Abstract A53: SmgGDS splice variants interact with K-Ras through association with both the polybasic region and the CAAX motif, and promote the malignant phenotype in lung, breast, and pancreatic cancers

SmgGDS is a chaperone protein that binds Ras and Rho family members that have a C-terminal polybasic region (PBR). We reported that the splice variants SmgGDS-607 and SmgGDS-558 regulate the prenylation and membrane trafficking of PBR-containing small GTPases. SmgGDS-607 interacts with non-prenylated small GTPases and regulates their entry into the prenylation pathway. In contrast, SmgGDS-558 interacts with prenylated small GTPases and promotes their trafficking to cell membranes. In the current study, we investigated the physical interaction of SmgGDS splice variants with K-Ras, and tested the ability of SmgGDS splice variants to promote the malignant phenotype of cancer cell lines expressing oncogenic K-Ras. Because SmgGDS-607 binds K-Ras before it is prenylated, we hypothesized that SmgGDS-607 recognizes the C-terminal CAAX motif of K-Ras. This CAAX motif consists of the sequence CVIM, and the C-terminal methionine causes newly synthesized K-Ras to interact with the farnesyltransferase and become farnesylated. However, newly synthesized K-Ras will alternatively interact with geranylgeranyltransferase-I and become geranylgeranylated in the presence of farnesyltransferase inhibitors (FTIs), or if the C-terminal methionine is mutated to leucine. To test the recognition of the K-Ras CAAX motif by SmgGDS-607, we examined binding of SmgGDS-607 to K-Ras that has the wildtype CAAX sequence (K-Ras-CVIM) or a mutant CAAX sequence (K-Ras-CVIL). We show that SmgGDS-607 associates more with K-Ras-CVIL than with K-Ras-CVIM in cells, indicating that SmgGDS-607 interacts with non-prenylated K-Ras that is destined to become geranylgeranylated rather than farnesylated. This conclusion is further supported by our finding that FTIs increase binding of wildtype K-Ras-CVIM to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. These findings indicate that SmgGDS-607 will preferentially interact with non-prenylated K-Ras that is destined to enter the geranylgeranylation pathway caused either by FTIs or mutation of the CAAX motif. In contrast to SmgGDS-607, we found that SmgGDS-558 interacts robustly with both farnesylated and geranylgeranylated K-Ras, as well as with oncogenic K-Ras(G12V). Because SmgGDS-558 interacts with prenylated and oncogenic K-Ras, we hypothesized that SmgGDS-558 plays a greater role than SmgGDS-607 in promoting the malignancy of cancers expressing oncogenic K-Ras. Consistent with this hypothesis, we found that the RNAi-mediated depletion of SmgGDS-558, but not SmgGDS-607, significantly diminishes proliferation, induces a G1 arrest, diminishes expression of the cell cycle promoter Cyclin D1, and increases expression of the cell cycle inhibitor p27 in human cancer cell lines expressing oncogenic K-Ras, including pancreatic (MiaPaCa-2 and PANC-1), lung (NCI-H23), and breast (MDA-MB-231) cancer cell lines. Interestingly, similar responses occurred after depletion of SmgGDS-558 in MCF-7 breast cancer cells but not in NCI-H1703 lung cancer cells, which are both cell lines expressing wildtype K-Ras. These results support our model that SmgGDS-558 promotes malignancy in multiple types of cancers by interacting with prenylated PBR-containing small GTPases, including K-Ras. We are currently investigating how SmgGDS interactions with small GTPases are disrupted by peptides corresponding to the PBR and CAAX motifs of K-Ras and other small GTPases, to further define the mechanisms utilized by SmgGDS to promote malignancy. Citation Format: Nathan J. Schuld, Andrew D. Hauser, Jeffrey S. Vervacke, Ellen L. Lorimer, Mark D. Distefano, Carol L. Williams. SmgGDS splice variants interact with K-Ras through association with both the polybasic region and the CAAX motif, and promote the malignant phenotype in lung, breast, and pancreatic cancers. [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 A53. doi: 10.1158/1557-3125.RASONC14-A53