Weak expression of focal adhesion kinase (pp125FAK) in patients with cervical cancer is associated with poor disease outcome.

PURPOSE: The pp125 focal adhesion kinase (FAK) plays a pivotal role in tumor cell signaling. FAK expression has been linked to tumor cell invasion and metastasis, but data on cervical cancer are inconclusive. Our goal was to investigate FAK expression in cervical cancer and to assess whether its expression correlates with prognosis. EXPERIMENTAL DESIGN: FAK expression was examined using immunohistochemistry with sections from 162 resected cervical cancer specimens. Kaplan-Meier survival curves were used to determine the significance of FAK expression in the prognosis of cervical cancer patients. RESULTS: Specific FAK expression was found in the tumor cells, whereas normal cervical epithelium showed barely any FAK expression. Of 162 invasive cervical cancer specimens, 55 (34%) revealed weak expression of FAK, whereas moderate and strong expression was found in 63 (39%) and 44 (27%) tumors, respectively. Patients with tumors expressing weak amounts of FAK were characterized by a significantly poorer overall survival compared with those with moderate and high intratumoral FAK expression (P = 0.002). Weak expression of FAK correlated with pelvic lymph node metastasis (P = 0.026) and recurrent disease (P = 0.013). Multivariate Cox regression analysis revealed decreased FAK expression and pelvic lymph node metastasis to be significant independent factors predictive of poor disease outcome (hazard ratio, 0.36; P = 0.005; hazard ratio, 2.38; P = 0.018, respectively). CONCLUSIONS: Weak expression of FAK in invasive cervical cancer is a strong independent predictor of poor patient outcome. Further studies are warranted to elucidate whether FAK expression analysis is a suitable tool identifying patients at high risk even at an early clinical stage.     

The focal adhesion kinase (P125FAK) is constitutively active in human malignant melanoma

Malignant melanoma cells show high aggressiveness and metastatic potential. Tumor cells as they become more metastatic, gradually lose their dependence on both adhesion and serum. Thus, in the process of tumor progression cells undergo series of changes that allow them to adapt to different tissue milieu. This implies that during this process, points on the integrin pathway may become constitutively activated. In the present study we investigated the possible role of FAK, being one of the key members of the integrin-signaling pathway, in the multistep progression towards a malignant phenotype in human melanoma. In our study we show that in melanoma cells there is neither an increase in the amount of FAK nor in its phosphorylation capacity, but rather in its levels of constitutive activation. Indeed, in all melanoma cells tested and not in nevus and neuroblastoma cells, we observed various degrees of constitutive activation of FAK. Our results also suggest that FAK constitutive activation is regulated at least in part by the cytoskeleton, implying that steps along the integrin signaling pathway involving FAK could be among the oncogenic mechanisms that operate in melanoma and may account for the highly aggressive, anchorage independent phenotype of this tumor.     

Inhibition of migration of human glioblastoma cells by cerivastatin in association with focal adhesion kinase (FAK)

To investigate the biological effect of cerivastatin on glioblastoma cells, we exposed them to various concentrations of cerivastatin. Cerivastatin exhibited dual effects on glioblastoma cells in a dose-dependent manner. Immunofluorescence microscopy showed disruption of actin stress fibers and focal adhesion plaques even at nanomolar concentrations. Matrigel assay demonstrated marked inhibition of glioblastoma cell invasion. Immunoblot analysis using a phosphospecific antibody against focal adhesion kinase (FAK) showed that inhibition of migration was associated with the down-regulation of tyrosine phosphorylation of FAK. Our data suggest that cerivastatin may be beneficial for combination therapy with conventional anti-cancer drugs by inhibiting the invasion of glioblastoma.     

Hepatocyte growth factor induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin and enhances cell-matrix interactions

This study examined the effects of hepatocyte growth factor/scatter factor (HGF/SF) on the adhesion of HT115 (human colon) and MDA MB 231 (human breast) tumour cells to an extracellular matrix, Matrigel, together with the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Treatment of cells with HGF increased cell adhesion to matrix. Following adhesion to Matrigel, FAK and paxillin were quickly phosphorylated and located to the focal adhesion area. HGF/SF increased both tyrosine phosphorylation of FAK and paxillin and also increased formation of focal adhesion. HGF also induced an increased spreading on matrix. It is concluded therefore that HGF/SF stimulated FAK and paxillin phosphorylation resulting in an increased tumour-matrix adhesion.     

Specific induction of pp125 focal adhesion kinase in human breast cancer

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT-PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51+/-0.84 (mean+/-standard deviation), which was strongly induced to 2.91 (+/-1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0+/-0.63 vs 2.27+/-0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.     

黏着斑激酶对肝癌细胞生物学行为的影响

目的：探讨降低黏着斑激酶（FAK）的表达对FAK高表达的人肝癌细胞株SMMC－7721恶性生物学行为影响。方法用Western杂交法比较不同肝癌细胞株FAK含量的差别,构建FAK反义质粒,转染至FAK高表达的细胞株,研究其多种细胞生物学行为的变化。结果SMMC－7721FAK含量比正常人肝细胞L02明显增高,转染FAK反义质粒后、SMMC－7721细胞生长受到抑制,软琼脂培养克隆形成率下降;细胞周期分析,S期细胞下降15％;黏附能力下降;细胞表面整连蛋白含量不变。结论在SMMC－7721中存在FAK过表达,降低FAK表达能部分逆转该肝癌细胞株的恶性表型。     

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

IntroductionFocal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.MethodsWe analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.ResultsElevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).ConclusionsImmunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.     

Focal adhesion kinase expression in human neuroblastoma: immunohistochemical and real-time PCR analyses.

PURPOSE: The focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase important in signaling between cells and their extracellular matrix. Studies have shown that FAK expression is up-regulated in several human tumors and is related to tumor progression. We recently found an increase in p125(FAK) expression in human neuroblastoma cells lines and wished to determine its expression in human neuroblastoma specimens and evaluate for a possible correlation between p125(FAK) expression and known prognostic factors for neuroblastoma. We hypothesized that p125(FAK) expression would be up-regulated in advanced human neuroblastomas. EXPERIMENTAL DESIGN: Using immunohistochemical techniques with monoclonal antibody 4.47 specific for p125(FAK) expression, we analyzed 70 formalin-fixed, paraffin-embedded human neuroblastoma specimens for p125(FAK) staining. In addition, real-time PCR was used to determine the abundance of FAK mRNA in 17 matched human neuroblastoma mRNA specimens. RESULTS: FAK staining was present in 51 of the 70 tumor specimens (73%). Immunohistochemical staining of p125(FAK) in the ganglion-type tumor cells correlated with advanced International Neuroblastoma Staging System tumor stages and FAK mRNA abundance. In addition, p125(FAK) staining was significantly increased in stage IV tumors with amplification of the N-MYC oncogene. CONCLUSIONS: These novel findings provide evidence that FAK is expressed by advanced-stage neuroblastoma and provide a rationale for targeting FAK in the treatment of this tumor.     

Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase

Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein. In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF. The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions. Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells. GDNF also induced reorganization of the actin cytoskelton. Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3' kinase (PI-3' kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3' kinase. In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho. These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3' kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase.     

Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK)

Doxycycline has been found to induce apoptosis and to inhibit the growth of a variety of tumor cells, in addition to its use as an antibiotic. However, the mechanism of its actions, especially at the molecular level, remains unknown and needs to be resolved. A crucial step possibly lies in the early period of doxycycline administration, which leads to a series of cascading effects depicting the consequential biological action of doxycycline on tumor cells. The present study focuses on the early-stage effects of doxycycline administration, specifically at the stages of treatment (before 16 h). In this paper, we report that doxycycline inhibits the adhesion and migration of melanoma cells. Afterwards, the cells undergo apoptosis (aniokis). Remarkably, doxycycline also inhibits the expression and phosphorylation of focal adhesion kinase (FAK), a protein tyrosine kinase involved in the regulation of cell adhesion and migration. We further demonstrate that doxycycline down-regulates the activities of MMP-2 and MMP-9, and its effects are stronger than those of an Integrin β1 antibody. Finally, we suggest that doxycycline might exert its anti-tumor effects by inhibiting FAK signaling pathway. These results provide an insight into the possible mechanisms that underlie the multiple drug actions of doxycycline. The potential use of doxycycline in anti-tumor treatment is promising and warrants further studies.     

HGF induces FAK activation and integrin-mediated adhesion in MTLn3 breast carcinoma cells.

Expression of hepatocyte growth factor (HGF) and its tyrosine kinase receptor, c-Met, is positively correlated with breast carcinoma progression. We found that in invasive and metastatic MTLn3 breast carcinoma cells, HGF stimulated both initial adhesion to and motility on the extracellular matrix (ECM) ligands laminin 1, type I collagen, and fibronectin. Next, analysis with function-perturbing antibodies showed that adhesion to the different ECM proteins was mediated through specific beta1 integrins. In MTLn3 cells, HGF induced rapid tyrosine phosphorylation and activation of both c-Met and focal adhesion kinase (FAK). Cell anchorage and adhesion to the ECM substrates was required for HGF-induced FAK activation, since HGF failed to trigger tyrosine phosphorylation of FAK in suspended cells. Our results provide evidence that the 2 signaling pathways, integrin/ECM and c-Met/HGF, cooperate synergistically to induce FAK activation in an adhesion-dependent manner, leading to enhanced cell adhesion and motility. Moreover, we found that a FRNK (the FAK-related non-kinase)-like molecule is expressed in MTLn3 cells. Since FRNK acts as a competitive inhibitor of FAK function, our results suggest that a FRNK-like protein could facilitate disassembly of focal adhesions and likely be responsible for the HGF-induced scattering and motility of MTLn3 cells.     

The direct effect of Focal Adhesion Kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

Background  

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.     

Effect of focal adhesion kinase (FAK) downregulation with FAK antisense oligonucleotides and 5-fluorouracil on the viability of melanoma cell lines

Inhibition of focal adhesion kinase (FAK), a non-receptor tyrosine kinase linked to tumour cell survival, causes cell rounding, loss of adhesion and apoptosis in human cancer cell lines. In this study, we tested antisense oligonucleotide inhibitors of FAK, in combination with 5-fluorouracil (5-FU), to increase its sensitivity in human melanoma cell lines. Antisense oligonucleotides directed to the 5' mRNA sequence of FAK and missense control oligonucleotides were used. In BL melanoma cells, treatment with FAK antisense oligonucleotide was associated with a 2.5-fold increase in cell death compared with treatment with control oligonucleotide (33+/-2% vs. 13+/-3%, P<0.0001). 5-FU alone had no effect on BL cells (4.4% cell death, P=0.15). The addition of 5-FU after antisense oligonucleotide resulted in a significant synergistic increase in cell death equal to 69+/-2% compared with treatments with antisense oligonucleotide alone, 5-FU alone and control oligonucleotide (P<0.0001). Similar results were found in the C8161 melanoma cell line. In both cell lines, reduction in cell viability was accompanied by an increased loss of adhesion and increased apoptosis that was proportional to the decrease in viability. Treatment with antisense oligonucleotide plus 5-FU resulted in significantly decreased p125FAK expression in both C8161 and BL melanoma cell lines, demonstrated by Western blot analyses. These data show that the downregulation of FAK by antisense oligonucleotide combined with 5-FU chemotherapy results in a greater loss of adhesion and greater apoptosis in melanoma cells than treatment with either agent alone, suggesting that the combination may be a potential therapeutic agent for human melanoma in vivo.     

p190RhoGEF (Rgnef) promotes colon carcinoma tumor progression via interaction with focal adhesion kinase

Focal adhesion kinase (FAK) functions downstream of integrins and growth factor receptors to promote tumor cell motility and invasion. In colorectal cancer, FAK is activated by amidated gastrin, a protumorigenic hormone. However, it is unclear how FAK receives signals from the gastrin receptor or other G-protein-coupled receptors that can promote cell motility and invasion. The Rho guanine-nucleotide exchange factor p190RhoGEF (Rgnef) binds FAK and facilitates fibroblast focal adhesion formation on fibronectin. Here we report that Rgnef mRNA and protein expression are significantly increased during colorectal tumor progression. In human colon carcinoma cells, Rgnef forms a complex with FAK and upon gastrin stimulation, FAK translocates to newly-forming focal adhesions where it facilitates tyrosine phosphorylation of paxillin. short hairpin (shRNA)-mediated knockdown of Rgnef or FAK, or pharmacological inhibition of FAK activity, is sufficient to block gastrin-stimulated paxillin phosphorylation, cell motility, and invadopodia formation in a manner dependent upon upstream cholecystokinin-2 receptor expression. Overexpression of the C-terminal region of Rgnef (Rgnef-C, amino acid 1,279-1,582) but not Rgnef-CΔFAK (amino acid 1,302-1,582 lacking the FAK binding site) disrupted endogenous Rgnef-FAK interaction and prevented paxillin phosphorylation and cell motility stimulated by gastrin. Rgnef-C-expressing cells formed smaller, less invasive tumors with reduced tyrosine phosphorylation of paxillin upon orthotopic implantation, compared with Rgnef-CΔFAK-expressing cells. Our studies identify Rgnef as a novel regulator of colon carcinoma motility and invasion, and they show that a Rgnef-FAK linkage promotes colon carcinoma progression in vivo.     

Mda-9/syntenin promotes human brain glioma migration through focal adhesion kinase (FAK)-JNK and FAK-AKT signaling

Invasion is usually recognized as the main reason for the high recurrence and death rates of glioma and restricts the efficacy of surgery and other therapies. Therefore, we aimed to investigate the mechanism involved in promotion effects of mda-9/syntenin on human glioma cell migration. The wound healing method was used to test the migration ability of human glioma cells CHG-5 and CHG-hS, stably overexpressing mda-9/syntenin. Western blotting was performed to determine the expression and phosphorylation of focal adhesion kinase (FAK) and JNK in CHG-5 and CHG-hS cells. The migration ability of CHG-hS cells was significantly higher than that of CHG-5 cells in fibronectin (FN)-coated culture plates. Phosphorylation of FAK on tyrosine 397, 576, and 925 sites was increased with time elapsed in CHG-hS cells. However, phosphorylated FAK on the tyrosine 861 site was not changed. Phosphorylated Src, JNK and Akt levels in CHG-hS cells were also significantly upregulated. Phosphorylation of JNK and Akt were abolished by the specific inhibitors SP600125 and LY294002, respectively. and the migration ability of CHG-hS cells was decreased, indicating that the JNK and PI3K/Akt pathways play important roles in regulating mda-9/syntenin-induced human brain glioma migration. Our results indicate Mda- 9/syntenin overexpression could activate FAK-JNK and FAK-Akt signaling and then enhance the migration capacity of human brain glioma cells.     

p22/WAF1 expression in human colorectal carcinoma: association with p53, transcription factor AP-2 and prognosis

p21/WAF1 expression was studied in a series of 162 colorectal carcinoma patients and its relation to p53- and activator protein (AP)-2 expressions and to stage as well as survival was assessed. p21 expression was moderate or intense in 33% of the tumours, and 53% of the tumours had moderate or strong p53 staining intensity. Eighty-nine percent of the tumours showed a weak cytoplasmic AP-2 signal. As expected, p21 and p53 stainings were inversely related to each other (P < 0.001). There was a significant positive association between p21 and AP-2 expression levels (P= 0.01). p21 intensity and percentage were higher in Dukes' A and B stages (P< 0.001). The cancer-related survival and recurrence-free survival (RFS) rates were significantly lower among patients with a low signal for p21 (P< 0.001) and low p21 percentage in tumour epithelium (P < 0.001). High p53 staining intensity in tumour epithelium predicted poor survival (P = 0.01) and RFS (P = 0.003). In the multivariate analysis, p21 percentage distribution independently predicted cancer-related survival in all cases, and p21 expression intensity in T1-4/N0-3/M0 and T1-3/N0/M0 cases. p21 percentage distribution was an independent predictor of RFS in all and T1-3/N0/M0 cases. AP-2 staining did not reach any prognostic significance. These results suggest that the immunohistochemical detection of cyclin-dependent kinase inhibitor p21 could be used to predict more precisely the outcome of colorectal cancer patients.     

Changes in the levels of integrin and focal adhesion kinase (FAK) in human melanoma cells following 532 nm laser treatment.

Despite the increase in laser therapy, concern remains that sublethal treatment of pre-malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the beta1 integrin in laser vs. non-laser treated cells. We treated cells with laser generated from a frequency doubled Q-switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with beta1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4- and 11.2-fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both alpha3 and 4 integrins were up-regulated, by approximately 4-fold and 7- to 9-fold, respectively. In addition, the beta1 integrin was proteolysed in both cell lines and the levels of FAK associated with beta1 was increased (2.1- and 2.7-fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology.     

Mutation of Y925F in focal adhesion kinase (FAK) suppresses melanoma cell proliferation and metastasis

This study focused on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, in the proliferation, adhesion, and invasion of melanoma cells in vitro and in vivo. We found that the Y925F-mutation of FAK in B16F10 melanoma cells suppressed metastasis in an experimental model, which correlated well with decreased extracellular matrix dependent proliferative capability, adhesive, migrational, and invasive capabilities. Transduction of the mutation Y925F resulted in a down-regulation of the phosphorylation of Erk, the expression of VEGF, and the association of FAK with paxillin. The results provide clear evidence that 925Y of FAK is critical for melanoma metastasis and this phosphorylation site will be an anti-metastatic target.