p190RhoGAP is the convergence point of adhesion signals from α5β1 integrin and syndecan-4

The fibronectin receptors α5β1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α5β1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α5β1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α5β1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion

p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling

Plexins are transmembrane receptors for semaphorins, guiding cell migration and axon extension. Plexin activation leads to the disassembly of integrin-based focal adhesive structures and to actin cytoskeleton remodelling and inhibition of cell migration; however, the underlying molecular mechanisms are unclear. We consistently observe a transient decrease of cellular RhoA-GTP levels upon plexin activation in adherent cells. One of the main effectors of RhoA downregulation is p190, a ubiquitously expressed GTPase activating protein (GAP). We show that, in p190-deficient fibroblasts, the typical functional activities mediated by plexins (such as cell collapse and inhibition of integrin-based adhesion) are blocked or greatly impaired. Notably, the functional response can be rescued in these cells by re-expressing exogenous p190, but not a mutant form specifically lacking RhoGAP activity. We furthermore demonstrate that semaphorin function is blocked in epithelial cells, primary endothelial cells and neuroblasts upon treatment with small interfering RNAs that knockdown p190 expression. Finally, we show that p190 transiently associates with plexins, and its RhoGAP activity is increased in response to semaphorin stimulation. We conclude that p190-RhoGAP is crucially involved in semaphorin signalling to the actin cytoskeleton, via interaction with plexins

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

BACKGROUND: Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS: We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS: Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS: A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib

Rnd Proteins Function as RhoA Antagonists by Activating p190 RhoGAP

AbstractBackground: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA. However, effector molecules of Rnd proteins with such properties have not been identified.Results: Here we identify p190 RhoGAP (p190), the most abundant GAP for RhoA in cells, as an interactor with Rnd proteins and show that this interaction is mediated by a p190 region that is distinct from the GAP domain. Using Rnd3-RhoA chimeras and Rnd3 mutants defective in p190 binding, as well as p190-deficient cells, we demonstrate that the cellular effects of Rnd expression are mediated by p190. We moreover show that Rnd proteins increase the GAP activity of p190 toward GTP bound RhoA and, finally, demonstrate that expression of Rnd3 leads to reduced cellular levels of RhoA-GTP by a p190-dependent mechanism.Conclusions: Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA

Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib

Non-small cell lung cancers (NSCLCs) with activating mutations in the kinase domain of the epidermal growth factor receptor (EGFR) demonstrate dramatic, but transient, responses to the reversible tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Some recurrent tumors have a common secondary mutation in the EGFR kinase domain, T790M, conferring drug resistance, but in other cases the mechanism underlying acquired resistance is unknown. In studying multiple sites of recurrent NSCLCs, we detected T790M in only a small percentage of tumor cells. To identify additional mechanisms of acquired resistance to gefitinib, we used NSCLC cells harboring an activating EGFR mutation to generate multiple resistant clones in vitro. These drug-resistant cells demonstrate continued dependence on EGFR and ERBB2 signaling for their viability and have not acquired secondary EGFR mutations. However, they display increased internalization of ligand-activated EGFR, consistent with altered receptor trafficking. Although gefitinib-resistant clones are cross-resistant to related anilinoquinazolines, they demonstrate sensitivity to a class of irreversible inhibitors of EGFR. These inhibitors also show effective inhibition of signaling by T790M-mutant EGFR and killing of NSCLC cells with the T790M mutation. Both mechanisms of gefitinib resistance are therefore circumvented by irreversible tyrosine kinase inhibitors. Our findings suggest that one of these, HKI-272, may prove highly effective in the treatment of EGFR-mutant NSCLCs, including tumors that have become resistant to gefitinib or erlotinib

Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management

Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression

Introduction: Rho signaling regulates key cellular processes including proliferation, survival, and migration, and it has been implicated in the development of many types of cancer including breast cancer. P190B Rho GTPase activating protein (RhoGAP) functions as a major inhibitor of the Rho GTPases. P190B is required for mammary gland morphogenesis, and overexpression of p190B in the mammary gland induces hyperplastic lesions. Hence, we hypothesized that p190B may play a pivotal role in mammary tumorigenesis. Methods: To investigate the effects of loss of p190B function on mammary tumor progression, p190B heterozygous mice were crossed with an MMTV-Neu breast cancer model. Effects of p190B deficiency on tumor latency, multiplicity, growth, preneoplastic progression and metastasis were evaluated. To investigate potential differences in tumor angiogenesis between the two groups, immunohistochemistry to detect von Willebrand factor was performed and quantified. To examine gene expression of potential mediators of the angiogenic switch, an angiogenesis PCR array was utilized and results were confirmed using immunohistochemistry. Finally, reciprocal transplantation of tumor fragments was performed to determine the impact of stromal deficiency of p190B on tumor angiogenesis. Results: P190B deficiency reduced tumor penetrance (53% of $p190B^{+/-}Neu$ mice vs. 100% of $p190B^{+/+}Neu$ mice formed tumors) and markedly delayed tumor onset by an average of 46 weeks. Tumor multiplicity was also decreased, but an increase in the number of preneoplastic lesions was detected indicating that p190B deficiency inhibited preneoplastic progression. Angiogenesis was decreased in the p190B heterozygous tumors, and expression of a potent angiogenic inhibitor, thrombospondin-1, was elevated in $p190B^{+/-}Neu$ mammary glands. Transplantation of $p190B^{+/-}Neu$ tumor fragments into wild-type recipients restored tumor angiogenesis. Strikingly, $p190B^{+/+}Neu$ tumor fragments were unable to grow when transplanted into $p190B^{+/-}Neu$ recipients. Conclusions: These data suggest that p190B haploinsufficiency in the epithelium inhibits MMTV-Neu tumor initiation. Furthermore, p190B deficiency in the vasculature is responsible, in part, for the inhibition of MMTV-Neu tumor progression

Rho GTPase function in flies: insights from a developmental and organismal perspective.

Morphogenesis is a key event in the development of a multicellular organism and is reliant on coordinated transcriptional and signal transduction events. To establish the segmented body plan that underlies much of metazoan development, individual cells and groups of cells must respond to exogenous signals with complex movements and shape changes. One class of proteins that plays a pivotal role in the interpretation of extracellular cues into cellular behavior is the Rho family of small GTPases. These molecular switches are essential components of a growing number of signaling pathways, many of which regulate actin cytoskeletal remodeling. Much of our understanding of Rho biology has come from work done in cell culture. More recently, the fruit fly Drosophila melanogaster has emerged as an excellent genetic system for the study of these proteins in a developmental and organismal context. Studies in flies have greatly enhanced our understanding of pathways involving Rho GTPases and their roles in development