Andrew D. Hauser

An Adenosine-Mediated Signaling Pathway Suppresses Prenylation of the GTPase Rap1B and Promotes Cell Scattering

Inhibition of adenosine receptors could reduce metastasis by enhancing prenylation-dependent signaling that promotes cell-cell adhesion. Signal to Scatter An early step in metastasis is the dissociation of cancer cells from the primary tumor mass. When localized to cell membranes, the small guanosine triphosphatase (GTPase) Rap1B promotes cell-cell adhesion, a function that was blocked by a signaling pathway identified by Ntantie et al. Activation of adenosine A2B receptors reduced the prenylation of Rap1B, a posttranslational modification that enables Rap1B to localize to cell membranes, and resulted in reduced cell-cell adhesion. Because tumors release adenosine, these findings suggest that inhibition of adenosine receptors could reduce cancer cell metastasis by enabling the prenylation and cell membrane localization of Rap1B, thereby promoting cell-cell adhesion. During metastasis, cancer cells acquire the ability to dissociate from each other and migrate, which is recapitulated in vitro as cell scattering. The small guanosine triphosphatase (GTPase) Rap1 opposes cell scattering by promoting cell-cell adhesion, a function that requires its prenylation, or posttranslational modification with a carboxyl-terminal isoprenoid moiety, to enable its localization at cell membranes. Thus, signaling cascades that regulate the prenylation of Rap1 offer a mechanism to control the membrane localization of Rap1. We identified a signaling cascade initiated by adenosine A2B receptors that suppressed the prenylation of Rap1B through phosphorylation of Rap1B, which decreased its interaction with the chaperone protein SmgGDS (small GTPase guanosine diphosphate dissociation stimulator). These events promoted the cytosolic and nuclear accumulation of nonprenylated Rap1B and diminished cell-cell adhesion, resulting in cell scattering. We found that nonprenylated Rap1 was more abundant in mammary tumors than in normal mammary tissue in rats and that activation of adenosine receptors delayed Rap1B prenylation in breast, lung, and pancreatic cancer cell lines. Our findings support a model in which high concentrations of extracellular adenosine, such as those that arise in the tumor microenvironment, can chronically activate A2B receptors to suppress Rap1B prenylation and signaling at the cell membrane, resulting in reduced cell-cell contact and promoting cell scattering. Inhibiting A2B receptors may be an effective method to prevent metastasis.

The role of Rac1 in the regulation of NF-κB activity, cell proliferation, and cell migration in non-small cell lung carcinoma.

The small GTPase Rac1 regulates many cellular processes, including cytoskeletal reorganization, cell migration, proliferation, and survival. Additionally, Rac1 plays a major role in activating NF-κB-mediated transcription. Both Rac1 and NF-κB regulate many properties of the malignant phenotype, including anchorage-independent proliferation and survival, metastasis, and angiogenesis. Despite these findings, the roles of Rac1and NF-κB in non-small cell lung carcinoma, a leading cause of cancer deaths, have not been thoroughly investigated. Here, we compared the effects of Rac1 siRNA to that of the Rac1 inhibitor NSC23766 on multiple features of the NSCLC malignant phenotype, including NF-κB activity. We show that the siRNA-mediated silencing of Rac1 in lung cancer cells results in decreased cell proliferation and migration. The decrease in proliferation was observed in both anchorage-dependent and anchorage-independent assays. Furthermore, cells with decreased Rac1 expression have a slowed progression through the G1 phase of the cell cycle. These effects induced by Rac1 siRNA correlated with a decrease in NF-κB transcriptional activity. Additionally, inhibition of NF-κB signaling with BAY 11–7082 inhibited proliferation; indicating that the loss of cell proliferation and migration induced by the silencing of Rac1 expression may be attributed in part to loss of NF-κB activity. Interestingly, treatment with the Rac1 inhibitor NSC23766 strongly inhibits cell proliferation, cell cycle progression, and NF-κB activity in lung cancer cells, to an even greater extent than the inhibition induced by Rac1 siRNA. These findings indicate that Rac1 plays an important role in lung cancer cell proliferation and migration, most likely through its ability to promote NF-κB activity, and highlight Rac1 pathways as therapeutic targets for the treatment of lung cancer.

Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells

Aggressive dissemination and metastasis of pancreatic ductal adenocarcinoma (PDAC) results in poor prognosis and marked lethality. Rho monomeric G protein levels are increased in pancreatic cancer tissue. As the mechanisms underlying PDAC malignancy are little understood, we investigated the role for cAMP in regulating monomeric G protein regulated invasion and migration of pancreatic cancer cells. Treatment of PDAC cells with cAMP elevating agents that activate adenylyl cyclases, forskolin, protein kinase A (PKA), 6‐Bnz‐cAMP, or the cyclic nucleotide phosphodiesterase inhibitor cilostamide significantly decreased migration and Matrigel invasion of PDAC cell lines. Inhibition was dose‐dependent and not significantly different between forskolin or cilostamide treatment. cAMP elevating drugs not only blocked basal migration, but similarly abrogated transforming‐growth factor‐β‐directed PDAC cell migration and invasion. The inhibitory effects of cAMP were prevented by the pharmacological blockade of PKA. Drugs that increase cellular cAMP levels decreased levels of active RhoA or RhoC, with a concomitant increase in phosphorylated RhoA. Diminished Rho signaling was correlated with the appearance of thickened cortical actin bands along the perimeter of non‐motile forskolin or cilostamide‐treated cells. Decreased migration did not reflect alterations in cell growth or programmed cell death. Collectively these data support the notion that increased levels of cAMP specifically hinder PDAC cell motility through F‐actin remodeling.

Mitochondria-targeted nitroxides exacerbate fluvastatin-mediated cytostatic and cytotoxic effects in breast cancer cells.

Mito-CP11, a mitochondria-targeted nitroxide formed by conjugating a triphenylphosphonium cation to a five-membered nitroxide, carboxy-proxyl (CP), has been used as a superoxide dismutase (SOD) mimetic. In this study, we investigated the antiproliferative and cytotoxic properties of submicromolar levels of Mito-CP11 alone and in combination with fluvastatin, a well known cholesterol lowering drug, in breast cancer cells. Mito-CP11, but not CP or CP plus the cationic ligand, methyl triphenylphosphonium (Me-TPP+), inhibited MCF-7 breast cancer cell proliferation. Mito-CP11 had only minimal effect on MCF-10A, non-tumorigenic mammary epithelial cells. Mito-CP11, however, significantly enhanced fluvastatin-mediated cytotoxicity in MCF-7 cells. Mito-CP11 alone and in combination with fluvastatin inhibited nuclear factor kappa-B activity mainly in MCF-7 cells. We conclude that mitochondria-targeted nitroxide antioxidant molecules (such as Mito-CP11) that are non-toxic to non-tumorigenic cells could enhance the cytostatic and cytotoxic effects of statins in breast cancer cells. This strategy of combining mitochondria-targeted non-toxic molecules with cytotoxic chemotherapeutic drugs may be successfully used to enhance the efficacy of antitumor therapies in breast cancer treatment.

The chaperone protein SmgGDS interacts with small GTPases entering the prenylation pathway by recognizing the last amino acid in the CAAX motif.

Background: SmgGDS-607 and SmgGDS-558 regulate GTPase movement through the prenylation pathway. Results: The specificity of SmgGDS for GTPases depends on the GTPase CAAX sequence and the cellular context. Conclusion: SmgGDS-607 binds to nonprenylated GTPases that end in a leucine and enter the geranylgeranylation pathway. Significance: The identification of SmgGDS-607 as a novel CAAX-binding protein will accelerate the development of more effective cancer therapeutics. Ras family small GTPases localize at the plasma membrane, where they can activate oncogenic signaling pathways. Understanding the mechanisms that promote membrane localization of GTPases will aid development of new therapies to inhibit oncogenic signaling. We previously reported that SmgGDS splice variants promote prenylation and trafficking of GTPases containing a C-terminal polybasic region and demonstrated that SmgGDS-607 interacts with nonprenylated GTPases, whereas SmgGDS-558 interacts with prenylated GTPases in cells. The mechanism that SmgGDS-607 and SmgGDS-558 use to differentiate between prenylated and nonprenylated GTPases has not been characterized. Here, 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 nonprenylated GTPases that will become geranylgeranylated than with nonprenylated GTPases that will become farnesylated. These binding relationships similarly occur with nonprenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors increase the binding of WT K-Ras to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of K-Ras and Rap1B, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but the specificity of the SmgGDS splice variants for prenylated versus nonprenylated GTPases is diminished in vitro. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared with SmgGDS-558.

The SmgGDS Splice Variant SmgGDS-558 Is a Key Promoter of Tumor Growth and RhoA Signaling in Breast Cancer

Breast cancer malignancy is promoted by the small GTPases RhoA and RhoC. SmgGDS is a guanine nucleotide exchange factor that activates RhoA and RhoC in vitro. We previously reported that two splice variants of SmgGDS, SmgGDS-607, and SmgGDS-558, have different characteristics in binding and transport of small GTPases. To define the role of SmgGDS in breast cancer, we tested the expression of SmgGDS in breast tumors, and the role of each splice variant in proliferation, tumor growth, Rho activation, and NF-κB transcriptional activity in breast cancer cells. We show upregulated SmgGDS protein expression in breast cancer samples compared with normal breast tissue. In addition, Kaplan–Meier survival curves indicated that patients with high SmgGDS expression in their tumors had worse clinical outcomes. Knockdown of SmgGDS-558, but not SmgGDS-607, in breast cancer cells decreased proliferation, in vivo tumor growth, and RhoA activity. Furthermore, we found that SmgGDS promoted a Rho-dependent activation of the transcription factor NF-κB, which provides a potential mechanism to define how SmgGDS-mediated activation of RhoA promotes breast cancer. This study demonstrates that elevated SmgGDS expression in breast tumors correlates with poor survival, and that SmgGDS-558 plays a functional role in breast cancer malignancy. Taken together, these findings define SmgGDS-558 as a unique promoter of RhoA and NF-κB activity and a novel therapeutic target in breast cancer. Implications: This study defines a new mechanism to regulate the activities of RhoA and NF-κB in breast cancer cells, and identifies SmgGDS-558 as a novel promoter of breast cancer malignancy. Mol Cancer Res; 12(1); 130–42. ©2013 AACR.

The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases.

The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

SmgGDS-558 regulates the cell cycle in pancreatic, non-small cell lung, and breast cancers

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and 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 multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (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.

Erratum: The tumor-suppressive small GTPase DiRas1 binds the noncanonical guanine nucleotide exchange factor SmgGDS and antagonizes SmgGDS interactions with oncogenic small GTPases (Journal of Biological Chemistry (2016) 291 (6534-6545))

Carmen Bergom, Andrew D. Hauser, Amy Rymaszewski, Patrick Gonyo, Jeremy W. Prokop, Benjamin C. Jennings, Alexis J. Lawton, Anne Frei, Ellen L. Lorimer, Irene Aguilera-Barrantes, Alexander C. Mackinnon, Jr., Kathleen Noon, Carol A. Fierke, and Carol L. Williams Ref. 40 was missing from the reference list. The information for Ref. 40 should be as follows: Ogita, Y., Egami, S., Ebihara, A., Ueda, N., Katada, T., and Kontani, K. (2015) Di-Ras2 protein forms a complex with SmgGDS protein in brain cytosol in order to be in a low affinity state for guanine nucleotides. J. Biol. Chem. 290, 20245–20256. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 20, p. 10948, May 13, 2016

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