Dominique Grall

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein‐coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth‐responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin‐insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre‐treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4‐beta‐phorbol‐12,13‐dibutyrate has no effect. Rather, thrombin‐induced Shc phosphorylation is enhanced in cells depleted of phorbol ester‐sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant‐negative effect on thrombin‐stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein‐coupled receptors.

ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells

Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin β subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of α5β1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in α5β1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on β1 and β3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of α5β1-competent matrix-forming adhesions.

Molecular dissection of the ILK-PINCH-parvin triad reveals a fundamental role for the ILK kinase domain in the late stages of focal-adhesion maturation

Integrin-linked kinase (ILK) and cytoplasmic adaptors of the PINCH and parvin families form a ternary complex, termed IPP, that localizes to integrin adhesions. We show here that deletion of the genes encoding ILK or PINCH1 similarly blocks maturation of focal adhesions to tensin-rich and phosphotyrosine-poor fibrillar adhesions (FBs) by downregulating expression or recruitment of tensin and destabilizing α5β1-integrin–cytoskeleton linkages. As IPP components are interdependent for integrin targeting and protein stability, functional dissection of the complex was achieved by fusing ILK, PINCH, parvin or their individual motifs to the cytoplasmic tail of β3 integrin, normally excluded from FBs. Using this novel gain-of-function approach, we demonstrated that expression of the C-terminal kinase domain of ILK can restore tensin recruitment and prompt focal-adhesion maturation in IPP-null cells. Debilitating mutations in the paxillin- or ATP-binding sites of ILK, together with α-parvin silencing, revealed a determinant role for ILK-parvin association, but not for direct paxillin binding, in this function. We propose a model in which the C-terminal domain of ILK promotes integrin sorting by reinforcing α5β1-integrin–actin linkage and controls force transmission by targeting tensin to maturing adhesions.

Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin‐linked kinase (ILK) is one such partner of β1 integrins that participates in dynamic rearrangement of cell‐matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane‐targeted green fluorescent protein (ILK‐GFP‐F). ILK‐GFP‐F is highly enriched in cell‐matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2–5 µm) and elongated adhesions (>5 µm). ILK‐GFP‐F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP‐bound Rac‐1. Conversely, ILK knock‐down by siRNA transfection decreases active Rac‐1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor βPIX. Further, expression of a dominant negative βPIX mutant reversed the increase in active Rac‐1 levels of ILK‐GFP‐F‐expressing cells, thus placing βPIX in the pathway leading from ILK to Rac‐1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK‐depleted cells, suggesting that an additional ILK‐dependent signal is required for cell spreading.—Boulter, E., Grall, D., Cagnol, S., and Van Obberghen‐Schilling, E. Regulation of cell‐matrix adhesion dynamics and Rac‐1 by integrin linked kinase. FASEB J. 20, E640‐E651 (2006)

Modulation of Rho GTPase activity in endothelial cells by selective proteinase-activated receptor (PAR) agonists

Summary.  The proteinase‐activated receptors (PAR) PAR1 and PAR2 mediate responses to thrombin and trypsin‐like proteases, respectively. Both receptors are expressed on endothelial cells where they have been reported to transduce a similar set of intracellular responses. In cultured human umbilical vein endothelial cells (HUVEC), we observed a marked difference in shape changes induced by PAR‐activating peptides (PAR‐APs); unlike PAR1‐AP, PAR2‐AP failed to stimulate cell rounding. Objectives were to shed light on the mechanisms underlying PAR‐mediated cytoskeletal responses. We examined the activation of the Rho family GTPases in HUVEC using highly selective PAR1‐ and PAR2‐APs to do this. Both peptides induced a robust and transient activation of RhoA, with the time course of activation being more sustained for the PAR1‐AP. Interestingly, divergent effects on Rac activity were observed. Addition of PAR1‐AP inhibited basal Rac activity as well as the phosphorylation of the Rac effector, p21‐activated kinase (PAK). In contrast, PAR2‐AP induced a modest activation of Rac, phosphorylation of PAK and translocation of cortactin from the cytosol to membrane ruffles, a Rac‐dependent event. In vivo, only PAR1‐AP rapidly enhanced vascular permeability in a mouse skin assay. We conclude that the differential regulation of the Rac/PAK pathway by PAR1 and PAR2 agonists in endothelial cells points toward distinct roles for these receptors in the control of vascular permeability and blood vessel remodeling.

An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression

Most normal cells require both mitogens and integrin-mediated attachment for growth. It is generally accepted that the p42/p44 MAP kinase module, which can be activated by both growth factors and adhesion, plays a critical role in G0 to S phase progression of quiescent cells. Studies on various cultured fibroblasts have shown that removal of anchorage leads to cell cycle arrest in G1 and it has been proposed that adhesion-dependent G1 progression requires the joint regulation of p42/p44 MAP kinase by integrins and growth factors. In quiescent CCL39 lung fibroblasts, MAP kinase activation in response to serum becomes compromised when cells are placed in suspension. Under these conditions, serum-stimulated cells arrest their growth in mid-G1 with reduced cyclin D1 expression and increased p21Cip/Waf1 expression, as compared to their attached counterparts. To determine whether a casual link exists between sub-optimal activation of MAP kinase in non-adherent cells and the observed G1 block, we used a variant of CCL39 stably expressing an estrogen-inducible activated-Raf-1 construct (ΔRaf-1:ER). We found that even strong and sustained activation of MAP kinase with estradiol, in addition to serum, is not able to boost cyclin D1 expression levels or stimulate hyperphosphorylation of pRb in suspended CCL39-ΔRaf-1:ER cells. These results indicate that p42/p44 MAP kinase activation is not a limiting factor for G1 to S phase transit in absence of anchorage. Thus, at least one adhesion-mediated signalling event, distinct from MAP kinase activation is required for maximal cyclin D1 induction and hyperphosphorylation of pRb.

Autocrine fibronectin directs matrix assembly and crosstalk between cell−matrix and cell−cell adhesion in vascular endothelial cells

Cellular fibronectin (cFN) variants harboring extra FN type 3 repeats, namely extra domains B and A, are major constituents of the extracellular matrix around newly forming blood vessels during development and angiogenesis. Their expression is induced by angiogenic stimuli and their assembly into fibrillar arrays is driven by cell-generated tension at α5β1 integrin-based adhesions. Here, we examined the role and functional redundancy of cFN variants in cultured endothelial cells by isoform-selective RNA interference. We show that FN fibrillogenesis is a cell-autonomous process whereby basally directed secretion and assembly of cellular FN are tightly coupled events that play an important role not only in signaling at cell–matrix adhesions but also at cell–cell contacts. Silencing of cFN variants differentially affects integrin usage, cell spreading, motility and capillary morphogenesis in vitro. cFN-deficient cells undergo a switch from α5β1- to αvβ3-based adhesion, accompanied by a Src-regulated disruption of adherens junctions. These studies identify a crucial role for autocrine FN in subendothelial matrix assembly and junctional integrity that provides spatially and temporally restricted control of endothelial plasticity during angiogenic blood vessel remodeling.

Fibronectin expression in glioblastomas promotes cell cohesion, collective invasion of basement membrane in vitro and orthotopic tumor growth in mice

Glioblastoma multiforme (GBM) are highly invasive and angiogenic malignancies with a median survival time from diagnosis of <15 months. Previous work has revealed robust overexpression of fibronectin (FN) mRNA in GBM, although immunohistochemical staining of FN in these tumors is typically associated with the angiogenic vasculature. Here we sought to examine the expression of tumor cell FN and address its possible involvement in the invasive phenotype of GBM. We found that FN was expressed and assembled into fibrillar arrays in human tumors and in established GBM lines. Cultured cells spontaneously formed dense cellular networks and spheroid-like domes. Depletion of FN by targeted-short hairpin RNA expression disrupted matrix assembly and multicellular network organization by exerting profound effects on cell adhesion and motility. Although FN depletion enhanced persistent directional migration of single cells, it compromised collective invasion of spheroids through a laminin-rich matrix and sensitized cells to ionizing radiation. In orthotopic grafts, FN depletion significantly reduced tumor growth and angiogenesis. Together our results show that FN produced by the tumor cells has a role in GBM pathophysiology and they provide insights into the implications that targeting FN interactions may have for combating this dreaded disease.

Epidermal Growth Factor Receptor Protein Detection in Head and Neck Cancer Patients: A Many-Faceted Picture

Purpose: Epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). Despite intensive biomarker studies, a consensual method for assessing EGFR protein expression is still lacking. Here we set out to compare three EGFR detection methods in tumor specimens from HNSCC patients. Experimental Design: Tumors were prospectively excised from a series of 79 high-risk HNSCC patients enrolled in a GORTEC-sponsored clinical trial. EGFR expression was determined using a ligand-binding assay on membranes, Western blotting (WB) on membranes and total homogenates, and immunohistochemistry (IHC) on tissue microarrays. In addition, phosphorylated EGFR (pEGFR) was measured by WB on membranes. Results: Distributions and ranges of tumor EGFR expression were method dependent. Moderate positive correlations (Spearman coefficient r ≈ 0.50) were observed between EGFR expression measured by the binding assay and WB or IHC. pEGFR levels positively and significantly correlated with total EGFR expression measured by WB or ligand binding, but not by IHC. The highest correlation (r = 0.85) was observed between EGFR and pEGFR levels, both measured by WB on membranes. Interestingly, the fraction of phosphorylated receptor (pEGFR/EGFR both measured by WB on membranes) significantly declined with increasing tumor EGFR expression, by all assessment methods used. Conclusion: This study shows significant correlations between EGFR detection methods. The observed relationships between EGFR and pEGFR indicate that high-throughput pEGFR/EGFR analyses merit further investigations and consideration for routine use in patient samples. Clin Cancer Res; 18(5); 1313–22. ©2012 AACR.

Contrasted outcomes to gefitinib on tumoral IGF1R expression in head and neck cancer patients receiving postoperative chemoradiation (GORTEC trial 2004-02).

Purpose: Intermediate/high-risk operated patients with head and neck cancer may benefit from the addition of EGF receptor (EGFR) inhibitor gefitinib to chemoradiation. This study was designed to assess improved outcomes and identify predictive biomarkers. Experimental Design: Patients provided informed consent for tumor biomarker analyses and, when eligible, were further enrolled in the therapeutic CARISSA multicenter randomized phase II trial of postoperative irradiation with cisplatin + gefitinib (GORTEC 2004-02-NCT00169221). Results: Seventy-nine patients were included in the biomarker study, whereas 27 did not meet prerequisites for randomization between gefitinib and placebo. Two-year disease-free survival (DFS) rate was 65.0% and did not differ between randomized patients treated with gefitinib or placebo (P = 0.85). The similarity of DFS curves between nonrandomized patients (n = 27), randomized patients without gefitinib (n = 27), and randomized patients receiving gefitinib (n = 25), and similar histoclinical parameter distributions for all groups, allowed us to conduct statistical analyses on the entire population. On multivariate analysis, elevated expression of PAK1 by Western blotting, CD31 and membranous insulin-like growth factor 1 receptor (IGF1R) both by immunohistochemistry was significantly associated with shorter DFS. There was a significant interaction between IGF1R and gefitinib. Gefitinib abolished the prognostic discriminative power of high IGF1R expression; patients with elevated IGF1R expression benefited from gefitinib whereas those with low IGF1R fared worse. Conclusion: Gefitinib treatment affords no significant clinical benefit on DFS in an unselected population of patients with head and neck cancer. Our results point to the potential advantage of personalizing treatment for gefitinib based on tumoral IGF1R expression. This should foster confirmatory analyses in trials involving EGFR-targeting agents. Clin Cancer Res; 18(18); 5123–33. ©2012 AACR.