Abstract
Simple SummaryThe 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.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.
Highlights
There are three human isoforms of the RAS oncogene—HRAS, KRAS, and NRAS—that collectively represent the most frequently activated oncogene in human cancer [1]
We have previously modeled this phenotype in mice, where we showed that induced expression of an oncogenic kras mutant in rassf1a+/– mice increased both the frequency of tumor formation and tumor size compared to rassf1a+/+ littermates [21]
We identified the protein Disabled homolog 2 interacting protein (DAB2IP), which bound to the
Summary
There are three human isoforms of the RAS oncogene—HRAS, KRAS, and NRAS—that collectively represent the most frequently activated oncogene in human cancer [1]. RAS possesses a highly potent transforming power, capable of activating several classical mitogenic pathways such as RAF-MEK-ERK, Phosphatidylinositol-3 kinase (PI3K)-AKT, and RAL guanine nucleotide exchange factor (RALGEF)-RAL [2]. The RAS GTPase cycle is tightly regulated; activating GDP-GTP exchange and inactivating GTP hydrolysis are controlled by two families of regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively [4]. RAS is rendered constitutively active by two main mechanisms: activating mutations and disruption of its regulators. Hyperactivating point mutations occur at RAS loci in up to 30% of tumors across all tissue types [1,5]. Overexpression/activating mutations in GEFs, or more commonly in the receptor systems that control them, and suppression/mutation of GAPs have been reported [6,7,8,9]
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