Tyrosine Phosphorylation Of p130Cas Research Articles

Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling.

Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRβ) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility.

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo.

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

Neuropilin-1 (NRP-1) and Magnetic Nanoparticles, a Potential Combination for Diagnosis and Therapy of Gliomas

In this review, Neuropilin-1 (NRP-1) has been focused as a novel molecular target for potential treatment of gliomas. The properties of NRP-1 were described briefly. The role of NRP-1 in gliomas was explored in details, including relationships of NRP-1 expression and glioma prognosis, Sema3A-NRP-1 signaling in gliomas, NRP-1 signaling and VEGF/VEGFR, PlGF, TGF-β, PDGF, LD22-4 of FGF2, autocrine of HGF/SF, p130Cas tyrosine phosphorylation and integrin-associated tumor microenvironment in gliomas, NRP-1 intracellular trafficking, NRP-1 and glioma stem-like cells, as well as magnetic nanoparticles related to targeting gliomas. NRP-1, a multifunctional-receptors protein, would mediate diverse cellular signaling pathways in gliomas, and might potentially act as a novel therapeutic target. In future, magnetic nanoparticles coated with NRP-1 may play a vital role on diagnosis and therapy of gliomas.

Selective interaction of CaMKIIδ2 and Fyn mediates vascular smooth muscle (VSM) cell motility

Our previous study demonstrated that CaMKIIδ2 and Fyn physically interact via CaMKIIδ2's C ‐terminal “tail”. We also reported that this interaction between CaMKIIδ2 and Fyn positively mediates VSM cell motility. The purpose of this study is to better understand the mechanistic relationship between CaMKIIδ2 and Fyn and to clarify their cooperative role in migrating VSM cells. With immunofluorescence, we show that SiRNA depletion of CaMKIIδ in VSM cells attenuated PDGF‐dependent translocation of Fyn to peripheral membranes. Similarly, overexpression of CaMKIIδ6, which does not interact with Fyn, also reduced Fyn's ability to translocate to the plasma membrane in response to PDGF. Furthermore, overexpression of CaMKIIδ6 reduced VSM cell motility in both a scratch wound assay and a transwell assay. The rate of focal adhesion protein recruitment to focal adhesions is directly linked to cell motility. Using TIRF/FRAP in cells overexpressing GFP‐tagged Paxillin, we show that SiRNA mediated loss of CaMKIIδ2 or Fyn disrupted the kinetics of focal adhesion turnover. Stimulation of VSM cells with PDGF results in tyrosine phosphorylation of P130Cas and Paxillin, both known substrates of Fyn. Along with preventing Fyn translocation to the plasma membrane, overexpression of CaMKIIδ6 inhibited this PDGF‐dependent phosphorylation of P130Cas and Paxillin. Therefore, we conclude that CaMKIIδ2 “delivers” Fyn to peripheral membranes proximal to focal adhesions where Fyn modulates focal adhesion dynamics and subsequent VSM cell motility.

Super‐resolution Imaging Shows Spatial Regulation of p130Cas Phosporylation within Focal Adhesions

Cell motility is critical for physiological processes such as tissue development and remodeling as well as pathologies such as cancer metastasis. Cell motility is influenced by its surrounding matrix through biochemical and mechanical stimuli. p130Cas is a signaling molecule that has been proposed to be a mechanosensor, which responds to mechanical stimuli and mediates cell motility during the early stage of lamellipodial protrusions. p130Cas localizes to focal adhesions (FAs) and binds with its effectors via tyrosine phosphorylation of its substrate domain. Previous studies have suggested that mechanical stimuli increase p130Cas tyrosine phosphorylation and these phosphotyrosines recruit SH2‐domain containing effectors like Crk, that initiate a signaling cascade activating the small Rho‐GTPase Rac1 leading to lamellipodial protrusions.Due to spatial heterogeneity of stresses within FAs, we hypothesized that p130Cas phosphorylation is not uniform across a single FA. Spatial differences across such small structures as FAs cannot be resolved using biochemical assays or standard microscopy. We used a super resolution imaging technique, PALM (photo‐activated localization microscopy) to study the differences in p130Cas phosphorylation within individual FAs using SH2 domains of Crk as probes. Our results show punctate localization of SH2 probes at single FAs compared to uniform distribution of p130Cas.Our data suggest that p130Cas activity is spatially regulated. In the future we will look at correlations between mechanical stress within FAs and p130Cas activity. Our results will help elucidate the mechanism of mechanical regulation of p130Cas phosphorylation, and may help understand how cells sense and respond to external mechanical stimuli.

Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells.

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca2+ concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [3H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2–9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling.

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]

Ocular hypotensive efficacy of Src-family tyrosine kinase inhibitors via different cellular actions from Rock inhibitors

We investigated the effects of Src-family tyrosine kinase (SFK) inhibitors on intraocular pressure (IOP) and trabecular meshwork (TM) cells. The SFK inhibitors, PP2, PP1, and damnacanthal, significantly lowered IOP from baseline following intracameral injection in ocular normotensive rabbits, and PP2 decreased trans-epithelial electrical resistance (TEER) of TM cell layers in a dose-dependent manner ranging from 0.1 μM to 100 μM. The maximal efficacy of PP2 on TEER was a reduction to 71.7% relative to the vehicle-treated group at 100 μM. PP2 decreased the adhesion of TM cells to culture surfaces either uncoated with specific ECM proteins dose-dependently or coated with extracellular matrix proteins such as laminin I, fibronectin, collagen type I and basement membrane extraction. Tyrosine phosphorylation of focal adhesion kinase and p130cas was decreased by PP2. On the other hand, major changes in actin staining of TM cells were not able to be detected after PP2 treatment, although quantitative analysis showed that PP2 induced some morphological changes which were in the different direction to those caused by Y-27632, a Rock inhibitor. Y-27632 at 10 μM increased the permeability of TM cell layers, but did not induce changes in the adhesion of TM cells. These results suggest that SFK inhibitors lower IOP, at least partly, by acting on TM cells in a manner that is distinct from Rock inhibitors.

P130Cas controls the susceptibility of cancer cells to TGF-β-induced growth inhibition

Transforming growth factor-beta (TGF-β) suppresses the initiation of tumorigenesis by causing arrest at the G1 phase of the cell cycle. The loss of the antiproliferative function of TGF-β is a hallmark of many cancers. Here we report that p130Cas plays a role in determining the cellular responsiveness to TGF-β-induced growth inhibition in some cancer cells. An analysis of the tyrosine phosphorylation levels of p130Cas revealed higher levels of phosphorylation in cancer cell lines (MCF7 and A375) than in corresponding normal cell lines (MCF10A and MEL-STV). In contrast to normal cells, the cancer cells showed resistance to not only TGF-β-induced Smad3 phosphorylation and p21 expression, but also growth inhibition. However, silencing p130Cas using siRNA was sufficient to restore Smad3 phosphorylation and p21 expression, as well as the susceptibility to TGF-β-induced growth inhibition. Interestingly, the stable overexpression of p130Cas accelerated TGF-β-induced epithelial–mesenchymal transition. Our results suggest that elevated expression and tyrosine phosphorylation of p130Cas contributes to the resistance to TGF-β-induced growth inhibition, and thus to the initiation and progression of human cancers that harbor an active integrin signal.

LPA Stimulates the Phosphorylation of p130Cas via G i2 in Ovarian Cancer Cells

Ovarian cancer is the most deadly gynecological cancer, with previous studies implicating lysophosphatidic acid (LPA) in the progression of approximately 90% of all ovarian cancers. LPA potently stimulates the tyrosine phosphorylation of p130Cas, a scaffolding protein, which, upon phosphorylation, recruits an array of signaling molecules to promote tumor cell migration. Our work presented here identifies Gαi2 as the major G protein involved in tyrosine phosphorylation of p130Cas in a panel of ovarian cancer cells consisting of HeyA8, SKOV3, and OVCA429. Our results also indicate that the G12 family of G proteins that are also involved in LPA-mediated migration inhibits tyrosine phosphorylation of p130Cas. Using p130Cas siRNA, we demonstrate that p130Cas is a necessary downstream component of LPA Gαi2-induced migration and collagen-1 invasion of ovarian cancer cells. Considering the fact that LPA stimulates invasive migration through the coordination of multiple downstream signaling pathways, our current study identifies a separate unique signaling node involving p130Cas and Gαi2 in mediating LPA-mediated invasive migration of ovarian cancer cells.

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.

Enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides GD2/GD3

The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2-negative (GD3/GD2-) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2- cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside-expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3- and/or GD2-expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular-targeted therapy.

Introduction: Crk Signaling and Cancer- The First 25 Years

Introduction: Crk Signaling and Cancer- The First 25 Years

Abstract 461: Involvement of α9β1 integrin-iNOS pathway in matrix metalloproteinase-9 and/or urokinase plasminogen activator receptor-mediated glioma cell migration

Abstract Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide (NO) production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we aimed to investigate the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Immunohistochemical analysis of the glioblastoma multiforme (GBM) tissue array revealed the prominent presence of iNOS in several clinical GBM samples. In addition, immunofluorescence analysis of U251 glioma cell lines and 5310 glioma xenograft cells showed a prominent expression of iNOS. Transcriptional inactivation of MMP-9 and/or uPAR by respective shRNA reduced iNOS expression in both U521 and 5310 glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in these glioma cells after transfection with MMP-9- or uPAR-overexpressing plasmids. Wound healing and spheroid migration assays showed a significant inhibition of the migration potential of MMP-9- or uPAR-overexpressed U251 glioma cells after treatment with L-NAME, an inhibitor of iNOS. Similarly, Matrigel invasion assay revealed a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed U251 and 5310 glioma cells after L-NAME treatment. Further, we noticed a prominent reduction of iNOS expression in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Integrin α9β1 regulates iNOS activity via Src tyrosine kinase; Src coordinates subsequent signaling pathways through activation of FAK and tyrosine phosphorylation of the adaptor protein p130Cas. Western blot analysis of MMP-9 and/or uPAR shRNA treated U251 and 5310 glioma cells revealed significant reductions in the protein expression of cSrc, phosphoSrc, FAK and p130Cas after simultaneous downregulation of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of these glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels. Therefore, our results highlight the possible involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 461. doi:1538-7445.AM2012-461

Abstract LB-24: LPA-mediated oncogenic signaling in ovarian cancer involves p130Cas

Abstract Ovarian cancer is the most deadly gynecological cancer and the need for new therapies is evidenced by the fact no new drugs targeting ovarian cancer have been identified. Recent studies have shown that lysophosphatidic acid (LPA), a lipid growth factor, is implicated in the genesis and progression of ovarian cancer, yet the majority of the mechanisms through which LPA promotes cancer genesis and progression remain to be elucidated. Therefore, the underlying theme of our research is to identify novel LPA-activated signaling nodes that can be targeted for therapy in ovarian cancer. Subjecting the lysates from HeyA8 ovarian cancer cells to a high-throughput antibody array analysis, we identified that LPA potently stimulates the tyrosine-phosphorylation of p130Cas, a scaffolding protein, which, upon phosphorylation, recruits an array of signaling molecules that promotes tumor cell proliferation, survival and metastasis. The antibody-array data was validated via immunoprecipitation of p130Cas and immunoblotting with a phospho-tyrosine specific antibody in a panel of ovarian cancer cells consisting of HeyA8, OVCA429, OVCA432 and SKOV3, establishing cell-type independent activation of p130Cas by LPA. Analyzing further, we have identified that tyrosine-410 of p130Cas is phosphorylated in response to LPA. Several past studies, including ours, have shown oncogenic signaling by LPA involves the activation of LPA receptors that are coupled to the α-subunits of the heterotrimeric G proteins, Gi, Gq, G12, and/or G13. To define the specific α-subunit that mediates LPA-induced p130Cas-phosphorylation, we monitored Tyr-410 phosphorylation of p130Cas in response to 20 μM of LPA (20 min) in HeyA8 cells in which the expression of Gαi2, Gαq, Gα12, or Gα13 was silenced by specific shRNAs. Our results indicated that only the silencing of Gαi2 abrogated LPA-mediated Tyr-410 phosphorylation of p130Cas. Since Gαi2 has been shown to play a critical role in LPA-LPAR coupled cell migration, we hypothesized that the LPA-LPAR-Gαi2-p130Cas signaling axis is involved in the invasive migration of ovarian cancer cells. To test this hypothesis, we examined whether the knocking down of p130Cas, via p130Cas-specific siRNA, attenuated LPA-mediated migration of ovarian cancer cells. Our results indicated that the silencing of p130Cas significantly inhibited LPA-stimulated migration of ovarian cancer cells by 58%. Thus, our study is the first to establish LPA-LPAR-Gαi2-mediated stimulation of p130Cas is involved in invasive migration of ovarian cancer cells. Overall, the results presented in our current study point to p130Cas as a critical signaling node in pathological LPA signaling and that by targeting LPA receptors, LPA production and/or the downstream kinases that phosphorylate p130Cas, it may be possible to disable the oncogenic signaling cascades activated by p130Cas in ovarian cancer and enhance the survival of ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-24. doi:1538-7445.AM2012-LB-24

Regulation of Focal Adhesions by Flightless I Involves Inhibition of Paxillin Phosphorylation via a Rac1-Dependent Pathway

Flightless I (Flii) is an actin-remodeling protein that influences diverse processes including cell migration and gene transcription and links signal transduction with cytoskeletal regulation. Here, we show that Flii modulation of focal adhesions and filamentous actin stress fibers is Rac1-dependent. Using primary skin fibroblasts from Flii overexpressing (Flii(Tg/Tg)), wild-type, and Flii deficient (Flii(+/-)) mice, we show that elevated expression of Flii increases stress fiber formation by impaired focal adhesion turnover and enhanced formation of fibrillar adhesions. Conversely, Flii knockdown increases the percentage of focal complex positive cells. We further show that a functional effect of Flii at both the cellular level and in in vivo mouse wounds is through inhibiting paxillin tyrosine phosphorylation and suppression of signaling proteins Src and p130Cas, both of which regulate adhesion signaling pathways. Flii is upregulated in response to wounding, and overexpression of Flii inhibits paxillin activity and reduces adhesion signaling by modulating the activity of the Rho family GTPases. Overexpression of constitutively active Rac1 GTPase restores the spreading ability of Flii(Tg/Tg) fibroblasts and may explain the reduced adhesion, migration, and proliferation observed in Flii(Tg/Tg) mice and their impaired wound healing, a process dependent on effective cellular motility and adhesion.

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

Neuropilin-1 Signaling through p130Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.

Ganglioside GD3 Enhances Adhesion Signals and Augments Malignant Properties of Melanoma Cells by Recruiting Integrins to Glycolipid-enriched Microdomains

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin when treated with fetal calf serum than GD3- cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin beta1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin beta1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin beta1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.