Focal Adhesion Kinase Y397 Phosphorylation Research Articles

Role of the Ste20-like kinase SLK in podocyte adhesion.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

Knee Extensors Muscle Plasticity Over a 5-Years Rehabilitation Process After Open Knee Surgery.

We investigated molecular and cellular parameters which set metabolic and mechanical functioning of knee extensor muscles in the operated and contralateral control leg of 9 patients with a chronically insufficient anterior cruciate ligament (ACL; 26.6 ± 8.3 years, 8 males, 1 female) after open reconstructive surgery (week 0), after ambulant physiotherapy under cast immobilization (week 9), succeeding rehabilitation training (up to week 26), and subsequent voluntary physical activity (week 260). Clinical indices of knee function in the operated leg were improved at 52 weeks and remained at a comparable level at week 260. CSA of the quadriceps (-18%), MCSA of muscle fibers (-24%), and capillary-to-fiber ratio (-24%) in m. vastus lateralis from the ACL insufficient leg were lower at week 0 than reference values in the contralateral leg at week 260. Slow type fiber percentage (-35%) and mitochondrial volume density (-39%) were reduced in m. vastus lateralis from the operated leg at weeks 9 and 26. Composition alterations in the operated leg exceeded those in the contralateral leg and, with the exception of the volume density of subsarcolemmal mitochondria, returned to the reference levels at week 260. Leg-specific deterioration of metabolic characteristics in the vasti from the operated leg was reflected by the down-regulation of mitochondrial respiration complex I-III markers (-41–57%) at week 9. After rehabilitation training at week 26, the specific Y397 phosphorylation of focal adhesion kinase (FAK), which is a proxy for mechano-regulation, was elevated by 71% in the operated leg but not in the contralateral leg, which had performed strengthening type exercise during ambulant physiotherapy. Total FAK protein and Y397 phosphorylation levels were lowered in both legs at week 26 resulting in positive correlations with mitochondrial volume densities and mitochondrial protein levels. The findings emphasize that a loss of mechanical and metabolic characteristics in knee extensor muscle remains detectable years after untreated ACL rupture, which may be aggravated in the post-operative phase by the deterioration of slow-oxidative characteristics after reconstruction due to insufficient load-bearing muscle activity. The reestablishment of muscle composition subsequent to years of voluntary physical activity reinforces that slow-to-fast fiber transformation is reversible in humans.

Plectin protects podocytes from adriamycin-induced apoptosis and F-actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway.

Podocyte injury is an early pathological change characteristic of various glomerular diseases, and apoptosis and F‐actin cytoskeletal disruption are typical features of podocyte injury. In this study, we found that adriamycin (ADR) treatment resulted in typical podocyte injury and repressed plectin expression. Restoring plectin expression protected against ADR‐induced podocyte injury whereas siRNA‐mediated plectin silencing produced similar effects as ADR‐induced podocyte injury, suggesting that plectin plays a key role in preventing podocyte injury. Further analysis showed that plectin repression induced significant integrin α6β4, focal adhesion kinase (FAK) and p38 MAPK phosphorylation. Mutating Y1494, a key tyrosine residue in the integrin β4 subunit, blocked FAK and p38 phosphorylation, thereby alleviating podocyte injury. Inhibitor studies demonstrated that FAK Y397 phosphorylation promoted p38 activation, resulting in podocyte apoptosis and F‐actin cytoskeletal disruption. In vivo studies showed that administration of ADR to rats resulted in significantly increased 24‐hour urine protein levels along with decreased plectin expression and activated integrin α6β4, FAK, and p38. Taken together, these findings indicated that plectin protects podocytes from ADR‐induced apoptosis and F‐actin cytoskeletal disruption by inhibiting integrin α6β4/FAK/p38 pathway activation and that plectin may be a therapeutic target for podocyte injury‐related glomerular diseases.

A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles.

Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca2+ -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca2+ sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation. Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of Nω -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation.

The Role of FAK in the Secretion of MMP9 after CD147 Stimulation in Macrophages.

To investigate whether focal adhesion kinase (FAK) can participate in the secretion of matrix metalloproteinase 9 (MMP9) after CD147 stimulation in THP-1 induced macrophages; thus, to explore the potential treatment perspectives for acute coronary syndrome (ACS).Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages. To confirm the peak mRNA and protein expression of FAK and MMP9 after the stimulation of CD147, the macrophages were divided into 5 groups (0, 3, 6, 9, and 12 hours), with 0 hours group as control group. To investigate the role of FAK in the secretion of MMP9, with stimulation of CD147 for 9 hours, FAK inhibitor 14 was used to inhibit FAK Y397 phosphorylation. The mRNA and protein expressions were quantified by qRT-PCR and western blotting, respectively. (1) Relative mRNA expression of FAK and MMP9 were both significantly up-regulated (all P < 0.05) after stimulation of CD147, FAK peaked at 9 hours (3.908 ± 0.106 versus 1, P < 0.05), whereas MMP9 peaked at 6 hours (2.522 ± 0.062 versus 1, P < 0.05). (2) Relative protein expression of FAK, pFAK, and MMP9 were all significantly increased after CD147 stimulation (all P < 0.05), FAK (1.930 ± 0.024 versus 1, P < 0.05) and pFAK (1.737 ± 0.021 versus 1, P < 0.05) peaked at 9 hours, whereas MMP9 peaked at 6 hours (1.527 ± 0.033 versus 1, P < 0.05). (3) CD147 up-regulates FAK, pFAK, and MMP9 mRNA and protein expressions in a dose-dependent manner. (4) FAK inhibitor 14 significantly reduced the relative protein expression level of pFAK (0.077 ± 0.012 versus 1, P < 0.05) and MMP9 (0.133 ± 0.012) at 9 hours after CD147 stimulation.The results demonstrated that FAK Y397 phosphorylation was involved in the secretion of MMP9 after CD147 stimulation in macrophages and may play a role in the regulation of ACS.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets

: Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called 'shear-induced platelet aggregation'. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of αIIbβ3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbβ3-antagonist Abciximab. Abnormally high shear rates (5000 s) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin αIIbβ3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s on immobilized fibrinogen, but in platelets exposed to 5000 s over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

Abstract TMEM-027: TARGETING RGNEF (P190RHOGEF/ARHGEF28) IMPAIRS OVARIAN TUMOR INITIATION AND PROGRESSION

Abstract Rho GTPase regulatory and integrin signaling pathways are associated with poor high-grade serous ovarian carcinoma (HGSOC) patient survival1. Focal adhesion kinase (FAK) transmits signals from integrins and promotes HGSOC tumor progression through mechanisms involving tumor-stromal cell interactions2. We previously found that the Rho guanine nucleotide exchange factor Rgnef (also named p190RhoGEF or Arhgef28) activates FAK as well as RhoA GTPase activity in mouse fibroblasts3. Here we find that Rgnef protein expression is elevated in Stage 3-4 HGSOC and that Rgnef knockout prevents spontaneous ovarian tumor formation in the MISIIR-T-antigen (TAg) induced C57BL/6 mouse model. At 17 weeks, Rgnef-/-;TAg ovarian tumors were significantly smaller (p&amp;lt;0.001) than Rgnef+/+;TAg controls. Primary Rgnef-/-;TAg and Rgnef+/+;TAg tumor cells were isolated and expanded ex vivo. Although loss of Rgnef did not alter growth as 3D spheroids, Rgnef-/-;TAg orthotopic (ovarian bursa) and intraperitoneal tumors were significantly smaller (p&amp;lt;0.01) than Rgnef+/+;TAg tumors in MISIIR-TAg-Low C57BL/6 syngeneic mice. Notably, FAK activation (as measured by FAK Y397 phosphorylation) was decreased in Rgnef-/-;TAg ascites-associated tumor cells. Together, these studies show that Rgnef loss impedes tumor growth independently of stromal Rgnef status and supports the notion that an Rgnef-FAK signaling linkage facilitates ovarian tumor initiation and progression. 1.Zhang, H., et al. Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer. Cell 166 (2016). 2.Sulzmaier, F.J., Jean, C. &amp; Schlaepfer, D.D. FAK in cancer: mechanistic findings and clinical applications. Nat. Rev. Cancer 14, 598-610 (2014). 3.Miller, N.L., et al. A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation. J. Cell Sci. 126, 5074-5085 (2013). Citation Format: Elizabeth G. Kleinschmidt, Nichol L.G. Miller, Isabelle Tancioni, Denise C. Connolly, David D. Schlaepfer. TARGETING RGNEF (P190RHOGEF/ARHGEF28) IMPAIRS OVARIAN TUMOR INITIATION AND PROGRESSION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-027.

Focal Adhesion Kinase Regulatory Interactions Quantified by TIRF-FRET

Focal adhesion kinase related non-kinase (FRNK) is an inhibitor of focal adhesion kinase (FAK)-dependent cell growth, survival, and migration pathways. In vascular smooth muscle cells, both FAK and FRNK are upregulated after vascular injury, suggesting balanced stimulation and inhibition of growth during recovery. Here we propose a novel mechanism by which FRNK can inhibit FAK: direct binding of FRNK to FAK forming an inhibitory complex within focal adhesions. This hypothesis is supported by co-immunoprecipitation of FAK and FRNK. In addition, total internal reflection fluorescence (TIRF)-mode fluorescence resonance energy transfer (FRET) measurements suggest that FRNK binds directly to FAK within focal adhesions of live vascular smooth muscle cells. We hypothesized that this interaction might be regulated by serine phosphorylation of FRNK S217, a site we found to be basally highly phosphorylated at S217 by extracellular signal-regulated kinase 1/2 (ERK). To determine the significance of S217 phosphorylation, we generated a non-phosphorylatable mutant FRNK by mutating S217 to alanine. S217A-FRNK exhibited significantly higher amounts of FRET compared to wild type FRNK, suggesting increased interaction with FAK. Progressive acceptor photobleaching analysis revealed that the FRNK-FAK complex consisted of FRNK and multiple FAK proteins in a high order hetero-oligomer. To determine the functional role of the proposed regulatory site we performed an apoptosis assay using flow cytometry. S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type FRNK. Additionally, we determined that S217A FRNK was a more potent inhibitor of FAK signaling by examining FAK Y397 phosphorylation. We conclude that dephosphorylation of FRNK at S217 during response to injury results in increased FRNK-FAK complex formation, thereby inhibiting FAK signaling.

Tyrosine 397 phosphorylation is critical for FAK-promoted Rac1 activation and invasive properties in oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is a common cancer worldwide. Despite advances in diagnosis and therapy, treatment options for patients with metastatic OSCC are few, due in part to the limited understanding of the molecular events involved in the invasion and metastasis of OSCC. In this study, we investigated the expression of focal adhesion kinase (FAK) and its tyrosine 397 phosphorylation (pY397) in the tissue specimens of OSCC. The roles of pY397 in regulating the activities of Rac1 and cortactin and the invasive properties of OSCC cells were further determined. Results from immunohistochemical analyses in 9 benign, 19 premalignant, and 19 malignant oral tissues showed that the immunoreactivity of FAK was observed in 5 benign (56%), 19 premalignant (100%), and 18 malignant tissues (95%), whereas the immunoreactivity of pY397 was only found in 1 of 9 (11%) benign lesions but was observed in 9 premalignant (47%) and 12 malignant (63%) lesions. Compared with the low-invading SCC4 cells, the high-invading OECM-1 cells exhibited higher levels of FAK expression and pY397, correlating with higher levels of GTP-bound Rac1 and cortactin phosphorylation. Manipulation of FAK expression or Y397 phosphorylation in SCC4, FaDu, OECM-1, or HSC-3 cells regulated their Rac1 activities and invasive properties. Furthermore, treatment of NSC23766, a Rac1-specific inhibitor, in OECM-1 and HSC-3 cells led to reduced invasive properties. Nevertheless, knockdown of FAK expression or suppression of pY397 had no effect on the cortactin activity in OECM-1 cells. The data collectively suggest that pY397 plays critical roles in the FAK-promoted Rac1 activation and invasive properties in OSCC cells. Thus, the inhibition of FAK phosphorylation at Y397 or Rac1 activity can serve as a therapeutic strategy for treating patients with metastatic OSCC.

Novel Mechanisms of FRNK Inhibition of FAK in Vascular Smooth Muscle Cells

The nonreceptor protein tyrosine kinase focal adhesion kinase (FAK) is an important regulator of growth and migration in smooth muscle cells that acts as a kinase and a scaffolding protein. FAK-related nonkinase (FRNK) is an endogenously expressed truncated form of FAK that inhibits FAK signaling through an unknown mechanism. Traditionally, FRNK has been thought to inhibit FAK by displacing it from focal adhesion binding sites. We propose an additional parallel mechanism wherein FRNK binds directly to FAK, inhibiting downstream signaling. Initial co-immunoprecipitation experiments suggested FAK and FRNK interact. We quantified fluorescence resonance energy transfer (FRET) from FAK to FRNK using acceptor photobleaching and fluorescence lifetime measurements. Both approaches used total internal reflection fluorescence (TIRF) microscopy for selective illumination. Acceptor photo-bleaching experiments indicated 5.2±1.2% FRET between CER-FRNK and YFP-FAK. Fluorescence lifetime FRET experiments revealed 8.6±0.9% FRET between GFP-FAK and mCherry-FRNK. Furthermore, we show that FAK and FRNK localize to specific locations within focal adhesions. FRNK appears to be localized preferentially towards the proximal side of the focal adhesion. We hypothesize that this polarization is induced by strain on the focal adhesion from the attached actin stress fiber. Lastly, we have identified a phosphorylation site on FRNK, serine at position 217, that appears to regulate FRNK inhibition. A phosphomimetic mutant with serine 217 mutated to aspartic acid reduced FAK Y397 (activation site) phosphorylation by approximately 50% compared to wild type. The phosphomimetic mutant tagged with mCherry exhibited reduced FRET with GFP-FAK (5.4 ± 0.8) compared to wild type FRNK (8.6 ± 0.9%) suggesting decreased FAK binding or an altered conformation of the FAK/FRNK regulatory complex. We conclude that FRNK regulates FAK through a direct inhibitory interaction, and the inhibitory potency of FRNK is modulated by phosphorylation of serine 217.

Mechanisms of FRNK Inhibition of FAK in Vascular Smooth Muscle Cells

The nonreceptor protein tyrosine kinase focal adhesion kinase (FAK) is an important regulator of growth and migration in smooth muscle cells that acts as a kinase and a scaffolding protein. FAK-related nonkinase (FRNK) is an endogenously expressed truncated form of FAK that inhibits FAK signaling through an unknown mechanism. Traditionally, FRNK has been thought to inhibit FAK by displacing it from focal adhesion binding sites. We propose an additional parallel mechanism wherein FRNK binds directly to FAK, inhibiting downstream signaling. Initial co-immunoprecipitation experiments suggested FAK and FRNK interact. We quantified fluorescence resonance energy transfer (FRET) from FAK to FRNK using acceptor photobleaching and fluorescence lifetime measurements. Both approaches used total internal reflection fluorescence (TIRF) microscopy for selective illumination. Acceptor photo-bleaching experiments indicated 5.2±1.2% FRET between CER-FRNK and YFP-FAK. Fluorescence lifetime FRET experiments revealed 8.6±0.9% FRET between GFP-FAK and mCherry-FRNK. Furthermore, we show that FAK and FRNK localize to specific locations within focal adhesions. FRNK appears to be localized preferentially towards the proximal side of the focal adhesion. We hypothesize that this polarization is induced by strain on the focal adhesion from the attached actin stress fiber. Lastly, we have identified a phosphorylation site on FRNK, serine at position 217, that appears to regulate FRNK inhibition. A phosphomimetic mutant with serine 217 mutated to aspartic acid reduced FAK Y397 (activation site) phosphorylation by approximately 50% compared to wild type. The phosphomimetic mutant tagged with mCherry exhibited reduced FRET with GFP-FAK (5.4 ± 0.8) compared to wild type FRNK (8.6 ± 0.9%) suggesting decreased FAK binding or an altered conformation of the FAK/FRNK regulatory complex. We conclude that FRNK regulates FAK through a direct inhibitory interaction, and the inhibitory potency of FRNK is modulated by phosphorylation of serine 217.

A small molecule FAK kinase inhibitor, GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells

Focal adhesion kinase (FAK) hyperactivation is common in pancreatic ductal adenocarcinoma (PDAC). A small molecule, GSK2256098 (GlaxoSmithKline), has been developed to inhibit FAK activity through targeting the phosphorylation site of FAK, tyrosine (Y) 397. We sought to determine whether GSK2256098 inhibition of FAK Y397 phosphorylation attenuates PDAC-associated cell proliferation, motility and survival. Cultured PDAC cells were used as cellular models of GSK2256098-impaired abnormal growth. Western blot analysis, cell viability analysis, clonogenic survival, soft-agar and wound healing assays were performed. The responses of 6 PDAC cell lines in regards to FAK Y397 phosphorylation or activity to GSK2256098 treatments (0.1–10 μM) ranged from low (less than 20% inhibition) to high (more than 90% inhibition). The least and most sensitive cell lines (PANC-1 and L3.6P1) were selected for further analysis. GSK2256098 inhibition of FAK Y397 phosphorylation correlated with decreased levels of phosphorylated Akt and ERK in L3.6P1 cells. GSK2256098 decreased cell viability, anchorage-independent growth, and motility in a dose dependent manner. Current studies demonstrate that small molecule kinase inhibitors targeting FAK Y397 phosphorylation can inhibit PDAC cell growth. Assessments of FAK Y397 phosphorylation in biopsies may be used as a biomarker to select the subgroup of responsive patients and/or monitor the effects of GSK2256098 on FAK-modulated tumor growth during treatment.

β Integrins Mediate FAK Y397 Autophosphorylation of Resistance Arteries during Eutrophic Inward Remodeling in Hypertension

Human essential hypertension is characterized by eutrophic inward remodeling of the resistance arteries with little evidence of hypertrophy. Upregulation of αVβ3 integrin is crucial during this process. In order to investigate the role of focal adhesion kinase (FAK) activation in this process, the level of FAK Y397 autophosphorylation was studied in small blood vessels from young TGR(mRen2)27 animals as blood pressure rose and eutrophic inward remodeling took place. Between weeks 4 and 5, this process was completed and accompanied by a significant increase in FAK phosphorylation compared with normotensive control animals. Phosphorylated (p)FAK Y397 was coimmunoprecipitated with both β1- and β3-integrin-specific antibodies. In contrast, only a fraction (<10-fold) was coprecipitated with the β3 integrin subunit in control vessels. Inhibition of eutrophic remodeling by cRGDfV treatment of TGR(mRen2)27 rats resulted in the development of smooth-muscle-cell hypertrophy and a significant further enhancement of FAK Y397 phosphorylation, but this time with exclusive coassociation of pFAK Y397 with integrin β1. We established that phosphorylation of FAK Y397 with association with β1 and β3 integrins occurs with pressure-induced eutrophic remodeling. Inhibiting this process leads to an adaptive hypertrophic vascular response induced by a distinct β1-mediated FAK phosphorylation pattern.

Abstract 752: Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

Abstract Ovarian cancer spreads via cell shedding and growth within malignant ascites. Effective targeted therapies have not been developed for ovarian cancer. Ascites contains an abundance of matrix proteins, and spheroids maintain integrin receptor expression. Through databases analyses we find that elevated osteopontin (OPN), β5 integrin, and focal adhesion kinase (FAK) mRNA levels are associated with decreased overall survival of serous ovarian cancer patients treated with platinum and taxol. In ovarian tumor tissue arrays, increased FAK activation (FAK Y397 phosphorylation) correlated with elevated tumor grade in parallel with increased in β5 integin and OPN levels. FAK is a cytoplasmic tyrosine kinase that remains active in spheroids, and treatment of seven ovarian carcinoma cell lines with sub-micromolar levels of FAK inhibitor (PND-1186) identified sensitive (HEY and OVCAR8), intermediate (OVCAR3, ID8-IP, and IGROV1-IP), and resistant (SKOV3-IP and OVCAR10) cells to blockage of growth under anchorage-independent conditions. Genetic or pharmacological FAK inhibition within ID8-IP or HEY cells selectively prevents anchorage-independent growth in culture and tumor growth in mice with corresponding reductions in β5 integrin and OPN expression. β5 knockdown reduced HEY growth in soft agar, tumor growth in mice, FAK Y397 phosphorylation, and OPN expression in spheroids. Although FAK inhibitor resistant ovarian carcinoma cells (SKOV3-IP and OVCAR10) were associated with anchorage-independent Akt S473 phosphorylation, membrane-targeted and activated Akt expression in sensitive cells (HEY and OVCAR8) resulted in only a partial rescue of FAK inhibitor-associated growth block. These results support the hypothesis that OPN, αvβ5 integrins, and FAK may function as a signaling axis promoting ovarian tumor progression. Although Akt signaling pathway activation is a common event in serous ovarian cancer, our results suggest that this may not impart complete resistance to FAK inhibitor treatment. Supported by NIH CA102310 Citation Format: Isabelle Tancioni, Sean Uryu, Florian Sulzmaier, Nina Shah, Christine Lawson, Nichol L.G. Miller, Christine Jean, Xiao Lei Chen, Kristy K. Ward, David D. Schlaepfer. Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 752. doi:10.1158/1538-7445.AM2014-752

Abstract 3157: An Rgnef (p190RhoGEF/Arhgef28) signaling axis regulates ovarian cancer progression

Abstract Ovarian cancer is a leading cause of cancer death in women, is usually diagnosed in late stage, and currently has insufficient effective targeted therapies. The primary mode of metastasis is unique in that dissemination occurs via tumor cell shedding into the peritoneal cavity, survival and growth within ascites, and re-adhesion and proliferation of tumor cells at sites within the abdomen. Therefore, molecules and pathways involving cell adhesion and survival in non-adherent environments are of particular interest. Rgnef (p190RhoGEF/Arhgef28) is a Rho family guanine exchange factor that associates with focal adhesion kinase (FAK) and functions both as a downstream target of FAK tyrosine kinase activity and a regulator of FAK activity following integrin stimulation. FAK regulates cell adhesion, migration, and survival. Expression of FAK is associated with poor clinical outcome, its activity (as measured by FAK Y397 phosphorylation) is frequently increased in serous ovarian carcinomas, and it is currently under investigation as a therapeutic target in ongoing clinical trials. Immunohistochemical analysis of human tumor tissue arrays with antibodies specific for Rgnef and FAK pY397 reveal a positive correlation between Rgnef expression and FAK activation and increased stage/grade of serous-type ovarian cancer. Stable knockdown of Rgnef in human ovarian carcinoma cells grown as subcutaneous or intraperitoneal xenografts in mice suggest that Rgnef may play roles in both primary tumor growth and ascites-associated cell survival and spread. Here, we use a transgenic model of spontaneous ovarian cancer (MISIIR-T-Antigen) to test the role of Rgnef in ovarian cancer progression. Female Rgnef+/+;TAg+ and Rgnef-/-;TAg+ mice were monitored by ultrasound imaging from age 12 to 17 weeks before euthanasia. Rgnef-/-;TAg+ tumors were significantly smaller (p=0.0006) and contained fewer Ki67 positive cells than Rgnef+/+;TAg+ controls at 17 weeks. Tumors and ovarian carcinoma cells isolated from Rgnef-/-;TAg+ mouse ascites have reduced FAK pY397 and increased E-cadherin expression compared to controls, suggesting that loss of Rgnef results in a less aggressive tumor phenotype. These findings identify Rgnef as a component in signaling pathways promoting FAK activation and regulating ovarian tumor progression. Citation Format: Nichol L. G. Miller, Isabelle Tancioni, Sean Uryu, Elizabeth G. Kleinschmidt, Denise C. Connolly, David D. Schlaepfer. An Rgnef (p190RhoGEF/Arhgef28) signaling axis regulates ovarian cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2014-3157

Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells

The prostaglandin E₂ (PGE₂) EP1 receptor has been implicated in hepatocellular carcinoma (HCC) cell invasion. However, little is known about the mechanisms of EP1 receptor-mediated cell adhesion and migration. We previously showed that PGE₂ promotes cell adhesion and migration by activating focal adhesion kinase (FAK). The present study was designed to elucidate the association between the EP1 receptor and FAK activation in HCC cells and to investigate the related signaling pathways. The effects of PGE₂, EP1 agonist 17-phenyl trinor-PGE₂ (17-PT-PGE₂), PKC and EGFR inhibitors on FAK activation were investigated by treatment of Huh-7 cells. Phosphorylation of FAK Y397 and c-Src Y416 was investigated by western blotting. Cell adhesion and migration were analyzed by WST and transwell assays, respectively. Protein kinase C (PKC) activity was measured with a PKC assay kit. The results showed that 17-PT-PGE₂ (3 µM) increased FAK Y397 phosphorylation by more than 2-fold and promoted cell adhesion and migration in Huh-7 cells. In transfected 293 cells, expression of the EP1 receptor was confirmed to upregulate FAK phosphorylation, while the EP1 receptor antagonist sc-19220 decreased PGE₂-mediated FAK activation. PKC activity and c-Src Y416 phosphorylation were enhanced after 17-PT-PGE₂ treatment. Both PKC and c-Src inhibitor suppressed the 17-PT-PGE₂-upregulated FAK phosphorylation, as well as 17-PT-PGE₂-induced cell adhesion and migration. In addition, exogenous epidermal growth factor (EGF) treatment increased FAK phosphorylation. The EGF receptor (EGFR) inhibitor also suppressed 17-PT-PGE₂-upregulated FAK phosphorylation. Our study suggests that the PGE₂ EP1 receptor regulates FAK phosphorylation by activating the PKC/c-Src and EGFR signal pathways, which may coordinately regulate adhesion and migration in HCC.

Heparin plays a key regulatory role via a p53/FAK‐dependent signaling in melanoma cell adhesion and migration

Heparin and its various derivatives affect cancer progression in humans. In this study, we show that heparin uptaken intracellularly by melanoma cells activated a signaling cascade, which in turn inhibited melanoma cell adhesion and migration. The reduced ability of M5 cells to adhere onto the fibronectin (FN) substrate was directly correlated to a decrease in the expression of focal adhesion kinase (FAK), which is a key regulator of melanoma motility. Cell treatment with heparin caused a marked downregulation in FAK expression (P ≤ 0.01). This is followed by an analogous inhibition of both constitutive and FN-induced FAK Y397-phosphorylation (P ≤ 0.01). Moreover, heparin stimulated the p53 expression (P ≤ 0.001) of M5 cells and its increased accumulation in the nucleus. This favors a decrease in FAK promoter activation and explains the reduced FAK transcript and protein levels. In conclusion, the results of this study clearly demonstrate that the action of heparin in the regulation of melanoma cell adhesion and migration involves a p53/FAK/signaling pathway, which may be of importance in molecular targeted therapy of the disease.

Inhibition of focal adhesion kinase decreases tumor growth in human neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously, we have shown that the MYCN oncogene, the primary adverse prognostic indicator in neuroblastoma, regulates the expression of FAK in neuroblastoma. In this study, we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN+/MYCN- neuroblastoma cell lines, we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment, decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 treatment than their MYCN negative counterparts. In addition, we have shown that treatment with Y15 in vivo leads to less tumor growth in nude mouse xenograft models, again with the greatest effects seen in MYCN+ tumor xenografts. The results of the current study suggest that FAK and phosphorylation at the Y397 site plays a role in neuroblastoma cell survival, and that the FAK Y397 phosphorylation site is a potential therapeutic target for this childhood tumor.

Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism

Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells’ ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration. These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.

Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: A combined computational and experimental approach

The adhesion ligand arginine–glycine–aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).