Form Of Focal Adhesion Kinase Research Articles

Src Inhibitors Pyrazolo[3,4-d]pyrimidines, Si306 and Pro-Si306, Inhibit Focal Adhesion Kinase and Suppress Human Glioblastoma Invasion In Vitro and In Vivo.

Glioblastoma (GBM), as the most aggressive brain tumor, displays a high expression of Src tyrosine kinase, which is involved in the survival, migration, and invasiveness of tumor cells. Thus, Src emerged as a potential target for GBM therapy. The effects of Src inhibitors pyrazolo[3,4-d]pyrimidines, Si306 and its prodrug pro-Si306 were investigated in human GBM cell lines (U87 and U87-TxR) and three primary GBM cell cultures. Primary GBM cells were more resistant to Si306 and pro-Si306 according to the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. However, the ability of all GBM cells to degrade the extracellular matrix was considerably compromised after Si306 and pro-Si306 applications. Besides reducing the phosphorylation of Src and its downstream signaling pathway components, both compounds decreased the phosphorylated form of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) expression, showing the potential to suppress the aggressiveness of GBM. In vivo, Si306 and pro-Si306 displayed an anti-invasive effect against U87 xenografts in the zebrafish embryo model. Considering that Si306 and pro-Si306 are able to cross the blood–brain barrier and suppress the spread of GBM cells, we anticipate their clinical testing in the near future. Moreover, the prodrug showed similar efficacy to the drug, implying the rationality of its use in clinical settings.

Characterization of stitch adhesions: Fibronectin-containing cell-cell contacts formed by fibroblasts

Fibronectin is a multifunctional, extracellular matrix glycoprotein that exists either as an insoluble multimeric fibrillar component of the extracellular matrix or as a soluble monomer. Cells attach to fibronectin through transmembrane integrin receptors and form a variety of cell-matrix contacts. Here we show that primary fibroblasts can use fibronectin to organize a specific cell-cell contact – “stitch adhesions.” This contact is formed by short parallel fibronectin fibrils connecting adjacent cells above the level of the focal adhesions that attach the cells to the substrate. Stitch adhesions contain integrin α5β1 but not αVβ3, align with actin filament bundles, and contain talin, tensin, α-actinin, vinculin, paxillin and a phosphorylated form of focal adhesion kinase. This combination of components differs from the described constituents of the known cell adhesions. Stitch adhesions are organized when protein synthesis and secretion are inhibited by cycloheximide and exogenous fibronectin is provided to the cells. The adhesion stitches described here provide an attractive model system for studying fibronectin fibrillogenesis and the mechanisms governing the formation of cellular adhesions.

Calcium Mobilization in Unfavorable-Prognosis Chronic Lymphocytic Leukemia Patients Mediates Focal Adhesion Kinase (FAK) Cleavage, Thereby Its Activation

INTRODUCTIONChronic Lymphocytic Leukemia (CLL) is the most common leukemia in the western world and is characterized by the accumulation of monoclonal B cells, due to both increased proliferation and apoptosis resistance. Although in the last years with the introduction of new kinase inhibitors blocking the pathways mediated by B-cell receptor (BCR) signaling we got an astonishing progress in the comprehension and treatment of this disease, CLL is still an incurable disease and many characteristics of its pathogenesis still remain unclear. Signaling events downstream the BCR engagement are central for the progression of CLL. Focal Adhesion Kinase (FAK), one of the primary enzyme involved in the engagement of integrins and assembly of focal adhesions, plays a major role in cellular adhesion and metastasis of various cancers, being regulated by Calcium (Ca2+) flux and by Src-kinases (e.g. Lyn) through a Calpain-dependent manner following BCR triggering. FAK has been demonstrated to be over-expressed in many human cancers but a down-modulation of its expression has also been reported. Studies concerning FAK expression in CLL are lacking in the literature. However, since an interaction of FAK with molecules implicated in BCR signal transduction, such as the Src-kinase Lyn, has been demonstrated we hypothesize that this kinase could have a key role in CLL pathogenesis.METHODSFAK expression was analyzed in B-lymphocytes from 107 CLL patients and 10 healthy subjects by Western blotting (WB) and the obtained expression data were correlated with the clinical features of the patients. In 25 out of 107 patients studied, surface IgM and IgD expression has been evaluated by flow cytometry (FC). For Ca2+ mobilization assessment, 1x107 cells were incubated with 4μM Fluo-4-AM at 37°C for 30min and then analyzed by FC; after 30s of baseline acquisition, α-IgM F(ab‘)2 and α-IgD F(ab‘)2 (10μg/ml) were added and fluorescence intensity was recorded for 5min. Ionomycin was added as positive control. Phosphorylation at Tyr397 was assessed with a specific antibody. Leukemic B cells from patients were treated in vitro with 5μM Defactinib (FAK inhibitor) and the apoptosis induction was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP by WB.RESULTSBy WB analyses we demonstrated a slightly significant difference in FAK expression between patients and controls (p<0.05), the protein being particularly down-regulated in unmutated IGHV and del17p/del11q/12+ CLL patients. We observed that FAK down-modulation was limited to its whole form detected in WB at 125kDa, while bands related to FAK cleavage (92/94 and 84kDa) were detected also in those patients lacking full length-FAK. Cleaved-FAK is due to Calpain protease activity, when stimulated by the bond with Ca2+ ions. We then compared FAK expression with the capability of the cell to mobilize Ca2+ from intracellular stores, observing that patients with this capability had less amount of full length-FAK, which translated into a higher presence of cleaved/activated form of FAK. The cleavage bands infact were found phosphorylated at activatory Tyr397. Of note, only IGHV-unmutated patients showed these features. Lastly, we studied the effect of Defactinib, a specific FAK inhibitor, in CLL cells; this molecule was able to induce apoptosis in leukemic cell in a caspase-dependent way, as assessed by the presence of the cleaved PARP.CONCLUSIONSWe herein propose that full length-FAK down-modulation could be considered as a new marker of unfavorable prognosis. In our model, poor prognosis CLL patients (particularly IGHV unmutated ones) presenting Ca2+ mobilization, are more prone to activate Calpain, which in turn activates FAK. Together with data from the literature, our results suggest that CLL cells missing the full length-FAK, not only are unaffected by the lack of it, but they rather present a cleaved/activated form of FAK that could favor cell migration and metastatic invasion. Moreover, since Defactinib can induce apoptosis in CLL cells, should these data be confirmed by in vivo studies, this FAK inhibitor could represent a new therapeutic approach for CLL. DisclosuresTrentin:Gilead: Research Funding; Abbvie: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding.

Abstract 5868: Role of STAT3 vs AKT in cellular survival

Abstract Cell to cell adhesion is mediated by the cadherin family of proteins. We previously demonstrated that cadherin engagement (which is favored under conditions of confluence) triggers a dramatic increase in Rac protein levels, followed by an increase in IL6 secretion and Stat3 (Signal Transducer and Activator of Transcription-3) activation. This is critical for survival of both normal and neoplastic cells. On the other hand, cell adhesion to the substratum, ie the extracellular matrix at the focal adhesion sites, is mediated by the integrin receptors. The focal adhesion kinase (FAK) binds β1-integrins and this forces FAK in an open configuration and autophosphorylation at tyr397 which constitutes a binding site for Src-SH2. This results in phosphorylation of FAK at tyr 576, 577, 861 and 925. The FAK/Src complex then activates downstream effectors such as Ras/Erk and phosphatidylinositol-3 kinase (PI3k)/Akt. Our results demonstrate that mouse fibroblasts or epithelial cells placed in suspension (ie in the absence of anchorage to a substratum or cell to cell adhesion) have very low levels of activated Akt-pser473, but levels are increased dramatically upon subsequent aggregation, ie cadherin engagement. Interestingly, cells growing attached in monolayers had high Akt-pser473 levels at low densities, which increased only slightly with confluence. This pointed to the possibility that a kinase may be phosphorylating Akt upon attachment to the substratum. The results showed that FAK knockout cells have very low Akt-pser473 at low cell densities, while reintroduction of wt-FAK reinstated the high Akt-pser473 levels. Furthermore, expression of a mutant form of FAK where the 5 sites that are phosphorylated by Src are converted to phenylalanine, did not result in an increase in Akt-pser473 levels, demonstrating a Src requirement for Akt, pser473 phosphorylation. Conclusions: Cell survival signals are a prerequisite for tissue function, as well as drug resistance. In cultured, non-neoplastic cells grown to low densities the integrin/FAK/cSrc signal activates PI3k/Akt and promotes survival, in the absence of cadherin engagement. Interestingly, the integrin/FAK signal does not activate Stat3, despite the fact that mutationally activated Src is a potent Stat3 activator. Additional mechanisms, perhaps involving Jak and/or activated receptors may also be required for Stat3 activation in cells grown attached but at low densities. On the other hand, upon cadherin engagement at high cell densities, activation of both the cadherin/Stat3 and integrin/FAK/Akt pathways can promote survival. Therefore, co-ordinate activation of these complementary pathways could greatly enhance survival and growth of disseminated tumor cells at distant metastastic sites, and might promote resistance to chemotherapeutic agents. In this scenario, pharmacological inhibition of both FAK/Src and Stat3 would enhance patient survival. Note: This abstract was not presented at the meeting. Citation Format: Maximilian Niit, Joshua Rosen, Rozanne Arulanandam, Victoria Hoskin, Bruce Elliott, Leda Raptis. Role of STAT3 vs AKT in cellular survival [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 5868. doi:10.1158/1538-7445.AM2017-5868

PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin β1, which is the most highly expressed integrin in our cohort of uveal melanomas.We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin β1 on collagen I. Knockdown experiments confirmed integrin β1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin β1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin β1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin β1 in FAs on collagen I but not on fibronectin.This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

Translocation of BBAP from the cytoplasm to the nucleus reduces the metastatic ability of vemurafenib-resistant SKMEL28 cells.

To the best of our knowledge, the present study is the first to demonstrate that treatment of vemurafenib-resistant SKMEL28 (SKMEL28-R) cells with paclitaxel leads to a shift in localization of the E3-ligase BBAP from the cytoplasm to the nucleus, consequently decreasing the metastatic ability of this cell line. The present study revealed that the movement of BBAP from the cytoplasm to nucleus initiated a change in cell morphology. In addition, the translocation of BBAP led to a decrease of metastatic characteristics in SKMEL28‑R cells, including migration and invasion via downregulation of the phosphorylated form of focal adhesion kinase and N‑cadherin, as well as an upregulation of p21 and E-cadherin. The results of the present study suggested that BBAP may not only be a novel biomarker for melanoma, but also a novel therapeutic target for treatment of metastatic melanoma.

P0103 Antitumour activity of Citrus bergamia juice and its flavonoid fraction

<h3>Background</h3> Increasing interest in the antitumour activity of several fruit juices and vegetable extracts led us to investigate the antitumour effects of bergamot juice in <i>in vitro</i> models. <i>Citrus bergamia</i> Risso et Poiteau (bergamot) is a small tree cultivated almost exclusively along the southern coast of the Calabria region of Italy. Bergamot fruit is used mostly for the extraction of its essential oil widely used in perfume industry, whereas bergamot juice is considered secondary product. <h3>Methods</h3> We assessed the ability of bergamot juice to reduce the growth rate of different cancer cell lines, observing the maximal antiproliferative effect in neuroblastoma cells. We also focused on the flavonoid fraction of bergamot juice (FFBJ). <h3>Findings</h3> The SH-SY5Y growth inhibition elicited by bergamot juice was not due to a cytotoxic action or apoptosis. Instead, bergamot juice stimulated the arrest of the cell cycle in the G1 phase and determined a modification in cellular morphology, causing a marked increase in detached cells. The inhibition of the adhesive capacity on different physiological substrates and on the endothelial cell monolayer were correlated with impairment of actin filaments and reduction in the expression of the active form of focal adhesion kinase (FAK) that in turn caused inhibition of cell migration. <h3>Interpretation</h3> Our results suggest that the FFBJ inhibits HT-29 human colorectal carcinoma cell growth and induces apoptosis by multiple mechanisms. Molecular assays revealed that higher concentration of FFBJ increases production of reactive oxygen species, which causes loss in mitochondrial membrane potential and oxidative DNA damage. Lower concentrations of FFBJ inhibit MAPK pathways and alter apoptosis-related proteins, and in turn induce cell cycle arrest and apoptosis. In conclusion, we have shown that bergamot juice exerts important anti-cancer effects <i>in vitro</i> by different mechanisms involved in the regulation of cell growth, adhesiveness, migration, and invasion, depending on its concentration and cell lines.

Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.

Pre-osteoblast adhesion and interaction with extracellular matrix (ECM) proteins through integrin receptors result in activation of signaling pathways regulating osteoblast differentiation. Connective tissue growth factor (CTGF/CCN2) is a matricellular protein secreted into the ECM. Prior studies in various cell types have shown that cell adhesion to CTGF via integrin receptors results in activation of specific signaling pathways that regulate cell functions, such as differentiation and cytoskeletal reorganization. To date, there are no studies that have examined whether CTGF can serve as an adhesive substrate for osteoblasts. In this study, we used the MC3T3-E1 cell line to demonstrate that CTGF serves as an adhesive matrix for osteoblasts. Anti-integrin blocking experiments and co-immunoprecipitation assays demonstrated that the integrin αvβ1 plays a key role in osteoblast adhesion to a CTGF matrix. Immunofluorescence staining of osteoblasts cultured on a CTGF matrix confirmed actin cytoskeletal reorganization, enhanced spreading, formation of focal adhesions, and activation of Rac1. Alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining demonstrated that osteoblast attachment to CTGF matrix enhanced maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves integrin-mediated activation of specific signaling pathways, we performed Western blot, chromatin immunoprecipitation (ChIP) and qPCR assays. Osteoblasts cultured on a CTGF matrix showed increased total and phosphorylated (activated) forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Inhibition of ERK blocked osteogenic differentiation in cells cultured on a CTGF matrix. There was an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter, and in the expression of osteogenic markers regulated by Runx2. Collectively, the results of this study are the first to demonstrate CTGF serves as a suitable matrix protein, enhancing osteoblast adhesion (via αvβ1 integrin) and promoting cell spreading via cytoskeletal reorganization and Rac1 activation. Furthermore, integrin-mediated activation of ERK signaling resulted in increased osteoblast differentiation accompanied by an increase in Runx2 binding to the osteocalcin promoter and in the expression of osteogenic markers.

The diverse roles and multiple forms of focal adhesion kinase in brain.

Although it was originally characterized as a constituent of focal adhesions in fibroblasts, focal adhesion kinase (FAK) is now considered to be not only a mediator of adhesion processes but also a crucial regulator of guidance and a modulator of gene expression. FAK is the main transducer of the integrin signaling required to stabilize the actin cytoskeleton. However, additional activities have been described over the years. In the brain, FAK deserves particular attention as it is found in various alternatively spliced forms - these distributed in multiple subcellular compartments or bound to multiple partners. Moreover, its signaling involves not only phosphorylation but also ubiquitination and proteolysis. Several experimental cell models demonstrate that FAK increases or decreases migration, participates in differentiation and contributes to plasticity events. In addition, this kinase is linked to cell survival in cancer and apoptosis. This review focuses on the diversity of events involving brain-located forms of FAK.

New insights into FAK function and regulation during spermatogenesis.

Germ cell transport across the seminiferous epithelium during the epithelial cycle is crucial to spermatogenesis, although molecular mechanism(s) that regulate these events remain unknown. Studies have shown that spatiotemporal expression of crucial regulatory proteins during the epithelial cycle represents an efficient and physiologically important mechanism to regulate spermatogenesis without involving de novo synthesis of proteins and/or expression of genes. Herein, we critically review the role of focal adhesion kinase (FAK) in coordinating the transport of spermatids and preleptotene spermatocytes across the epithelium and the BTB, respectively, along the apical ectoplasmic specialization (ES) - blood-testis barrier - basement membrane (BM) functional axis during spermatogenesis. In the testis, p-FAK-Tyr&#179;&#8312;&#8311; and p-FAK-Tyr&#8308;&#8304;&#8311; are spatiotemporally expressed during the epithelial cycle at the actin-rich anchoring junction known as ES, regulating cell adhesion at the Sertoli-spermatid (apical ES) and Sertoli cell-cell (basal ES) interface. Phosphorylated forms of FAK exert their effects by regulating the homeostasis of F-actin at the ES, mediated via their effects on actin polymerization so that microfilaments are efficiently re-organized, such as from their "bundled" to "de-bundled/branched" configuration and vice versa during the epithelial cycle to facilitate the transport of: (i) spermatids across the epithelium, and (ii) preleptotene spermatocytes across the BTB. In summary, p-FAK-Tyr&#8308;&#8304;&#8311; and p-FAK-Tyr&#179;&#8312;&#8311; are important regulators of spermatogenesis which serve as molecular switches that turn "on" and "off" adhesion function at the apical ES and the basal ES/BTB, mediated via their spatiotemporal expression during the epithelial cycle. A hypothetical model depicting the role of these two molecular switches is also proposed.

A novel synthetic oleanane triterpenoid suppresses adhesion, migration, and invasion of highly metastatic melanoma cells by modulating gelatinase signaling axis

A methyl derivative natural triterpenoid amooranin (methyl-25-hydroxy-3-oxoolean-12-en-28-oate, AMR-Me) has been found to possess antiproliferative, proapoptotic, and antiinflammatory effects against established tumor cells. Large-scale synthesis of pure AMR-Me has eliminated the need of the natural phytochemical for further development of AMR-Me as an anticancer drug. Metastatic melanoma is a fatal form of cutaneous malignancy with poor prognosis and limited therapeutic options. It was hypothesized that antitumor pharmacological effect of AMR-Me could be linked to AMR-Me-mediated suppression of the metastatic potential of B16F10 murine melanoma. AMR-Me was assessed for its antimetastatic efficacy by cell adhesion, migration, and invasion assays in B16F10 cells. The signaling crosstalk was explored by gelatin zymography, Western blot, ELISA, and immunocytochemistry. The results elicited that AMR-Me was successful in restricting the adhesion, migration, and invasion of highly metastatic cells. The antimetastatic potential of this compound may be attributed to the reduced expression of membrane type 1 metalloproteinase (MT1-MMP) and matrix metalloproteinases (MMP-2 and MMP-9). AMR-Me was found to downregulate vascular endothelial growth factor (VEGF)/phosphorylated forms of focal adhesion kinase (pFAK397 )/Jun N-terminus kinase (pJNK)/extracellular signal-regulated kinase (pERK). This, in turn, inhibited transcription factor nuclear factor-κB (NF-κB) and transactivation of MMPs. Moreover, the activation of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) might have influenced the downmodulation of MT1-MMP, MMP-2, and MMP-9. AMR-Me suppresses the activity of MT1-MMP, MMP-2, and MMP-9 by downregulation of VEGF/pFAK397 /pJNK/pERK/NF-κB and activation of TIMP-1 and TIMP-2 in metastatic melanoma cell line, B16F10. AMR-Me has the potential as an effective anticancer drug for metastatic melanoma which is a dismal disease.

Ganoderiol A-Enriched Extract Suppresses Migration and Adhesion of MDA-MB-231 Cells by Inhibiting FAK-SRC-Paxillin Cascade Pathway

Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA), dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK) and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP) and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

P-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr(397), a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII-X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr(397) that regulates spermatid adhesion at the apical ES in vivo.

Mechanisms Underlying the Anti-Tumoral Effects of Citrus bergamia Juice

Based on the growing deal of data concerning the biological activity of flavonoid-rich natural products, the aim of the present study was to explore in vitro the potential anti-tumoral activity of Citrus Bergamia (bergamot) juice (BJ), determining its molecular interaction with cancer cells. Here we show that BJ reduced growth rate of different cancer cell lines, with the maximal growth inhibition observed in neuroblastoma cells (SH-SY5Y) after 72 hs of exposure to 5% BJ. The SH-SY5Y antiproliferative effect elicited by BJ was not due to a cytotoxic action and it did not induce apoptosis. Instead, BJ stimulated the arrest in the G1 phase of cell cycle and determined a modification in cellular morphology, causing a marked increase of detached cells. The inhibition of adhesive capacity on different physiologic substrates and on endothelial cells monolayer were correlated with an impairment of actin filaments, a reduction in the expression of the active form of focal adhesion kinase (FAK) that in turn caused inhibition of cell migration. In parallel, BJ seemed to hinder the association between the neural cell adhesion molecule (NCAM) and FAK. Our data suggest a mechanisms through which BJ can inhibit important molecular pathways related to cancer-associated aggressive phenotype and offer new suggestions for further studies on the role of BJ in cancer treatment.

Integrin α5β1 simultaneously controls EGFR-dependent proliferation and Akt-dependent pro-survival signaling in epidermoid carcinoma cells

To delineate distinctive role of the components of α5β1 integrin-EGFR axis in control of epidermoid carcinoma cell proliferation, we performed individual inhibition of α5β1 and EGFR via genetic and phamacological methods, respectively. We demonstrated that pharmacological inhibition of epidermal growth factor receptor (EGFR) significantly affected proliferation of A431 human cells by inducing the G0/G1 cell cycle arrest, whereas shRNA-mediated depletion of α5 subunit of α5β1 integrin led to a similar type of cell cycle arrest followed by significant apoptosis. Both treatments resulted in suppression of activated (phosphorylated) forms of focal adhesion kinase (FAK) and Erk. However, unlike EGFR inhibition, depletion of α5 led to substantial suppression of AKT activity. Accordingly, pharmacological inhibition of EGFR and AKT recapitulated detrimental effects caused by shRNA-mediated depletion of α5. Moreover, depletion of α5 led to a severe drop in the amounts of active EGFR. Thus, for the first time, we demonstrated that α5β1 integrin simultaneously maintains pro-survival signaling via continuous activation of AKT and up-regulates proliferation via activation of EGFR.

Expression and Clinical Significance of FAK and Src Proteins in Human Endometrial Adenocarcinoma

Focal Adhesion Kinase (FAK) is a protein tyrosine kinase, localised in the focal adhesions, which, upon activation interacts with Src, another tyrosine kinase, regulating several cellular signalling pathways. Both enzymes have been implicated in malignant transformation and disease progression. The aim of the present study was to evaluate the clinical significance of FAK and Src expression in cases of endometrial adenocarcinoma. The total (t) and the activated, phosphorylated (p) forms of FAK and Src proteins were assessed immunohistochemically in tumour specimens obtained from 43 endometrial adenocarcinoma patients and were statistically analyzed in relation to various clinicopathological parameters and tumour proliferative capacity, reflected by Ki-67 labelling index. t-FAK positivity was significantly correlated with FIGO disease stage (p = 0.031), and t-FAK overexpression with patients' age (p = 0.015). No statistically significant correlation was identified between t-FAK staining intensity, t-Src positivity, overexpression or staining intensity and any of the clinicopathological parameters tested. No significant correlation was found between neither the positivity nor the intensity of staining of either p-FAk or p-Src with any of the parameters under study. Nonetheless, important, but non-significant, trends were identified between t-FAK staining intensity, t-Src positivity and overexpression and patients' survival (log rank, p = 0.122, p = 0.090 and p = 0.057 respectively). Similarly, p-FAK and p-Src staining characteristics seemed to correlate, even though non-significantly, with patients' survival (log rank, p = 0.051 and p = 0.070 for p-FAK and p-Src expression, respectively; log rank, p = 0.134 and p = 0.110 for p-FAK and p-Src staining intensity, respectively). These results support an important potential role of FAK-Src signalling in endometrial malignant disease progress and render further research in this field a necessity.

224 MOLECULAR CONSEQUENCES OF MITOCHONDRIAL DNA MUTATIONS IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010224 MOLECULAR CONSEQUENCES OF MITOCHONDRIAL DNA MUTATIONS IN PROSTATE CANCER Courtney Plattner, Carrie Sun, Takara Scott, Rebecca Arnold, and John Petros Courtney PlattnerCourtney Plattner More articles by this author , Carrie SunCarrie Sun More articles by this author , Takara ScottTakara Scott More articles by this author , Rebecca ArnoldRebecca Arnold More articles by this author , and John PetrosJohn Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.282AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It is now known that mutations in the mitochondrial DNA (mtDNA) are associated with prostate cancer. These mutations can be either inherited or somatically acquired and may contribute to the racial disparities in prostate cancer, with blacks exhibiting a higher incidence of inherited mtDNA mutations than whites. The objective of this study was to identify molecular consequences of mtDNA mutations in prostate cancer that may contribute to the malignant phenotype. By understanding these specific molecular alterations it is hoped that novel therapeutic agents may be developed targeted at this pathway, perhaps based on genetic testing. METHODS We introduced a single mtDNA base mutation that alters an amino acid into a human prostate cancer cell line. The ATP6 gene mutation T8993G results in the amino acid substitution Leu156Arg and hyperpolarizes the inner mitochondrial membrane. These cells (along with their wild type counterparts) were studied in vivo as nude mouse xenografts and in vivo for cell and molecular biologic alterations. Quantitative western analysis was used to measure changes in protein expression that resulted as a consequence of the mutation. Superoxide anion and hydrogen peroxide levels were measured by fluorescence using reagents dihydroethidium (DHE) and dichlorofluorescein (DCF) respectively. RESULTS The introduction of the 8993 mtDNA mutation increases tumor growth rates in nude mice and reactive oxygen production in these tumors. In the mutant cell lines there was a significant increase in the phosphorylated (active) form of focal adhesion kinase (pFAK) and fibroblast growth factor-1 (FGF1) compared to wild type prostate cancer cells. Cancer cells had significantly less PEDF (SERPINF1) protein than wild type cells. The mtDNA mutation also caused increased reactive oxygen, both superoxide and peroxide. CONCLUSIONS Mutations of the mitochondrial genome are common in clinical prostate cancer and may exert their carcinogenic effects via production of mitochondrially generated reactive oxygen. The T8993G mtDNA mutation causes prostate cancer cells to generate more ROS, and induces a coordinated change in three key cancer related proteins favoring the malignant phenotype. This suggests the potential future clinical translation of prostate cancer pharmaceuticals directed against mitochondrial ROS and/or specific proteins (FGF, pFAK, PEDF) based on mtDNA genotyping of patients or their tumors. Atlanta, GA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e88 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Courtney Plattner More articles by this author Carrie Sun More articles by this author Takara Scott More articles by this author Rebecca Arnold More articles by this author John Petros More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.

Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by α1-integrins during colon cancer cell invasion

Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44V Interaction Enhances Survival in Gastrointestinal Cancer Cells

Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44(V) in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44(V) isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-D-lysin, and preincubation of cells with anti-integrin beta(1) antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44(V) interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer.