M. Lee Schmidt

NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2

NORE1A is a Ras senescence effector that modulates HIPK2-dependent posttranslational modifications of p53.

Ras signaling through RASSF proteins.

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).

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

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-RafV600E-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.

The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair

ABSTRACT 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.

Cell cycle restriction is more important than apoptosis induction for RASSF1A protein tumor suppression.

Background: The RASSF1A tumor suppressor regulates apoptosis and the cell cycle. Results: Loss of microtubule association does not affect the proapoptotic function of RASSF1A but abolishes its ability to modulate the cell cycle and suppress transformation. Conclusion: Regulation of the cell cycle supersedes the proapoptotic effects of RASSF1A for suppression of the transformed phenotype. Significance: This study provides further and unexpected insights into RASSF1A function. 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.

Ras Regulates SCFβ-TrCP Protein Activity and Specificity via Its Effector Protein NORE1A

Background: NORE1A is a Ras effector with a poorly defined function. Results: NORE1A forms a direct, Ras-regulated complex with β-TrCP and activates the SCFβ-TrCP ubiquitin ligase complex to promote β-catenin degradation. Conclusion: Ras controls the activity and specificity of SCFβ-TrCP via NORE1A. Significance: We identify a novel Ras signaling pathway, defective in many tumors, that allows Ras to control specific protein stability. 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 SCFβ-TrCP ubiquitin ligase complex. This interaction allows Ras to stimulate the ubiquitin ligase activity of SCFβ-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 SCFβ-TrCP, is not sensitive to NORE1A-promoted degradation. We identify a completely new signaling mechanism for Ras that allows for the specific regulation of SCFβ-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.

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication

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).

The role of the NORE1A tumor suppressor in Oncogene-Induced Senescence

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.

Abstract A06: NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb

In addition to driving growth and transformation, activated forms of Ras are potent inducers of oncogene induced senescence (OIS). OIS appears to be a major barrier that must be overcome to permit Ras driven tumorigenesis. The signaling pathways utilized by Ras to induce senescence, and how they are subverted during tumor development remain poorly characterized. NORE1A is a member of the RASSF family of tumor suppressors. It binds directly to activated Ras via the Ras effector domain and acts as a Ras death effector. Frequent loss of NORE1A expression is observed in many tumor types and hereditary genetic defects in NORE1A predispose carriers to cancer. We have found that NORE1A can connect Ras to the induction of p21CIP1 and cell cycle arrest. Therefore, we sought to determine if NORE1A might play a role in Ras induced senescence. We now show that NORE1A is a potent mediator of Ras induced senescence. Knockdown of NORE1A suppresses the ability of Ras to induce senescence in multiple cell systems and enhances Ras driven transformation. We have identified two novel signaling pathways activated by Ras/NORE1A. First, NORE1A forms a Ras regulated, endogenous complex with the kinase HIPK2. HIPK2 can phosphorylate p53 to induce apoptosis or recruit acetyltransferases to acetylate p53 to induce senescence signaling. NORE1A suppresses HIPK2 apoptotic post-translational modifications of p53 but activates pro-senescence post-translational modifications. NORE1A also binds and destabilizes mdm2 to enhance the stability of nuclear p53. Primary human tumors show a close correlation between the expression levels of NORE1A and acetylated p53. In addition to p53, we show that NORE1A also links Ras to the regulation of Retinoblastoma (Rb) protein. NORE1A promotes a potent, Ras dependent stabilization of the Rb protein. It also promotes dephosphorylation of RB, an activating event. Thus, NORE1A is a double-barreled Ras senescence effector which connects Ras to two of the most important senescence regulating tumor suppressors in the cell. Loss of NORE1A activity in tumors is usually due to epigenetic inactivation or aberrant protein degradation by calpains. Both of these mechanisms, in principal, may be subject to clinical intervention to restore NORE1A function. This may provide a novel approach to antagonizing Ras driven tumors. Citation Format: Howard Donninger, Diego Calvisi, Thibaut Barnoud, M. Lee Schmidt, Geoffrey J. Clark. NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A06. doi: 10.1158/1557-3125.RASONC14-A06

RASSF1A Deficiency Enhances RAS-Driven Lung Tumorigenesis

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.