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 C-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. Here, 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 G-protein dissociation stimulator). These events promoted the cytosolic and nuclear accumulation of non-prenylated Rap1B and diminished cell-cell adhesion, resulting in cell scattering. We found that non-prenylated 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.
Regulatory T cells (Tregs), in particular CD4+ Foxp3+ T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8+ Foxp3+ T cells that are induced early during GVHD, constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4+ Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4+ induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8+ and CD4+ iTregs had accelerated GVHD mortality compared to animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4+ and CD8+ T cells that secreted IFN-γ and IL-17. The presence of CD8+ iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4+ Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8+ iTregs could compensate for CD4+ iTregs. These studies define a novel population of CD8+ Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.
The localization of prenylated Ras at the plasma membrane promotes activation of Ras by receptor tyrosine kinases and stimulates oncogenic signaling by mutant Ras. The Nogo-B receptor (NgBR) is a transmembrane receptor that contains a conserved hydrophobic pocket. Here, we demonstrate that the NgBR promotes the membrane accumulation of Ras by directly binding prenylated Ras at the plasma membrane. We show that NgBR knockdown diminishes the membrane localization of Ras in multiple cell types. NgBR overexpression in NIH-3T3 fibroblasts increases membrane-associated Ras, induces the transformed phenotype in vitro, and promotes the formation of fibrosarcoma in nude mice. NgBR knockdown in human breast cancer cells reduces Ras membrane localization, inhibits EGF-stimulated Ras signaling, and diminishes tumorigenesis of xenografts in nude mice. Our data demonstrate that NgBR is a unique receptor that promotes accumulation of prenylated Ras at the plasma membrane and promotes EGF pathways.
Author contributions S.N. and W.G. conceived and designed the overall project. S.S. and C.I.U. assisted with selecting the family, gathering the clinical histories and collecting DNA samples under human subject IRB-approved protocols. S.N., W.G. and I.L. designed the WGS analysis. I.L. performed the WGS analysis and candidate variant filtering. S.N., J.W., A.J.K., J.E.H., A.G.C. and J.H. designed and generated the zebrafish rabl3 mutant lines and performed the cancer studies. J.R.H. and S.N. performed zebrafish histology preparation and analysis. J.D.M. performed and analyzed the AP-MS experiments and CompPASS suite protein interactomics. S.N., W.G. and C.W. conceived and designed the in vitro immunoprecipitation, prenylation assays and HEK293T cell proliferation assays, and P.G., A.B., E.L. and B.U. performed these experiments. S.N. and O.M. designed and performed RASless MEF experiments. J.W.P. performed protein structural modeling. B.C.J. and C.A.F. designed and performed purification of recombinant protein. J.A.P., S.G. and J.D.M. assisted with mass spectrometry analysis. Y.H. assisted with RNA-seq data analysis. M.B.G. performed the zebrafish μCT and bone histomorphometric analysis. O.M., X.W. and J.D.M. provided assistance with tissue culture experiments. C.A.C. and J.A.R. provided analysis of clinical exome sequencing data. C.A.C. and I.L. provided analysis of variants in the Exome Aggregation Consortium.
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 Smg-GDS-mediated NF-B transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-B 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.
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