Integrin Signaling Molecules Research Articles

Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the β2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Coating of Polyurethane Scaffold With Arabinogalactan Leads to Increase of Adhesion to Fibroblast Cells by Integrin Molecules Pathway

The purpose of this study was to determine the effect of polyurethane blood vein scaffold (PUBVS) and polyurethane blood vein scaffold impregnated with arabinogalactan (PUBVSIAG) on the cell attachment by integrin signaling molecules. After exposure of HFF cells to PUBVS and PUBVSIAG, both cell attachment and cell viability were determined. Also, the expression of integrin signaling genes was evaluated by both Real-time PCR and western blot. This study showed that both PUBVSIAG and arabinogalactan 5% had the same toxicity in all incubation times. Significant differences were shown between the viability of HFF cells when exposed to PUBVS and PUBVSIAG (P<0.001). At all incubation times, PUBVSIAG had higher attached cells compared with PUBVS (P<0.001). In case of PUBVSIAG, the expression of alpha integrin, beta integrin, fibronectin, and laminin was higher than PUBVS (P<0.001). Western blot analysis confirmed the high expression of integrin molecules. In vivo test showed the reconstruction of new micro-vessels.

DNA Damage Repair Pathway Alterations in Multiple Myeloma Predict Poor Prognosis, but Correlate with Sensitivity to IGF1R-PI3K-mTOR and HDAC Inhibitors

IntroductionAlthough several novel drugs have recently been approved or are in development for multiple myeloma (MM), there are few molecular indicators to guide treatment selection. In addition, the impact of recurrent myeloma alterations on drug response is often unclear. To address these limitations and elucidate genotype to phenotype relationships in myeloma, we comprehensively analyzed 100 MM samples and compared genomic, transcriptomic, and cytogenetic information to ex vivo drug response profiles and clinical outcome of individual MM patients. Our results reveal novel insights on i) drug response and resistance mechanisms, ii) biomarkers for drug response, and iii) potential treatment combinations to overcome drug resistance.MethodsBone marrow aspirates were collected from MM patients (n=100; newly diagnosed n=34; relapsed/refractory n=66) and healthy individuals (n=14). CD138+ plasma cells were enriched from the mononuclear cell fraction by immunomagnetic bead selection. Cells were screened against 142 oncology drugs tested in a 10,000-fold concentration range and 12 different drug combinations Somatic alterations were identified by exome sequencing of DNA from CD138+ cells and skin biopsies from each patient (n=85). RNA sequencing derived read counts from CD138+ cells of MM samples (n=67) were used for differential gene expression. Karyotype was determined by fluorescence in situ hybridization.ResultsFor most drugs tested, no significant difference in response was observed between samples from newly diagnosed and relapsed refractory patients except for signal transduction inhibitors targeting IGF1R-PI3K-mTOR, MAPK and HSP90. A positive correlation was observed between mutational burden and sensitivity to targeted therapies. The median number of somatic alterations was 118 in sensitive compared to 50 in resistant samples. 14% of the samples exhibited a multidrug resistant phenotype and were resistant to proteasome inhibitors, immunomodulatory drugs and glucocorticoids. 30% of the resistant samples were from del(17p) patients. In addition, gene expression analysis revealed elevated expression of cell adhesion and integrin signaling molecules including ITGB3, ITGA2B, VCL, TLN1, MMP8, MMP9, plus ABCC3, which encodes a transporter protein shown to be associated with multidrug resistance. A combination of the protein kinase C inhibitor bryostatin-1 and pan-BCL2 inhibitor navitoclax was highly effective against the resistant samples. 26% of the patient samples harbored mutations in genes involved in DNA damage repair signaling, namely TP53, TP73, ATM and BAX, in a mutually exclusive pattern. In addition, patients with these mutations had a high relapse rate and poor overall survival (HR=7.2,95%CI 3.2-16.08). Interestingly, CD138+ cells from these patients showed activation of IGF1R-PI3K-mTOR signaling and were highly susceptible to inhibitors targeting this signaling axis. These samples were also highly sensitive to HDAC inhibitors. While no strong correlation between RAS pathway mutations (NRAS, KRAS, NF1, BRAF) and MEK inhibitor sensitivity was observed, samples with clonal RAS mutations tended to be more sensitive to MEK inhibitors compared to samples with subclonal mutations.SummaryOur results suggest that drug resistance in myeloma may occur either via accumulation of somatic alterations or via cell adhesion mediated cytoprotection. Driver alterations in DNA damage signaling pathways were found to contribute to poor prognosis, but samples with these mutations showed enhanced sensitivity to IGF1R-PI3K-mTOR and HDAC inhibitors. Using genomic and transcriptomic data we identified molecular events that may shape the drug response landscape and found drug combinations that can overcome resistance mechanisms. Our results demonstrate that molecular information and ex vivo drug profiling may be useful to develop tailored treatment strategies and guide treatment decision, especially for relapsed/refractory myeloma patients. DisclosuresSilvennoinen:Sanofi: Honoraria, Other: Lecture fee; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fee; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Pfizer: Research Funding; Celgene: Research Funding.

Beta 1-integrin ligation and TLR ligation enhance GM-CSF-induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties.

Retinoic acid (RA)–producing CD103+ mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) play crucial roles in gut immunity. GM-CSF and RA contribute to the expression of the RA-producing enzyme ALDH1A2. However, additional signals appeared to be required for inducing ALDH1A2high mature DCs from immature DCs. We found here that TLR ligands (Ls) and immobilized E-cadherin could provide such signals in FLT3-L–generated bone marrow (BM)–derived DCs after treatment with GM-CSF and the RA receptor agonist Am80. The TLR-L-treated DCs produced proinflammatory cytokines unlike normal ALDH1A2high MLN-DCs, whereas the E-cadherin-treated DCs did not. Immobilized VCAM-1 and semaphorin 7 A exerted effects similar to those of E-cadherin. Soluble anti-integrin β1 antibodies or inhibitors of integrin signaling molecules suppressed the effects of these immobilized proteins, whereas immobilized anti-integrin β1 antibodies enhanced the GM-CSF/Am80-induced ALDH1A2 expression without inducing proinflammatory cytokines. Sequential stimulation of splenic pre-DCs with GM-CSF/Am80 and immobilized E-cadherin or anti-integrin β1 antibody also induced differentiation to mature DCs with high ALDH activity. The E-cadherin-treated BM-DCs induced gut-tropic Foxp3+ T cells and alleviated DSS–induced colitis, whereas the TLR-L-treated DCs aggravated DSS–induced colitis. The results suggest that integrin β1-mediated signals contribute to the differentiation and maturation of RA-producing anti-inflammatory DCs.

Procyanidin B2 3,3″-di-O-gallate inhibits endothelial cells growth and motility by targeting VEGFR2 and integrin signaling pathways.

Targeting angiogenesis, one of the hallmarks of carcinogenesis, using non-toxic phytochemicals has emerged as a translational opportunity for angioprevention and to control advanced stages of malignancy. Herein, we investigated the inhibitory effects and associated mechanism/s of action of Procyanidin B2-3,3″-di- O-gallate (B2G2), a major component of grape seed extract, on human umbilical vein endothelial cells (HUVECs) and human prostate microvascular endothelial cells (HPMECs). Our results showed that B2G2 (10-40 μM) inhibits growth and induces death in both HUVECs and HPMECs. Additional studies revealed that B2G2 causes a G1 arrest in cell cycle progression of HUVECs by down-regulating cyclins (D1 and A), CDKs (Cdk2 and Cdc2) and Cdc25c phosphatase and up-regulating CDK inhibitors (p21 and p27) expression. B2G2 also induced strong apoptotic death in HUVECs through increasing p53, Bax and Smac/Diablo expression while decreasing Bcl-2 and survivin levels. Additionally, B2G2 inhibited the growth factors-induced capillary tube formation in HUVECs and HPMECs. Interestingly, conditioned media (CCM) from prostate cancer (PCA) cells (LNCaP and PC3) grown under normoxic (~21% O2) and hypoxic (1% O2) conditions significantly enhanced the tube formation in HUVECs, which was compromised in presence of conditioned media from B2G2-treated PCA cells. B2G2 also inhibited the motility and invasiveness of both HUVECs and HPMECs. Mechanistic studies showed that B2G2 targets VEGFR2/PI3K/Akt and integrin signaling molecules which are important for endothelial cells survival, proliferation, tube formation and motility. Overall, we report that B2G2 inhibits several attributes of angiogenesis in cell culture; therefore, it warrants further investigation for efficacy for angioprevention and cancer control.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness

Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

Focal adhesion proteins as markers of malignant transformation and prognostic indicators in breast carcinoma

Focal adhesion kinase (FAK) is one of the central signaling molecules found at focal adhesion sites, which are specific areas on the cell membrane where cells attach to extracellular matrix proteins. Focal adhesion kinase interacts with multiple signaling and adaptor molecules and effects several signaling pathways. Overexpression of FAK and its substrate c-Src has been implicated in malignant transformation and acquisition of an invasive tumor phenotype of different tissues. Overexpression of the multidomain protein paxillin, which is also a FAK ligand and a c-Src substrate, has been associated with less malignant tumor behavior. The purpose of this study was to analyze the involvement of integrin signaling molecules FAK, c-Src, and paxillin in malignant transformation of the breast epithelium. Using phosphospecific antibodies FAK-pY(397) and Src-pY(416), we demonstrated that neither activation of FAK nor activation of c-Src correlates with development of invasive tumor properties. However, activation of both FAK and c-Src correlates with malignant transformation. We further demonstrated that overexpression of paxillin also correlates with malignant transformation and is a marker of a less invasive tumor phenotype. Using tissue microarray, we demonstrated that expression and activation of paxillin inversely correlated with lymph node metastases and lymphovascular invasion, respectively. No correlation between paxillin expression and activation and tumor grade, estrogen, progesterone, and Her2/Neu receptor expression was found. In summary, focal adhesion proteins FAK and c-Src can be used as markers of malignant transformation in epithelial cells but not invasive phenotype, whereas expression and activation of paxillin may represent a good prognosticator in breast carcinoma.

Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion.

Integrins can intercommunicate with cadherins. Here, we examined their possible relationship by use of small interfering RNA–mediated protein knockdown in HeLa cells. We found that a subset of integrin signaling molecules, namely Fak and paxillin, but not p130 Crk-associated substrate or proline-rich tyrosine kinase 2, participate in processes regulating N-cadherin–based cell–cell adhesion. Paxillin was found to be required primarily for the recruitment of Fak to robust focal adhesions. Our results suggest that at least some signals involving Fak are linked to a mechanism down-regulating Rac1 activity at the cell periphery, which appears to be important for the formation of N-cadherin–based adhesions in motile cells. Our analyses simultaneously exemplified the essential role of Fak in the maintenance of cell–cell adhesions in collective cell migration, a type of migration occurring in embryonic development and carcinoma invasion.

Modulation of integrins and integrin signaling molecules in the pressure-loaded murine ventricle.

Integrins are heterodimeric cell-surface receptors that link the extracellular matrix and the intracellular cytoskeleton and function as mechanotransducers. Signaling through integrins is important for cell growth, migration, and survival. Extracellular matrix is altered in the myocardium during hypertrophic induction and the transition to heart failure. The role of integrins in this process is poorly understood. Recently, integrin subunits have been identified that are dominantly expressed in striated muscle. We tested the hypothesis that since integrins are mechanotransducers, their expression and signaling would be modulated with murine left ventricular hemodynamic loading. The acute and chronic effects of pressure overload on changes in the expression of integrins, as well as related integrin-mediated signaling events were studied. Acute pressure loading increased phosphorylation of focal adhesion kinase, p42 and p44 extracellular signal-regulated kinase. Chronic loading: (1) increased expression of alpha1, alpha5, and beta1 integrin transcripts and (2) increased protein expression of integrin subunits which are dominantly expressed in striated muscle (alpha7 and beta1D) both by western blotting and immunofluorescent microscopy. These results show that adaptive responses of the myocardium to pressure overload include acute modulation of integrin-related signaling molecules and more chronic changes effect expression of integrin subunits, including ones dominantly expressed in muscle.