Deletion Of Focal Adhesion Kinase Research Articles

Remodeled Extracellular Matrix Guides Endothelial Cell Phenotype and Transcriptome to Fibrotic Niche

Impairment of endothelial barrier integrity causes vascular injury leading to acute lung injury (ALI), which, if not resolved, can compromise a patient’s lung functions and mortality. While it is known that both endothelial cells (EC) and their underlying extracellular matrix (ECM) are required for preserving lung homeostasis and functions, much of the efforts to understand mechanisms regulating these functions have focused mainly on EC. A fundamental unresolved question is whether lung-remodeled ECM formed due to leaky endothelium impacts EC functions, and if so, how? Herein, using lung ECM derived from a mouse lacking focal adhesion kinase (FAK), specifically in the endothelium (remodeled ECM), we assessed ECM components and their role in regulating EC functions and their relevance in vivo. Previously, we have shown that conditional deletion of FAK in endothelium increases EC permeability and induces vascular injury at homeostasis. Mass spectrometric analysis showed 805 proteins in control ECM (FAKfl/fl mice lung derived ECM) consisting of glycoproteins, matrisome-associated proteins, and secreted factors. However, the remodeled ECM had 722 proteins and was highly enriched with fibrillin 1, fibrinogen, and collagen I. Moreover, the remodeled ECM was two-fold stiffer than the control ECM. Remodeled ECM with its inherent composition recapitulates the 3D microenvironment, encouraging EC morphological alteration, increased cell stiffness, and enhanced focal adhesion (FA) formation compared to control ECM. In addition, RNA sequencing identified increased expression of genes linked with fibrosis, including integrin b8, TGFb2, GDF15, SMAD7, COL1A1, IL33, ID3, BMPR1A, and ADAMTS4 in EC seeded on remodeled ECM. Interestingly, fibrotic signaling observed in EC plated on remodeled ECM mimicked in vivo in EC-FAK−/− mice as they showed increased hydroxyproline content and collagen deposition in the lungs, indicating loss of EC-FAK as the crucial initiator of fibrotic signaling. In summary, studies for the first time show disrupted EC signaling as the instructor of fibrotic niche in the lungs for targeting pulmonary fibrosis. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell contractility and Focal Adhesion Kinase control circumferential arterial stiffness.

Arterial stiffening is a hallmark of aging and cardiovascular disease. While it is well established that vascular smooth muscle cells (SMCs) contribute to arterial stiffness by synthesizing and remodeling the arterial extracellular matrix, the direct contributions of SMC contractility and mechanosensors to arterial stiffness, and particularly the arterial response to pressure, remain less well understood despite being a long-standing question of biomedical importance. Here, we have examined this issue by combining use of pressure myography of intact carotid arteries, pharmacologic inhibition of contractility, and genetic deletion of SMC focal adhesion kinase (FAK). Biaxial inflation-extension tests performed at physiological pressures showed that acute inhibition of cell contractility with blebbistatin or EGTA altered vessel geometry and preferentially reduced circumferential, as opposed to axial, arterial stiffness in wild-type mice. Similarly, genetic deletion of SMC FAK, which attenuated arterial contraction to KCl, reduced vessel wall thickness and circumferential arterial stiffness in response to pressure while having minimal effect on axial mechanics. Moreover, these effects of FAK deletion were lost by treating arteries with blebbistatin or by inhibiting myosln light chain kinase. The expression of arterial fibrillar collagens, the integrity of arterial elastin, or markers of SMC differentiation were not affected by deletion of SMC FAK. Our results connect cell contractility and SMC FAK to the regulation of arterial wall thickness and directionally-specific arterial stiffening.

Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition

We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40–45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN−/− females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis.

Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear to be important. Given the dynamic environment of the lung, we examined the significance of mechanotransduction on both inflammatory and fibrotic signaling during IPF. Innovation: Mechanotransduction pathways have not been thoroughly examined in the context of lung disease, and pharmacologic approaches for IPF do not currently target these pathways. The interplay between mechanical strain and inflammation in pulmonary fibrosis remains incompletely understood. Approach: In this study, we used conditional KO mice to block mechanotransduction by knocking out Focal Adhesion Kinase (FAK) expression in fibroblasts, followed by induction of pulmonary fibrosis using bleomycin. We examined both normal human and human IPF fibroblasts and used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western Blot to evaluate the effects of FAK inhibitor (FAK-I) on modulating fibrotic and inflammatory genes. Results: Our data indicate that the deletion of FAK in mice reduces expression of fibrotic and inflammatory genes in lungs. Similarly, mechanical straining in normal human lung fibroblasts activates inflammatory and fibrotic pathways. The FAK inhibition decreases these signals but has a less effect on IPF fibroblasts as compared with normal human fibroblasts. Conclusion: Administering FAK-I at early stages of fibrosis may attenuate the FAK-mediated fibrotic response pathway in IPF, potentially mediating disease progression.

Focal adhesion kinase (FAK) promotes cholangiocarcinoma development and progression via YAP activation

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. Invitro cell line studies and invivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in invitro and invivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.

FAK signaling in cancer-associated fibroblasts promotes breast cancer cell migration and metastasis by exosomal miRNAs-mediated intercellular communication.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the major components of tumor microenvironment (TME) and play crucial roles in tumor development and metastasis. Here, we generated fibroblast-specific inducible focal adhesion kinase (FAK) knockout (cKO) mice in a breast cancer model to study potential role and mechanisms of FAK signaling in CAF to promote breast cancer metastasis in vivo. While not affecting primary tumor development and growth, FAK deletion significantly suppressed breast cancer metastasis in vivo. Analyses of CAFs derived from cKO mice as well as human CAFs showed that FAK is required for their activity to promote mammary tumor cell migration. We further showed that FAK ablation in CAFs decreased their exosome amount and functions to promote tumor cell migration and other activities, which could contribute to the reduced metastasis observed in cKO mice. Lastly, profiling of miRs from CAF exosomes showed alterations of several exosomal miRs in FAK-null CAFs, and further analysis suggested that miR-16 and miR-148a enriched in exosomes from FAK-null CAFs contribute to the reduced tumor cell activities and metastasis. Together, these results identify a new role for FAK signaling in CAFs that regulate their intercellular communication with tumor cells to promote breast cancer metastasis.

Vitronectin mitigates stroke-increased neurogenesis only in female mice and through FAK-regulated IL-6

Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN−/− mice at 14 d. In female VTN−/− mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN−/− mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

Inhibition of astrocyte FAK-JNK signaling promotes subventricular zone neurogenesis through CNTF.

Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK-JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF-/- mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF-/- littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK-JNK-CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.

FAK maintenance of endothelial mechanotransduction controls epigenetic repression of KLF2 and S1PR1 transcription

Sphingosine‐1‐phosphate (S1P) activation of sphingosine‐1‐phosphate receptor 1 (S1PR1) in endothelial cells (ECs) strengthens the endothelial barrier and prevents lung injury. However mechanisms regulating EC‐S1PR1 expression remains unclear. Recently, we showed that endothelial‐specific deletion of focal adhesion kinase (FAK) in mice severely disrupted lung vascular barrier function. Microarray analysis of FAK depleted cells showed a marked reduction in S1PR1 expression. Thus, we tested the hypothesis that impaired S1PR1 synthesis is responsible for defective endothelial barrier repair in EC‐FAK−/− mice. We confirmed that FAK deletion markedly reduced S1PR1 expression both at the mRNA and protein level in lungs and ECs whereas the expression of S1PR2 and S1PR3 and S1P generating enzymes, sphingosine kinases (SPHK1 and SPHK2) was not reduced. Consistently, FAK depleted ECs and mice conditionally lacking FAK in pulmonary ECs failed to anneal adherens junctions and showed impairment of endothelial barrier function under basal condition and even after S1P application. Rescuing S1PR1 expression in FAK null ECs restored barrier function in the pulmonary vessels of EC‐FAK null mice. Next, we screened for potential transcription factors binding to the S1PR1 promoter which included KLF2, Sox2, FoxP1, GATA1, GATA4 and ERG1. Intriguingly, we observed that rescuing KLF2 expression in FAK depleted ECs restored S1PR1 expression and endothelial barrier function to the levels observed in control cells. In other studies, we observed that KLF2 increased S1PR1 promoter activity while the S1PR1 cDNA lacking KLF2 binding sites failed to increase S1PR1 promoter activity, demonstrating that KLF2 can promote endothelial barrier function in the absence of EC‐FAK by transducing S1PR1. Because FAK maintains intracellular tension by balancing the activities of RhoA and Rac1 GTPases and KLF2 mRNA expression is regulated by methylation of CpG islands, we next address the possible role of increased cellular tension and DNA methyltransferases (DNMT) in suppressing KLF2 gene transcription and thereby S1PR1 expression in EC‐FAK null mice. We found that DNMT activity was elevated by 3‐fold in FAK depleted ECs in a RhoA‐dependent manner. Intriguingly, we found that depletion of FAK increased EC‐stiffness, which was associated with hypermethylation of KLF2 by DNMT3a. Interestingly, inhibition of RhoA in FAK depleted cells restored KLF2 and S1PR1 expression. Thus, our data describes the novel role of FAK in suppressing cellular tension driven‐epigenetic modification of KLF2 and S1PR1 and thereby vascular integrity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression.

Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.

Abstract 5882: FAK is required for the survival of tumor cells in MMTV-Wnt1 driven basal-like mammary tumors

Abstract Breast cancer is a heterogeneous disease. Stratification of patients based on the subtype of breast cancer is a key to successful treatment of breast cancer. Focal Adhesion Kinase (FAK), a cytoplasmic tyrosine kinase is over expressed and activated in several cancers including breast cancer. Our earlier studies have shown inhibition of FAK in MMTV-PyMT mouse mammary tumors that are classified as Luminal B subtype, delays tumor onset and reduces tumor growth. To address whether inhibition of FAK would be beneficial in basal-like mammary tumors, we generated a conditional deletion of FAK and a knock in mutation of FAK lacking its kinase activity, in MMTV-WNT1 mouse model, which classifies as basal-like. Similar to PyMT mammary tumors we found that loss of FAK or its kinase function delays tumor onset and tumor growth of basal like WNT1 mammary tumors. However unlike the PyMT tumors, the reduced tumor growth in WNT1 model is not due to decreased proliferation. Interestingly loss of FAK activity in WNT1 tumors results in accumulation of cleaved caspase 3, suggesting that loss of FAK activity results in compromised tumor cell survival. When we investigated the pathways through which FAK could affect survival, we found that loss of FAK activity reduces activation of AKT. Reduced AKT activation induces the expression of pro-apoptotic genes. In summary our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells. Hence inhibition of FAK could be beneficial for elimination of basal-like tumor cells. Citation Format: Ritama Paul, Syn K. Yeo, Ming Luo, Susan Waltz, Jun-Lin Guan. FAK is required for the survival of tumor cells in MMTV-Wnt1 driven basal-like mammary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5882. doi:10.1158/1538-7445.AM2017-5882

Lung epithelial cell focal adhesion kinase signaling inhibits lung injury and fibrosis.

Progressive pulmonary fibrosis is a devastating consequence of many acute and chronic insults to the lung. Lung injury leads to alveolar epithelial cell (AEC) death, destruction of the basement membrane, and activation of transforming growth factor-β (TGF-β). There is subsequent resolution of the injury and a coordinated and concurrent initiation of fibrosis. Both of these processes may involve activation of similar intracellular signaling pathways regulated in part by dynamic changes to the extracellular matrix. Matrix signaling can augment the profibrotic fibroblast response to TGF-β. However, similar matrix/integrin signaling pathways may also be involved in the inhibition of ongoing TGF-β-induced AEC apoptosis. Focal adhesion kinase (FAK) is an integrin-associated signaling molecule expressed by many cell types. We used mice with AEC-specific FAK deletion to isolate the epithelial aspect of integrin signaling in the bleomycin model of lung injury and fibrosis. Mice with AEC-specific deletion of FAK did not exhibit spontaneous lung injury but did have significantly greater terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive cells (18.6 vs. 7.1) per ×200 field, greater bronchoalveolar lavage protein (3.2 vs. 1.8 mg/ml), and significantly greater death (77 vs. 19%) after bleomycin injury compared with littermate control mice. Within primary AECs, activated FAK directly associates with caspase-8 and inhibits activation of the caspase cascade resulting in less apoptosis in response to TGF-β. Our studies support a model in which dynamic changes to the extracellular matrix after injury promote fibroblast activation and inhibition of epithelial cell apoptosis in response to TGF-β through FAK activation potentially complicating attempts to nonspecifically target this pathway for antifibrotic therapy.

Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation.

Paxillin and Hic-5 are homologous focal adhesion adaptor proteins that coordinate cytoskeletal rearrangements in response to integrin signaling, but their role(s) in cortical development are unknown. Here, we find that Hic-5-deficient mice are postnatal viable with normal cortical layering. Mice with a neural-specific deletion of paxillin are also postnatal viable, but show evidence of a cortical neuron migration delay that is evident pre- and perinatally, but is not detected at postnatal day 35 (P35). This phenotype is not modified by Hic-5 deficiency (double knockout). Specific deletion of paxillin in postmitotic neurons using Nex-Cre-mediated recombination as well as in utero electroporation of a Cre-expression construct identified a cell-autonomous requirement for paxillin in migrating neurons. Paxillin-deficient neurons have shorter leading processes that exhibited multiple swellings in comparison with control. Multiphoton imaging revealed that paxillin-deficient neurons migrate ∼30% slower than control neurons. This phenotype is similar to that produced by deletion of focal adhesion kinase (FAK), a signaling partner of paxillin, and suggests that paxillin and FAK function cell-autonomously to control migrating neuron morphology and speed during cortical development.

FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy.

Understanding the molecular mechanisms of liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. Focal adhesion kinase (FAK) regulates different cellular functions, including cell survival, proliferation and cell migration. The role of FAK in liver regeneration remains unknown. In this study, we found that Fak is activated and induced during liver regeneration after two-thirds partial hepatectomy (PHx). We used mice with liver-specific deletion of Fak and investigated the role of Fak in liver regeneration in 2/3 PHx model (removal of 2/3 of the liver). We found that specific deletion of Fak accelerates liver regeneration. Fak deletion enhances hepatocyte proliferation prior to day 3 post-PHx but attenuates hepatocyte proliferation 3 days after PHx. Moreover, we demonstrated that the deletion of Fak in liver transiently increases EGFR activation by regulating the TNFα/HB-EGF axis during liver regeneration. Furthermore, we found more apoptosis in Fak-deficient mouse livers compared to WT mouse livers after PHx. Conclusion: Our data suggest that Fak is involved in the process of liver regeneration, and inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients after liver transplantation.

Abstract 5048: Platelet FAK is a critical regulator of tumor growth after withdrawal of anti-angiogenic therapy

Abstract Objective: In most clinical trials, anti-angiogenic therapies have offered only modest improvements in progression-free survival without impacting overall survival. Following cessation of anti-angiogenic therapy, concerns have been raised about a possible rebound in tumor growth, but the underlying mechanisms are poorly understood. Therefore, in this study we aimed at comparing tumor growth and the effects on tumor microenvironment after therapy withdrawal compared to continuous treatment with anti-angiogenic agents. Methods: Mice were intraperitoneally injected with human or mouse ovarian cancer cells and were treated with the anti-angiogenic drugs pazopanib, bevacizumab or B20 for either a short-term with subsequent withdrawal or continuous therapy until necropsy. Immunohistochemical staining was used to evaluate platelet infiltration into the tumor microenvironment, tumor angiogenesis and vascular leakage. To assess the significance of focal adhesion kinase (FAK) in the process of platelet infiltration and tumor rebound after withdrawal of therapy, we either used the FAK inhibitor GSK2256098 or a mouse model with platelet-specific FAK deletion. Results: Cessation of therapy with pazopanib, bevacizumab and the cross-human and murine anti-VEGF antibody B20 was associated with up to 4-fold increased tumor growth in mouse models of ovarian cancer when compared to continuous treatment. Tumor outgrowth was associated with significant tumor hypoxia, increased tumor angiogenesis and vascular leakage. More importantly, we found 380% increased hypoxia-induced ADP production and 3-fold increased platelet infiltration into tumors where anti-angiogenic therapy was withdrawn. Lowering platelet levels significantly inhibited tumor rebound after withdrawal of anti-angiogenic therapy. Interestingly, FAK in platelets regulated their migration into tumor microenvironment and FAK knock-out specifically in platelets completely prevented the rebound tumor growth. Additionally, combined therapy with the FAK inhibitor GSK2256098 and the anti-angiogenic agents pazopanib and bevacizumab led to up to 5-fold reduced orthotopic tumors and likewise inhibited negative effects of withdrawal of anti-angiogenic therapy. Conclusions: Collectively, our results characterize a previously unknown role for platelets in the tumor microenvironment and provide a potential therapeutic benefit for FAK inhibitors in preventing rebound in tumor growth following discontinuation of anti-angiogenic agents. Additionally, dual targeting of FAK and VEGF could have important therapeutic implications for ovarian cancer management. Citation Format: Monika Haemmerle, Justin Bottsford-Miller, Sunila Pradeep, Morgan L. Taylor, Hyun-Jin Choi, Rebecca L. Stone, Min Soon Cho, Alpa M. Nick, Gabriel Lopez-Berestein, Vahid Afshar-Khargan, Anil K. Sood. Platelet FAK is a critical regulator of tumor growth after withdrawal of anti-angiogenic therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5048.

Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion.

Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

N-Cadherin Induction by ECM Stiffness and FAK Overrides the Spreading Requirement for Proliferation of Vascular Smooth Muscle Cells.

SUMMARYIn contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness-and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.