Faculty Bibliography

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein-coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B.

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.

Effective anti-rat sarcoma viral oncogene (RAS) therapies have remained the holy grail of cancer treatment. Mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) sustains tumorigenesis when linked to the plasma membrane (PM). The G protein-coupled receptor 31 (GPR31) is now identified to mediate KRAS membrane association and is crucial for proliferation, survival and macropinocytosis of KRAS-dependent cancer cells, suggesting that GPR31 is a druggable target for anti-RAS therapy.

The Ras-related (R-Ras) isoforms TC21, R-Ras and M-Ras are members of the Ras superfamily of small GTPases. R-Ras family proteins are frequently overexpressed in human cancers, and expression of activated mutants of these GTPases is sufficient to induce cell transformation. Unlike Ras, few activating mutations of R-Ras proteins have been reported in human cancer, and very little is known about the regulation of their activity. In this study, we report that TC21 and R-Ras are phosphorylated on a conserved serine, Ser186 and Ser201, respectively, in intact cells. This residue is located in the C-terminal hypervariable region of the proteins and is not conserved in M-Ras. We show that the MAP kinases ERK1/2 phosphorylate TC21 and R-Ras on this C-terminal serine residue both in vitro and in vivo. Phosphorylation of R-Ras proteins does not affect their subcellular localization or stability but rather stimulates their activation. Phosphorylation-defective mutants of R-Ras and TC21 are compromised in their ability to promote cancer cell adhesion and migration/invasion, respectively. Importantly, we show that phosphorylation of TC21 and R-Ras potentiates their tumorigenic activity in immunodeficient mice. Our results identify a novel regulatory mechanism of the small GTPases TC21 and R-Ras that controls their oncogenic potential.

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (Riam). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for Riam we show that the Rap1/Riam module is not required for T or B cell development but is essential for efficient adhesion to ICAM-1 and VCAM-1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in Riam deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lympyhocyte trafficking was accompanied by defective humoral immunity to T cell-dependent antigens. Platelet function was intact in Riam deficient animals. These in vivo results confirm a role for Riam in the regulation of some, but not all, leukocyte integrins and suggest that Riam-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

RAS proteins require membrane association for their biologic activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTI) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anticancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes, and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated posttranslational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. Clin Cancer Res; 21(8); 1819-27. (c)2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."