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.

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.

Alveolar Rhabdomyosarcoma (ARMS) patients carrying a chromosomal translocation of PAX3 and FOXO1 have the highest mortality rates, indicating the importance of understanding how this transcription factor influences gene expression. Many studies have been performed to identify PAX3‐FOXO1 target genes and while some overlapping gene sets have been identified, there exists great variation in the genes identified. The search for these target genes is hampered by a lack of understanding of the full DNA‐binding capabilities of this fusion protein. We have examined the truncated FOXO1 DNA‐binding domain in PAX3‐FOXO1 and found that it retains activity, but does not have the same sequence specifications for binding as full length FOXO1. A search of 1463 PAX3‐FOXO1 putative binding sites revealed that 37% contained both a PAX3 paired domain and FOXO1 recognition sequence within close proximity. The sequences containing the dual motif mapped to 160 genes with the most interesting of these known to be regulated by PAX3‐FOXO1 such as MET or upregulated in response to PAX3‐FOXO1 expression such as ABAT and PBK. Here we present additional data exploring the sequence variations on the PAX3 and FOXO1 recognition sequences found within Pax3‐FOXO1 putative binding sites as well as in vitro confirmation of the interaction. Grant Funding Source: Supported by NIH SC2GM095430