FAK Y397 Phosphorylation Research Articles

Abstract 1254: FAK activity regulates αvβ5 integrin and osteopontin expression to control breast and ovarian cancer anchorage-independent growth

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell motility and survival downstream of integrin receptors. FAK expression and activity are elevated during breast and ovarian cancer progression and this is associated with a poor clinical prognosis. Small molecule FAK inhibitors (FAK-I) block breast and ovarian tumor progression through a novel mechanism that is associated with the apoptosis under anchorage-deprived conditions. Metastatic cells can grow as anchorage-independent spheroids and we hypothesize that a spheroid micro-environment remains dependent on integrin- and FAK-associated signaling for survival. Here we show that intraperitoneal (ip) injection of murine ID8 ovarian carcinoma cells into mice, recovery of ascites-associated tumor cells (ID8ip) and growth ex vivo, yielded tumor cells with elevated levels of phosphorylated FAK (Y397 and Y576 phosphorylation), αvβ5 integrin, and osteopontin (OPN) matrix protein expression compared to parental ID8 cells. Analyses of paraffin-embedded breast and ovarian tumor sections revealed significantly higher pY397 FAK (p<0.05), β5 integrin (p<0.0001), and OPN expression (p<0.005) in stage II-III compared to stage I tumors. Treatment of ovarian ID8ip, human ovarian HEY carcinoma, or human MDA-MB-231 breast carcinoma cells with FAK-I inhibited cell growth, prevented FAK Y397 phosphorylation, reduced β5 integrin and OPN levels independent of effects on β1 or β3 integrins or changes in matrix proteins such as fibronectin. FAK-I impairs anchorage independent cell growth and orthotopic tumor growth but not growth of cells in plastic-adherent cell culture. Stable knockdown of either FAK or β5 integrin in HEY cells prevented anchorage-independent growth. Re-expression of wildtype or kinase-dead FAK within HEY cells verified the importance of FAK activity in promoting β5 integrin and OPN expression associated with enhanced tumor growth. Ongoing studies are focused on determining the molecular connections of FAK to the regulation of αvβ5 and OPN expression. Together, our studies support a novel role for a signaling loop involving OPN and αvβ5 integrin leading to FAK activation and reinforcement of OPN and β5 integrin expression within the spheroid and tumor micro-environment enhancing breast and ovarian cancer cell survival and tumor progression. Supported by Susan G. Komen for the Cure (KG111237) and NIH grant CA102310 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 1254. doi:1538-7445.AM2012-1254

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.

Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis

Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.

Abstract 1725: Neuroendocrine modulation of anoikis

Abstract Chronic stress and associated hormones are known to affect multiple systems including immunological and endocrinological functions, but the effects on cancer growth and progression are not fully understood. Here, we demonstrate that chronic stress and associated increases in catecholamines, protect tumor cells from anoikis and promote growth by activating focal adhesion kinase (FAK). These effects involve phosphorylation of FAKY397 associated with actin-dependent Src interaction with membrane-associated FAK. In human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in ovarian cancer patients. These findings further point at potential new therapeutic targets for cancer management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1725.

Mechanosensing machinery for cells under low substratum rigidity

Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates beta(1)-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered beta(1)-integrin (V737N), but not constitutively active beta(1)-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess beta(1)-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers beta(1)-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of beta(1)-integrin clustering enhances the interaction between beta(1)-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.

Early adhesion induces interaction of FAK and Fyn in lipid domains and activates raft-dependent Akt signaling in SW480 colon cancer cells

Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.

S1691 Endogenous Protein Kinase D Contributes to Gq-Coupled Receptor Induction of DNA Synthesis in Swiss 3T3 Cells

overexpressing prostaglandin dehydrogenase (18.PGDH) comprise a model with abolished basal PG production. Pyk2 expression, nearly absent in these cells, was reintroduced by transient transfection either as wild type or a kinase deficient (KD) mutant form. Pyk2 immunoprecipitates were used to phosphorylate a FAK regulatory domain segment with either wild type Y397 or mutant Y397F fused to GST. Total 32P incorporation In Vitro from 32P-ATP was measured by autoradiography. Y397 phosphorylation of GST-FAK In Vitro, and endogenous FAK in cell extracts, was detected by Western blot analysis with phosphospecific FAK Y397 antibody. Results: IEC-18 cells with siRNA-reduced Pyk2 expression exhibited similar levels of total FAK protein, but very low basal FAK Y397 phosphorylation in cell extracts. 18.PGDH cells, in comparison with parental IEC-18 cells, also exhibited selectively reduced constitutive FAK Tyr397 phosphorylation. Transfected Pyk2, but not Pyk2-KD, partially restored constitutive FAK Y397 phosphorylation in these cells. Pyk2, but not Pyk2-KD immunoprecipitates incorporated 32P into both GST-FAK and, to a lesser extent, GST-FAK(Y397F), but not GST. In particular, phospho-Y397Western blot confirmed that Pyk2 catalyzed dramatically increased FAK Y397 phosphorylation in GST-FAK. Conclusions: Taken together, these studies extend the observed correlation between Pyk2 downregulation and FAK Y397 phosphorylation in intestinal cells deprived of basal PG, indicating that Pyk2 can directly phosphorylate FAK Y397 as well as additional, as yet unidentified site(s) within the FAK regulatory domain.

MAP-kinase activity necessary for TGFβ1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by > 40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.

Activation of p38 MAPKα by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)- differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPKalpha small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 +/- 20.5% in DMSO-treated THP-1 macrophages and by 20.9 +/- 9% in primary monocytes (P < 0.05 each). SB-203580 (20 microM) reduced basal p38 MAPK phosphorylation by 34.7 +/- 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPKalpha siRNA reduced total p38 MAPK protein by 50-60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPKalpha siRNA reduced total FAK protein by 40-50%, and total ERK by 10-15%, but increased phosphorylated ERK 1.4 +/- 0.1-fold. p38 MAPKalpha siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAK-dependent inverse effect on p38 MAPKalpha that might subsequently downregulate ERK.

Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK.

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37 degrees C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 +/- 1.8 vs. 42.0 +/- 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 +/- 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 microM), an ERK activation inhibitor, increased basal phagocytosis (26.9 +/- 1.8 vs. 31.7 +/- 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation.

A novel mode for integrin-mediated signaling: tethering is required for phosphorylation of FAK Y397.

The common model for integrin mediated signaling is based on integrin clustering and the potential for that clustering to recruit signaling molecules including FAK and src. The clustering model for transmembrane signaling originated with the analysis of the EGF receptor signaling and remains the predominant model. The roles for substrate-bound ligand and ligand occupancy in integrin-mediated signaling are less clear. A kinetic model was established using HT1080 cells in which there was a linear relationship between the strength of adhesion, the proportion of alpha5beta1 integrin that could be chemically cross-linked, and the number of receptor-ligand bonds. This graded signal produced a similarly graded response measured by the level of specific phosphorylation of FAK Y397. FAK Y397 phosphorylation could also be induced by antibody bound to the substrate. In contrast, clustering of alpha5beta1 on suspended cells with either antibody to beta1 or by clustering of soluble ligand bound to alpha5beta1 induced the phosphorylation of FAK Y861 but not Y397. There were no differences in signaling when activating antibodies were compared with blocking antibodies, presence or absence of ligand. Only tethering of alpha5beta1 to the substrate was required for induction of FAK Y397 phosphorylation.

GRP receptor-induced phosphorylation of FAK-Y397 decreases cell distensibility and blebbing

GRP receptor-induced phosphorylation of FAK-Y397 decreases cell distensibility and blebbing

Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling.

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.