during lengthy adaptation to atmospheric oxygen (20% O2) in cell culture, limiting pre-clinical studies of patient tumor stromal interactions. Here we describe a microenvironment mimetic in vitro cell culturing system that incorporates elements of the in vivo lung environment, including lung fibroblast derived extracellular matrix and physiological hypoxia (5% O2). Using this system, we easily isolated and rapidly expanded stromal progenitors from patient lung tumor resections without complex sorting methods or growth supplements. These progenitor populations retained expression of pluripotency markers, secreted factors associated with cancer progression, and enhanced tumor cell growth and metastasis. An understanding of the biology of these progenitor cell populations in a TME-like environment may advance our ability to target these cells and limit their effects on promoting cancer metastasis.

Mutant K-RAS has been shown to have both tumor-promoting and -suppressing functions, and growing evidence suggests that the RASSF family of tumor suppressors can act as RAS apoptosis and senescence effectors. It has been hypothesized that inactivation of the RASSF1A tumor suppressor facilitates K-RAS–mediated transformation by uncoupling it from apoptotic pathways such as the Hippo pathway. In human lung tumors, combined activation of K-RAS and inactivation of RASSF1A is closely associated with the development of the most aggressive and worst prognosis tumors. Here, we describe the first transgenic mouse model for activation of K-RAS in the lung in a RASSF1A-defective background. RASSF1A deficiency profoundly enhanced the development of K-RAS–driven lung tumors in vivo. Analysis of these tumors showed loss of RASSF1A-uncoupled RAS from the proapoptotic Hippo pathway as expected. We also observed an upregulation of AKT and RALGEF signaling in the RASSF1A− tumors. Heterozygosity of RASSF1A alone mimicked many of the effects of RAS activation on mitogenic signaling in lung tissue, yet no tumors developed, indicating that nonstandard Ras signaling pathways may be playing a key role in tumor formation in vivo. In addition, we observed a marked increase in inflammation and IL6 production in RASSF1A-deficient tumors. Thus, RASSF1A loss profoundly affects RAS-driven lung tumorigenesis and mitogenic signaling in vivo. Deregulation of inflammatory pathways due to loss of RASSF1A may be essential for RAS-mediated tumorigenesis. These results may have considerable ramifications for future targeted therapy against RAS+/RASSF1A− tumors. Significance: A transgenic mouse model shows that suppression of RASSF1A dramatically enhances Ras-driven tumorigenesis and alters Ras signaling pathway activity. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/10/2614/F1.large.jpg. Cancer Res; 78(10); 2614–23. ©2018 AACR.

The Ras genes are the most frequently mutated oncogenes in human cancer. However, Ras biology is quite complex. While Ras promotes tumorigenesis by regulating numerous growth promoting pathways, activated Ras can paradoxically also lead to cell cycle arrest, death, and Oncogene-Induced Senescence (OIS). OIS is thought to be a critical pathway that serves to protect cells against aberrant Ras signaling. Multiple reports have highlighted the importance of the p53 and Rb tumor suppressors in Ras mediated OIS. However, until recently, the molecular mechanisms connecting Ras to these proteins remained unknown. The RASSF family of tumor suppressors has recently been identified as direct effectors of Ras. One of these members, NORE1A (RASSF5), may be the missing link between Ras-induced senescence and the regulation of p53 and Rb. This occurs both quantitatively, by promoting protein stability, as well as qualitatively via promoting critical pro-senescent post-translational modifications. Here we review the mechanisms by which NORE1A can activate OIS as a barrier against Ras-mediated transformation, and how this could lead to improved therapeutic strategies against cancers having lost NORE1A expression.

Ras is the most frequently activated oncogene found in human cancer, but its mechanisms of action remain only partially understood. Ras activates multiple signaling pathways to promote transformation. However, Ras can also exhibit a potent ability to induce growth arrest and death. NORE1A (RASSF5) is a direct Ras effector that acts as a tumor suppressor by promoting apoptosis and cell cycle arrest. Expression of NORE1A is frequently lost in human tumors, and its mechanism of action remains unclear. Here we show that NORE1A forms a direct, Ras-regulated complex with β-TrCP, the substrate recognition component of the SCF(β-TrCP) ubiquitin ligase complex. This interaction allows Ras to stimulate the ubiquitin ligase activity of SCF(β-TrCP) toward its target β-catenin, resulting in degradation of β-catenin by the 26 S proteasome. However, the action of Ras/NORE1A/β-TrCP is substrate-specific because IκB, another substrate of SCF(β-TrCP), is not sensitive to NORE1A-promoted degradation. We identify a completely new signaling mechanism for Ras that allows for the specific regulation of SCF(β-TrCP) targets. We show that the NORE1A levels in a cell may dictate the effects of Ras on the Wnt/β-catenin pathway. Moreover, because NORE1A expression is frequently impaired in tumors, we provide an explanation for the observation that β-TrCP can act as a tumor suppressor or an oncogene in different cell systems.

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.

RASSF1A is a microtubule-associated protein. This study provides evidence for RASSF1A regulating MT dynamics via segmental binding to provide local stabilization of the MT network, thus facilitating MT rescue. RASSF1A reconfigures the MT network through bundling of nearby MTs and provides a stable platform to maintain 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.

There are six core RASSF family proteins that contain conserved Ras Association domains and may serve as Ras effectors. They lack intrinsic enzymatic activity and appear to function as scaffolding and localization molecules. While initially being associated with pro-apoptotic signaling pathways such as Bax and Hippo, it is now clear that they can also connect Ras to a surprisingly broad range of signaling pathways that control senescence, inflammation, autophagy, DNA repair, ubiquitination and protein acetylation. Moreover, they may be able to impact the activation status of pro-mitogenic Ras effector pathways, such as the Raf pathway. The frequent epigenetic inactivation of RASSF genes in human tumors disconnects Ras from pro-death signaling systems, enhancing Ras driven transformation and metastasis. The best characterized members are RASSF1A and RASSF5 (NORE1A).

The RASSF1A tumor suppressor gene is frequently inactivated by promoter methylation in human tumors. The RASSF1A protein forms an endogenous complex with tubulin and promotes the stabilization of microtubules. Loss of RASSF1A expression sensitizes cells to microtubule destabilizing stimuli. We have observed a strong correlation between the loss of RASSF1A expression and the development of Taxol resistance in primary ovarian cancer samples. Thus, we sought to determine if RASSF1A levels could dictate the response to Taxol and whether an epigenetic therapy approach might be able to reverse the Taxol resistant phenotype of RASSF1A negative ovarian tumor cells. We found that knocking down RASSF1A expression in an ovarian cancer cell line inhibited Taxol-mediated apoptosis and promoted cell survival during Taxol treatment. Moreover, using a combination of small molecule inhibitors of DNA Methyl Transferase enzymes, we were able restore RASSF1A expression and Taxol sensitivity. This identifies a role for RASSF1A in modulating the tumor response to Taxol and provides proof of principal for the use of epigenetic therapy to overcome Taxol resistance.

The manipulation of the cationic lipid structures to increase polynucleotide binding and delivery properties, while also minimizing associated cytotoxicity, has been a principal strategy for developing next-generation transfection agents. The polar (DNA binding) and hydrophobic domains of transfection lipids have been extensively studied; however, the linking domain comprising the substructure used to tether the polar and hydrophobic domains has attracted considerably less attention as an optimization variable. Here, we examine the use of an oxime ether as the linking domain. Hydrophobic oxime ethers were readily assembled via click chemistry by oximation of hydrophobic aldehydes using an aminooxy salt. A facile ligation reaction delivered the desired compounds with hydrophobic domain asymmetry. Using the MCF-7 breast cancer, H1792 lung cancer and PAR C10 salivary epithelial cell lines, our findings show that lipoplexes derived from oxime ether lipids transfect in the presence of serum at higher levels than commonly used liposome formulations, based on both luciferase and green fluorescent protein (GFP) assays. Given the biological compatibility of oxime ethers and their ease of formation, this functional group should find significant application as a linking domain in future designs of transfection vectors.

The RASSF1A tumor suppressor binds and activates proapoptotic MST kinases. The Salvador adaptor protein couples MST kinases to the LATS kinases to form the hippo pathway. Upon activation by RASSF1A, LATS1 phosphorylates the transcriptional regulator YAP, which binds to p73 and activates its proapoptotic effects. However, although serving as an adaptor for MST and LATS, Salvador can also bind RASSF1A. The functional role of the RASSF1A/Salvador interaction is unclear. Although Salvador is a novel tumor suppressor in Drosophila and mice, its role in human systems remains largely unknown. Here we show that Salvador promotes apoptosis in human cells and that Salvador inactivation deregulates the cell cycle and enhances the transformed phenotype. Moreover, we show that although the salvador gene is seldom mutated or epigenetically inactivated in human cancers, it is frequently down-regulated posttranscriptionally. Surprisingly, we also find that although RASSF1A requires the presence of Salvador for full apoptotic activity and to activate p73, this effect does not require a direct interaction of RASSF1A with MST kinases or the activation of the hippo pathway. Thus, we confirm a role for Salvador as a human tumor suppressor and RASSF1A effector and show that Salvador allows RASSF1A to modulate p73 independently of the hippo pathway.

RASSF2 is a novel proapoptotic effector of K-Ras. Inhibition of RASSF2 expression enhances the transforming effects of K-Ras, and epigenetic inactivation of RASSF2 is frequently detected in mutant Ras-containing primary tumors. Thus, RASSF2 is implicated as a tumor suppressor whose inactivation facilitates transformation by disconnecting apoptotic responses from Ras. The mechanism of action of RASSF2 is not known. Here we show that RASSF2 forms a direct and endogenous complex with the prostate apoptosis response protein 4 (PAR-4) tumor suppressor. This interaction is regulated by K-Ras and is essential for the full apoptotic effects of PAR-4. RASSF2 is primarily a nuclear protein, and shuttling of PAR-4 from the cytoplasm to the nucleus is essential for its function. We show that RASSF2 modulates the nuclear translocation of PAR-4 in prostate tumor cells, providing a mechanism for its biological effects. Thus, we identify the first tumor suppressor signaling pathway emanating from RASSF2, we identify a novel mode of action of a RASSF protein, and we provide an explanation for the extraordinarily high frequency of RASSF2 inactivation we have observed in primary prostate tumors.

NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21CIP1 as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21CIP1 expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21CIP1 expression and promotes proliferation. The best characterized activator of p21CIP1 is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21CIP1 via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53. [Cancer Res 2009;69(11):4629–37]

The putative tumor suppressor NORE1A (RASSF5) is a member of the Ras association domain family and is commonly inactivated in human cancer. The closely related gene family member and functional collaborator RASSF1A is a bona fide tumor suppressor and is frequently involved in neuroblastoma. In the present study, we sought to investigate the role of NORE1A in human neuroblastoma. A panel of tumors (36 neuroblastomas and 4 ganglioneuromas) and neuroblastoma cell lines was assessed for NORE1A gene expression by Taqman quantitative RT-PCR. Promoter methylation was quantitatively determined by methylation sensitive pyrosequencing. The antitumourigenic role was functionally investigated in Nore1a transfected SK-N-BE (2) cells by fluorescent inhibition of caspase activity and BrdU incorporation assays. Neuroblastoma cells showed very low or absent NORE1A mRNA expression, which could not be reversed by trichostatin A or 5-aza-cytidine treatments. Neuroblastoma tumors showed suppressed NORE1A gene expression that was particularly pronounced in cases without MYCN amplification or 1p loss. Methylation of the NORE1A promoter was not observed in primary tumors and only one out of seven neuroblastoma cell lines displayed weak partial methylation. Transient expression of Nore1a resulted in enhanced apoptosis and delayed cell cycle progression. In conclusion NORE1A appears to be strongly suppressed in neuroblastic tumors and reconstitution of its expression diminishes the tumorigenic phenotype. Promotor methylation is not a common mechanism responsible for NORE1A transcriptional suppression in this tumor type.

RASSF1A (Ras association domain family 1 isoform A) is a recently discovered tumor suppressor whose inactivation is implicated in the development of many human cancers. Although it can be inactivated by gene deletion or point mutations, the most common contributor to loss or reduction of RASSF1A function is transcriptional silencing of the gene by inappropriate promoter methylation. This epigenetic mechanism can inactivate numerous tumor suppressors and is now recognized as a major contributor to the development of cancer.RASSF1A lacks apparent enzymatic activity but contains a Ras association (RA) domain and is potentially an effector of the Ras oncoprotein. RASSF1A modulates multiple apoptotic and cell cycle checkpoint pathways. Current evidence supports the hypothesis that it serves as a scaffold for the assembly of multiple tumor suppressor complexes and may relay pro-apoptotic signaling by K-Ras.

RASSF family proteins are tumor suppressors that are frequently downregulated during the development of human cancer. The best-characterized member of the family is RASSF1A, which is downregulated by promoter methylation in 40-90% of primary human tumors. We now identify and characterize a novel member of the RASSF family, RASSF6. Like the other family members, RASSF6 possesses a Ras Association domain and binds activated Ras. Exogenous expression of RASSF6 promoted apoptosis, synergized with activated K-Ras to induce cell death and inhibited the survival of specific tumor cell lines. Suppression of RASSF6 enhanced the tumorigenic phenotype of a human lung tumor cell line. Furthermore, RASSF6 is often downregulated in primary human tumors. RASSF6 shares some similar overall properties as other RASSF proteins. However, there are significant differences in biological activity between RASSF6 and other family members including a discrete tissue expression profile, cell killing specificity and impact on signaling pathways. Moreover, RASSF6 may play a role in dictating the degree of inflammatory response to the respiratory syncytial virus. Thus, RASSF6 is a novel RASSF family member that demonstrates the properties of a Ras effector and tumor suppressor but exhibits biological properties that are unique and distinct from those of other family members.

NORE1A (RASSF5) and RASSF1A are newly described Ras effectors with tumour suppressor functions. Both molecules are frequently inactivated in various cancers. In this study, we aimed to explore the potential involvement of NORE1A and RASSF1A in pheochromocytoma and abdominal paraganglioma tumorigenesis. A panel of 54 primary tumours was analysed for NORE1A and RASSF1A mRNA expression by TaqMan quantitative RT-PCR. Furthermore, NORE1A and RASSF1A promoter methylation was assessed by combined bisulphite restriction endonuclease assay and methylation-sensitive Pyrosequencing respectively. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A-transfected PC12 rat pheochromocytoma cells by fluorescent inhibition of caspase activity and soft agar assays. Significantly suppressed NORE1A and RASSF1A mRNA levels were detected in primary tumours compared with normal adrenal medulla (PNORE1A promoter was not observed in primary tumours. On the other hand, 9% (5/54) of the primary tumours examined showed RASSF1A promoter methylation greater than 20% as detected by Pyrosequencing. Methylation of the RASSF1A promoter was significantly associated with malignant behaviour (PNORE1A and RASSF1A are frequently suppressed in pheochromocytoma and abdominal paraganglioma. Silencing of NORE1A contributes to the transformed phenotype in these tumours.

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.

BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1 expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.

NORE1A is a Ras senescence effector that modulates HIPK2-dependent posttranslational modifications of p53., The Ras oncoprotein is a key driver of cancer. However, Ras also provokes senescence, which serves as a major barrier to Ras-driven transformation. Ras senescence pathways remain poorly characterized. NORE1A is a novel Ras effector that serves as a tumor suppressor. It is frequently inactivated in tumors. We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation. We show that Ras induces the formation of a complex between NORE1A and the kinase HIPK2, enhancing HIPK2 association with p53. HIPK2 is a tumor suppressor that can induce either proapoptotic or prosenescent posttranslational modifications of p53. NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53. Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence.

Magnetic nanoparticle-supported lipid bilayers (SLBs) constructed around core–shell Fe3O4–SiO2 nanoparticles (SNPs) were prepared and evaluated as potential drug carriers. We describe how an oxime ether lipid can be mixed with SNPs to produce lipid–particle assemblies with highly positive ζ potential. To demonstrate the potential of the resultant cationic SLBs, the particles were loaded with either the anticancer drug doxorubicin or an amphiphilic analogue, prepared to facilitate integration into the supported lipid bilayer, and then examined in studies against MCF-7 breast cancer cells. The assemblies were rapidly internalized and exhibited higher toxicity than treatments with doxorubicin alone. The magnetic SLBs were also shown to increase the efficacy of unmodified doxorubicin.

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf(V600E)-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.

Recent advances in the study of RASSF1A, the candidate tumor suppressor gene, indicate a possible role of RASSF1A in cell cycle regulation; however, very little is known regarding molecular mechanisms underlying this control. Using small interfering RNA to knockdown endogenous RASSF1A in the breast tumor cell line HB2 and in the cervical cancer cell line HeLa, we identify that a key player in cell cycle progression, cyclin A2, is concomitantly increased at both protein and mRNA levels. In A549 clones stably expressing RASSF1A, cyclin A2 levels were diminished compared with vector control. A known transcriptional regulator of cyclin A2, p120E4F (a repressor of cyclin A2), has been shown previously by our group to interact with RASSF1A. We show that levels of p120E4F are not affected by RASSF1A small interfering RNA in HB2 and HeLa cells. However, electrophoretic mobility shift assays indicate that knockdown of endogenous RASSF1A in HB2 and HeLa cells leads to a reduction in the binding capacity of p120E4F to the cyclin A2 promoter, whereas in the A549 clone stably expressing RASSF1A the binding capacity is increased. These data are further corroborated in vitro by the luciferase assay and in vivo by chromatin immunoprecipitation experiments. Together, these data identify the cyclin A2 gene as a cellular target for RASSF1A through p120E4F and for the first time suggest a transcriptional mechanism for RASSF1A-dependent cell cycle regulation.

Activated Ras proteins interact with a broad range of effector proteins to induce a diverse series of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras may also induce growth antagonistic effects such as senescence or apoptosis. It is now apparent that some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. To date, four members of this family have been identified (Nore1, RASSF1, RASSF2, and RASSF3). We now identify a fifth member of this group, RASSF4 (AD037). RASSF4 shows approximately 25% identity with RASSF1A and 60% identity with RASSF2. RASSF4 binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells. Thus, RASSF4 appears to be a new member of the RASSF family of potential Ras effector/tumor suppressors.

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.

Although Ras is a potent oncogene in human tumors it has the paradoxical ability to promote Oncogene Induced Senescence (OIS). This appears to serve as a major barrier to Ras driven transformation in vivo. The signaling pathways used by Ras to promote senescence remain relatively poorly understood, but appear to invoke both the p53 and the Rb master tumor suppressors. Exactly how Ras communicates with p53 and Rb has remained something of a puzzle. NORE1A is a direct Ras effector that is frequently downregulated in human tumors. We have now found that it serves as a powerful Ras senescence effector. Moreover, we have defined signaling mechanisms that allows Ras to control both p53 and Rb post-translational modifications via the NORE1A scaffolding molecule. Indeed, NORE1A can be detected in complex with both p53 and Rb. Thus, by coupling Ras to both tumor suppressors, NORE1A forms a major component of the Ras senescence machinery and serves as the missing link between Ras and p53/Rb.

Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.

Ras oncoproteins mediate multiple biological effects by activating multiple effectors. Classically, Ras activation has been associated with enhanced cellular growth and transformation. However, activated forms of Ras may also inhibit growth by inducing senescence, apoptosis, and differentiation. Induction of apoptosis by Ras may be mediated by its effector RASSF1, which appears to function as a tumor suppressor. We now show that the Ras effector Nore1, which is structurally related to RASSF1, can also mediate a Ras-dependent apoptosis. Moreover, an analysis of Nore1 protein expression showed that it is frequently down-regulated in lung tumor cell lines and primary lung tumors. Like RASSF1, this correlates with methylation of the Nore1 promoter rather than gene deletion. Finally, re-introduction of Nore1, driven by its own promoter, impairs the growth in soft agar of a human lung tumor cell line. Consequently, we propose that the Ras effector Nore1 is a member of a family of Ras effector/tumor suppressors that includes RASSF1.

Dysregulation of miRNAs and chronic inflammation are strongly implicated in the development of various malignancies, including prostate cancer (PCa). Previously, our lab identified several inflammatory and immune response sequence variants in CCL5 and CCR5 significantly modified PCa risk. Recently, we propose an oncogenic role for miR-186-5p based on its up-regulation in serum from PCa patients and metastatic PCa cell lines. Moreover, miR-186-5p inhibition reduced proliferation, anchorage independent growth and invasion in metastatic PC cells (PC3, MDA-PCA-2b). We hypothesize dysregulation of inflammatory and immune response markers may enhance immune surveillance leading to a reduced aggressive tumor phenotype. Following modulations in miRNA-186 levels in normal prostate epithelial (RWPE1) and metastatic PCa (PC3) cell lines, we evaluated alterations in mRNA expression using micro-array analysis. Ectopic expression of miRNA-186 in the RWPE1 resulted in a 1.3-2.6-fold down-regulation in TLR2, IRAK2, CCL20, IL1RAP, IL1RAP, IL15, and IL17RD (FDR p-value ≥7.8x10-06). Whereas, inhibition of miR-186 in PC3 cells corresponded with a 1.4-fold up-regulation of IL18R1 (p=0.0095). Notably, these inflammatory markers are involved in cell invasion (TLR2), apoptosis (IL17RD), tumorigenesis (TLR2, IRAK2) and autoimmune diseases (CCL20). Validation of these targets is underway using qRT-PCR, western blots and luciferase reporter assays. We will assess the mechanism by which these miR-186 targets suppress aggressive cancer behavior using knock-out and knock-in cell and ultimately animal models. Future studies may identify inflammatory targets that may guide immune therapies for the effective treatment of aggressive prostate cancer. Citation Format: Suman Suman, Dominique Z. Jones-Reed, M. L. Schmidt, Geoffrey J. Clark, Carolyn Klinge, Shirish Barve, Kevin S. Kimbro, La Creis R. Kidd. Alteration of miR-186 expression modifies inflammatory markers in normal epithelial and prostate cancer cell models [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 1483. doi:10.1158/1538-7445.AM2017-1483

RASSF1A may be the most frequently inactivated tumor suppressor identified in human cancer so far. It is a proapoptotic Ras effector and plays an important role in the apoptotic DNA damage response (DDR). We now show that in addition to DDR regulation, RASSF1A also plays a key role in the DNA repair process itself. We show that RASSF1A forms a DNA damage-regulated complex with the key DNA repair protein xeroderma pigmentosum A (XPA). XPA requires RASSF1A to exert full repair activity, and RASSF1A-deficient cells exhibit an impaired ability to repair DNA. Moreover, a cancer-associated RASSF1A single-nucleotide polymorphism (SNP) variant exhibits differential XPA binding and inhibits DNA repair. The interaction of XPA with other components of the repair complex, such as replication protein A (RPA), is controlled in part by a dynamic acetylation/deacetylation cycle. We found that RASSF1A and its SNP variant differentially regulate XPA protein acetylation, and the SNP variant hyperstabilizes the XPA-RPA70 complex. Thus, we identify two novel functions for RASSF1A in the control of DNA repair and protein acetylation. As RASSF1A modulates both apoptotic DDR and DNA repair, it may play an important and unanticipated role in coordinating the balance between repair and death after DNA damage.

Polymorphonuclear neutrophils (PMNs), the main effectors of the innate immune system, have rarely been considered as an anticancer therapeutic tool. However, recent investigations using animal models and preliminary clinical studies have highlighted the potential antitumor efficacy of PMNs. In the current study, we find that PMNs from some healthy donors naturally have potent cancer-killing activity against 4 different human cancer cell lines. The killing activity appears to be cancer cell-specific since PMNs did not kill primary normal epithelial cells or an immortalized breast epithelial cell line. Transfecting the immortalized mammary cells with plasmids expressing activated forms of the rat sarcoma viral oncogene homolog (Ras) and teratocarcinoma oncogene 21 (TC21) oncogenes was sufficient to provoke aggressive attack by PMNs. However, transfection with activated Ras-related C3 botulinum toxin substrate (Rac1) was ineffective, suggesting specificity in PMN-targeting of neoplastic cells. Furthermore, PMNs from lung cancer patients were also found to exhibit relatively poor cancer-killing activity compared to the cytolytic activity of the average healthy donor. Taken together, our results suggest that PMN-based treatment regimens may represent a paradigm shift in cancer immunotherapy that may be easily introduced into the clinic to benefit a subset of patients with PMN-vulnerable tumors.