Towards Targeting Endothelial Rap1B to Overcome Vascular Immunosuppression in Cancer.

The vascular endothelium, a specialized monolayer of endothelial cells (ECs), is crucial for maintaining vascular homeostasis by controlling the passage of substances and cells. In the tumor microenvironment, Vascular Endothelial Growth Factor A (VEGF-A) drives tumor angiogenesis, leading to endothelial anergy and vascular immunosuppression-a state where ECs resist cytotoxic CD8+ T cell infiltration, hindering immune surveillance. Immunotherapies have shown clinical promise. However, their effectiveness is significantly reduced by tumor EC anergy. Anti-angiogenic treatments aim to normalize tumor vessels and improve immune cell infiltration. Despite their potential, these therapies often cause significant systemic toxicities, necessitating new treatments. The small GTPase Rap1B emerges as a critical regulator of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) signaling in ECs. Our studies using EC-specific Rap1B knockout mice show that the absence of Rap1B impairs tumor growth, alters vessel morphology, and increases CD8+ T cell infiltration and activation. This indicates that Rap1B mediates VEGF-A's immunosuppressive effects, making it a promising target for overcoming vascular immunosuppression in cancer. Rap1B shares structural and functional similarities with RAS oncogenes. We propose that targeting Rap1B could enhance therapies' efficacy while minimizing adverse effects by reversing endothelial anergy. We briefly discuss strategies successfully developed for targeting RAS as a model for developing anti-Rap1 therapies.

Novel Pt(IV) complexes containing salvigenin ligand reverse cisplatin-induced resistance by inhibiting Rap1b-mediated cancer cell stemness in esophageal squamous cell carcinoma treatments.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that is highly susceptible to metastasis, recurrence and resistance, and few therapeutic targets have been identified and proven effective. Herein, we demonstrated for the first time that Rap1b can positively regulate ESCC cell stemness, as well as designed and synthesized a novel class of Pt(IV) complexes that can effectively inhibit Raplb. In vitro biological studies showed that complex-1 exhibited stronger cytotoxicity than cisplatin and oxaliplatin against a variety of ESCC cells, and effectively reversed cisplatin-induced resistance of TE6 cells by increasing cellular accumulation of platinum and inhibiting cancer cell stemness. Significantly, complex-1 also exhibited strong ability to reversal cisplatin-induced cancer cell resistance and inhibit tumor growth in TE6/cDDP xenograft mice models, with a tumor growth inhibition rate of 73.3 % at 13 mg/kg and did not show significant systemic toxicity. Overall, Rap1b is a promising target to be developed as an effective treatment for ESCC. Complex-1, as the first Pt(IV) complex that can strongly inhibit Rap1b, is also worthy of further in-depth study.

Rap2B promotes cell adhesion, proliferation, migration and invasion of human glioma.

PURPOSE: Rap2B, a member of the GTP-binding proteins, is generally up-regulated in numerous types of tumors. Nevertheless, the influence and regulatory mechanisms of Rap2B in gliomas are still not corroborated. Therefore, we analyzed the expression of Rap2B in glioma tissues and cells, and researched its significance in adhesion, proliferation, migration and invasion of the glioma cell line. METHODS: We analyzed the expression of Rap2B in different pathologic grades of glioma tissues by tissue microarray and immunohistochemistry. We assessed the expression of Rap2B in glioma tissue and non-tumor tissue by Western blot. And the expression of Rap2b protein in glioma cells and normal human astrocytes (NHA) was detected by Western blot. In addition, we disclosed the effect of Rap2B knockdown on cell adhesion, proliferation, migration and invasion by using cell attachment assay, CCK-8 assay, cell migration assay and Wound Healing assay, cell invasion assay, respectively. Western blot was used to detect the changes of expression level of NF-kB, MMP-2 and MMP-9 protein when downregulated the expression of Rap2B. RESULTS: The tissue microarray immunohistochemical results of glioma showed that the expression of Rap2B had no significant correlations between Rap2B expression and the clinicopathologic variables, including patient age (P = 0.352), gender (P = 0.858), WHO Grade (P = 0.693) and histology type (P = 0.877). Western blot analysis showed that the glioma tissue had a dramatically increase of Rap2B expression compared with the non-tumor tissues (P < 0.01). And the expression of Rap2B was markedly up-regulated in all 5 glioma cell lines compared with that in normal human astrocytes (NHA) (P < 0.01). We found that the ability of adhesion, proliferation, migration and invasion of glioma cells were significantly decreased after downregulated Rap2B expression compared with the control group (P < 0.05). In addition, Western blot results showed that the expression levels of NF-kB, MMP-2 and MMP-9 in the interference group were significantly lower than those in the negative control group (P < 0.05). CONCLUSIONS: Rap2B expression is up-regulated in glioma tissues and glioma cell lines. Knockdown of Rap2B inhibits glioma cells' adhesion and proliferation in vitro. Knockdown of Rap2B inhibits glioma cells' migration in vitro. Knockdown of Rap2B inhibits glioma cells' invasion and MMPs activity through NF-kB pathway.

Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells.

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF-small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor-Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element-binding protein-dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element-binding protein pathway mediating cell survival and the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor-Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

Rap2b siRNA significantly enhances the anticancer therapeutic efficacy of adriamycin in a gold nanoshell-based drug/gene co-delivery system.

Rap2b is a novel p53 target we have identified recently. Knockdown of Rap2b sensitizes HCT116 cells to adriamycin-induced apoptosis, indicating that Rap2b promotes adriamycin resistance in cancer cells. In the present study, we designed a nanostructure-based drug/gene delivery system to evaluate the potential of Rap2b siRNA as a therapeutic agent against human cancers. Specifically, after co-incubated with HCT116 cells, adriamycin- and Rap2b siRNA-loaded gold nanoshells were internalized. Subsequent laser irradiation promoted release of adriamycin and Rap2b siRNA from the nanoparticles. The laser-induced release of Rap2b siRNA decreased cellular expression of Rap2b and significantly enhanced the anticancer therapeutic efficacy of adriamycin in vitro and in vivo. In addition, laser irradiation of the nanoparticles might exert an additional thermal killing effect on cancer cells and further improved the anticancer efficacy of adriamycin. In summary, Rap2b siRNA is a potential enhancing agent for adriamycin-based anticancer therapeutics and the gold nanoshell-based drug/gene delivery system carrying both adriamycin and Rap2b siRNA provides a promising anticancer therapeutic strategy.

miR-9 and miR-124 synergistically affect regulation of dendritic branching via the AKT/GSK3ß pathway by targeting Rap2a.

A single microRNA (miRNA) can regulate expression of multiple proteins, and expression of an individual protein may be controlled by numerous miRNAs. This regulatory pattern strongly suggests that synergistic effects of miRNAs play critical roles in regulating biological processes. miR-9 and miR-124, two of the most abundant miRNAs in the mammalian nervous system, have important functions in neuronal development. In this study, we identified the small GTP-binding protein Rap2a as a common target of both miR-9 and miR-124. miR-9 and miR-124 together, but neither miRNA alone, strongly suppressed Rap2a, thereby promoting neuronal differentiation of neural stem cells (NSCs) and dendritic branching of differentiated neurons. Rap2a also diminished the dendritic complexity of mature neurons by decreasing the levels of pAKT and pGSK3ß. Our results reveal a novel pathway in which miR-9 and miR-124 synergistically repress expression of Rap2a to sustain homeostatic dendritic complexity during neuronal development and maturation.

Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.

A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKß, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2ß1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.

Rap2a is a novel target gene of p53 and regulates cancer cell migration and invasion.

The p53 transcription factor is a critical regulator of the cell cycle, DNA repair, and apoptosis. Recent evidences suggest that p53 may contribute to the regulation of cell invasion and migration. Rap2a, a member of the small GTPase superfamily, mediates diverse cellular events such as cell adhesion, migration and proliferation through various signaling pathways. In this study, we identify that Rap2a is a novel target of p53 and is induced upon DNA damage in a p53-dependent manner. Upon DNA damage, p53 directly binds to the promoter of Rap2a and activates its transcription. We show that Rap2a is significantly upregulated in many types of tumors. In addition, the ectopic expression of Rap2a enhances the migration and invasive ability of cancer cells and increases activities of matrix metalloproteinase MMP2 and MMP9. In contrast, the inactivation of Rap2a inhibits cell invasion and activities of MMP2 and MMP9. We also show that Rap2a regulates the phosphorylation level of Akt. Collectively, our results show that ectopic expression of Rap2a has a key role in enhancing migration, invasion and metastasis by upregulating p-Akt.

Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca(2+)-dependent activation of platelet Rap1b GTPase.

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbß3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.

Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

Histone deacetylase inhibitors upregulate Rap1GAP and inhibit Rap activity in thyroid tumor cells.

Increases in Rap activity have been associated with tumor progression. Although activating mutations in Rap have not been described, downregulation of Rap1GAP is frequent in human tumors including thyroid carcinomas. In this study, we explored whether endogenous Rap1GAP expression could be restored to thyroid tumor cells. The effects of deacetylase inhibitors and a demethylating agent, individually and in combination, were examined in four differentiated and six anaplastic thyroid carcinoma (ATC) cell lines. Treatment with the structurally distinct histone deacetylase (HDAC) inhibitors, sodium butyrate and trichostatin A, increased Rap1GAP expression in all the differentiated thyroid carcinoma cell lines and in four of the six ATC cell lines. The demethylating agent, 5-aza-deoxycytidine, restored Rap1GAP expression in one anaplastic cell line and enhanced the effects of HDAC inhibitors in a second anaplastic cell line. Western blotting indicated that Rap2 was highly expressed in human thyroid cancer cells. Importantly, treatment with HDAC inhibitors impaired Rap2 activity in both differentiated and anaplastic tumor cell lines. The mechanism through which Rap activity is repressed appears to entail effects on the expression of multiple Rap regulators, including RapGEFs and RapGAPs. These results suggest that HDAC inhibitors may provide a tractable approach to impair Rap activity in human tumor cells.

ADP ribosylation of Arg41 of Rap by ExoS inhibits the ability of Rap to interact with its guanine nucleotide exchange factor, C3G.

ExoS is a bifunctional type III cytotoxin that is secreted by Pseudomonas aeruginosa. The N-terminal domain comprises a RhoGAP activity, while the C-terminal domain comprises a ADP-ribosyltransferase activity. Previous studies showed that ExoS ADP ribosylated Ras at Arg41 which interfered with the ability of Ras to interact with its guanine nucleotide exchange factor. Rap and Ras share considerable primary amino acid homology, including Arg41. In this study, we report that ExoS ADP ribosylates Rap1b at Arg41 and that ADP ribosylation of Arg41 inhibits the ability of C3G to stimulate guanine nucleotide exchange. The mechanism responsible for this inhibition is one in which ADP-ribosylated Rap binds inefficiently to C3G, relative to wild type Rap. This identifies a second member of the Ras GTPase subfamily that can be ADP ribosylated by ExoS and indicates that ExoS can inhibit both Ras and Rap signaling pathways in eukaryotic cells.