SmgGDS is a novel CAAX-specific binding protein and is involved in pancreatic and lung cancer malignancy

The goal of this study is to identify the individual roles of the SmgGDS splice variants in pancreatic and lung cancer malignancy, and to develop the mechanistic understanding of how SmgGDS and the small GTPase K-Ras interact through the prenylation pathway. K-Ras and SmgGDS have both been identified as playing a role in the malignancy of multiple cancers, and SmgGDS-607 and SmgGDS-558 have been implicated in the regulation of K-Ras. Therefore, this study tested the hypothesis that the SmgGDS splice variants increase K-Ras signaling through interactions in the prenylation pathway and these SmgGDS-dependent interactions promote the malignant phenotype in NSCLC and pancreatic cancer.;In Specific Aim 1 we tested the hypothesis that SmgGDS-558 promotes the malignant phenotype and is a better therapeutic target than SmgGDS-607 in NSCLC and pancreatic cancer. To examine the roles of SmgGDS splice variants in malignancy, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 versus SmgGDS- 607 on proliferation, NF- B activity, and on cell cycle progression as well as the expression of cyclin D1, p27, and p21 in pancreatic and lung cancer cell lines. We show for the first time that SmgGDS promotes proliferation and NF- B activity in pancreatic cancer cells. Additionally, we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in some NSCLC cell lines, and all of the tested pancreatic cancer cell lines. Depleting both splice variants of SmgGDS in the cancer cell lines produced an alternative signaling profile compared to depleting SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 NSCLC xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.;In Specific Aim 2 we tested the hypothesis that K-Ras activity is increased more by SmgGDS-558 than by SmgGDS-607, and that SmgGDS-607 binds only to small GTPases that will become geranylgeranylated. However, our data from Specific Aim 2 does not support a role for SmgGDS-607 or SmgGDS-558 in the trafficking, localization, or activity of K-Ras. However, we provide evidence that SmgGDS-607 associates with GTPases through recognition of the last amino acid in the CAAX motif. We show that SmgGDS-607 forms more stable complexes in cells with non-prenylated GTPases that will become geranylgeranylated than with non-prenylated GTPases that will become farnesylated. These binding relationships similarly occur with non-prenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors (FTIs) increase the binding of WT K-Ras to SmgGDS-607, indicating a potential role for SmgGDs-607 in the alternate geranylgeranylation of K-Ras. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of Rap1B and K-Ras, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but additional cellular proteins may be needed to maintain the specificities of SmgGDS splice variants for prenylated and non-prenylated GTPases. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared to SmgGDS-558.;Taken together, the results of this research provide new mechanistic insights into the regulation of malignancy by SmgGDS, and the interactions of SmgGDS splice variants with K-Ras. (Abstract shortened by UMI.).