Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.

The Overexpressed FAK （Focal Adhesion Kinase） in Higher Grade Human Urothelial Tumors

Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kinase, which is recruited to focal adhesions and plays a key role in cell migration, proliferation and survival, could reflect the invasive capacity of bladder carcinomas. The aim of this study was to evaluate the FAK expression in cancer ceils as an important prognostic factor of the evolution of bladder carcinomas. Tumor and paired peritumoral biopsies were obtained during transurethral endoscopic resection or cystectomy of bladder tumors in 280 patients at the Urology Unit of the Mustapha Hospital of Algiers and the Hospital of Tizi-Ouzou （Algeria）. The authors studied FAK expression in samples from bladder carcinomas at different stages of malignant transformation by western blot analysis using a specific anti-FAK antibody. Western blot is one of the most common laboratory techniques; it is used to detect the presence of a specific protein in a complex mixture extracted from cells. A weak increase in FAK expression was observed in tumors of grade 1 and 2 （1.65; 2.99） as compared to healthy tissues; it became particularly important in grade 3 tumors; the authors show that FAK levels significantly increased gradually according to the tumor stage.

Progress in researches about focal adhesion kinase in gastrointestinal tract

Focal adhesion kinase(FAK)is a 125-kDa non-receptor protein tyrosine.Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases,resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation.FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract.FAK also plays an important role in the restitution,cell survival and apoptosis and carcinogenesis of the gastrointestinal tract.FAK is over-expressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells.FAK has been proposed as a potential target in cancer therapy.Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells,indicating a high potential for application in cancer therapy.

Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Inhibition of focal adhesion kinase enhances antitumor response of radiation therapy in pancreatic cancer through CD8+ T cells

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.

Effect of focal adhesion kinase on cytoskeletal arrangement of HepG2 cells induced by hypoxia

Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.

Inhibiting focal adhesion kinase:A potential target for enhancing therapeutic efficacy in colorectal cancer therapy

Focal adhesion kinase(FAK) is a major integrin- dep-endent tyrosine phosphorylated protein, recently, FAK association with colorectal cancer(CRC) has gained at-tention. The various cancer-promoting mechanisms that associated with FAK can be implicated in the progression of CRC. The interactions between structural features of FAK and various kinases could be closely related to growth, survival, and metastasis in CRC cells. These interactions include human epithelial growth factor re-ceptor, c-Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and Src. Such interactions can trigger the survival signaling of CRC cells and are also involved signaling downstream of phosphatidylinositol 3-kinase, AKT, and the extracellular regulated kinase. Based on this scientific background, many pharmaceutical companies are taking efforts to develop FAK inhibitors to treat solid cancer including CRC. Although the anti-cancer efficacies have been noted in many studies, the commercial drugs have not been deve-loped yet. Therefore, the FAK research on CRC is expec-ted to gain momentum and be highly appreciated as a potential field for developing the new drugs. Therefore, the studies on FAK that effect on the progression of human CRC s would be possible to suggest various app-roaches to CRC treatment, and FAK could be a potential target as an anticancer candidate for CRC therapies.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma

Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

Clinical significance of upregulated Rho GTPase activating protein 12 causing resistance to tyrosine kinase inhibitors in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.

根皮素通过抑制FAK改善肾小管间质纤维化

目的探究根皮素能否通过抑制黏着斑激酶(FAK)改善肾小管间质纤维化。方法肾小管上皮细胞NRK-52E经TGF-β1(10 ng/mL)诱导建立肾纤维化细胞模型,并构建单侧输尿管梗阻(UUO)肾间质纤维化小鼠模型。通过免疫荧光及Western blot法检测细胞及小鼠肾脏组织中α-SMA、FN的蛋白表达水平,明确根皮素体内外抗肾纤维化作用;检测实验小鼠血清中肌酐(Cr)和尿素氮(BUN)水平,分析根皮素的肾保护作用;以苏木精-伊红(HE)染色和Masson染色观察根皮素对UUO小鼠肾脏病理学改变和胶原纤维沉积的影响;Western blot检测TGF-β1诱导的NRK-52E细胞和UUO小鼠肾组织中FAK、p-FAK的蛋白表达水平;通过分子对接预测根皮素与FAK蛋白的潜在结合模式和结合能力;使用FAK抑制剂PND-1186,探究根皮素改善肾间质纤维化的作用机制。结果根皮素可逆转TGF-β1所致的肾小管上皮细胞及UUO小鼠肾组织中α-SMA、FN的蛋白表达水平升高,且显著降低UUO小鼠血清中Cr、BUN水平,改善UUO小鼠肾小管扩张、炎性细胞浸润及间质胶原沉积;根皮素可以显著抑制UUO小鼠肾组织中FAK、p-FAK蛋白的表达水平,分子对接提示根皮素与FAK蛋白之间具有良好相互作用。根皮素与PND-1186均可改善TGF-β1诱导的NRK-52E细胞纤维化。结论根皮素通过抑制FAK信号通路在体内外发挥抗肾间质纤维化作用。

通关藤对胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平的影响

目的探讨通关藤对胃癌小鼠类固醇受体共激活因子(Steroid receptor coactivator,Src)/黏着斑激酶(Focaladhesion kinase,FAK)信号通路及Th1/Th2相关因子水平的影响。方法将SPF级成年雌雄各半C57BL/6小鼠60只,根据随机数字表法分为空白对照组、模型组(注射生理盐水)、环磷酰胺组(注射环磷酰胺)、通关藤低、中、高剂量组(分别注射0.1 ml、0.2 ml、0.4 ml通关藤注射液),每组各10只。干预1次/2 d,于干预第21天时处死所有小鼠,颈部取血。测量小鼠体质量以及胸腺、脾、肿瘤质量。HE染色观察小鼠瘤体病理形态变化。流式细胞术检测小鼠CD4^(+)、CD8^(+)以及CD4^(+)/CD8^(+)水平,ELISA法检测白细胞介素-2(Interleukin-2,IL-2)、白细胞介素-4(Interleukin-4,IL-4)、白细胞介素-10(Interleukin-10,IL-10)、干扰素-γ(Interferon-γ,INF-γ)水平,Western blot法检测Src、磷酸化Src(p-Src)、FAK、磷酸化FAK(p-FAK)蛋白表达水平。结果与模型组比较,通关藤各干预组脾指数、胸腺指数水平升高,且随通关藤剂量增加,脾指数、胸腺指数水平、抑瘤率均升高,差异有统计学意义(P<0.05)。环磷酰胺组脾指数和胸腺指数明显低于各通关藤剂量组,差异有统计学意义(P<0.05);环磷酰胺组和通关藤高剂量组抑瘤率明显高于通关藤低、中剂量组,差异有统计学意义(P<0.05)。HE染色结果显示,模型组肿瘤细胞排列整齐且密集,而环磷酰胺组和通关藤各剂量组肿瘤细胞密度有所减少,且分布不均,并呈不同程度的肿瘤细胞坏死灶。与模型组比较,通关藤各干预组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05);相较于环磷酰胺组,通关藤低、中、高剂量组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,IL-2、INF-γ水平升高越明显,IL-4、IL-10水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,CD4^(+)、CD4^(+)/CD8^(+)水平升高越明显,CD8^(+)水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组p-Src/Src、p-FAK/FAK蛋白水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组p-Src/Src、p-FAK/FAK蛋白水平升高,差异有统计学意义(P<0.05),且通关藤干预剂量越高,p-Src/Src、p-FAK/FAK蛋白水平降低越明显。结论通关藤可调节胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平,从而发挥抑癌作用,且剂量越高,效果越显著。

RGS4 promotes the progression of gastric cancer through the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition

BACKGROUND Regulator of G protein signaling(RGS)proteins participate in tumor formation and metastasis by acting on theα-subunit of heterotrimeric G proteins.The speci-fic effect of RGS,particularly RGS4,on the progression of gastric cancer(GC)is not yet clear.AIM To explore the role and underlying mechanisms of action of RGS4 in GC develop-ment.METHODS The prognostic significance of RGS4 in GC was analyzed using bioinformatics based public databases and verified by immunohistochemistry and quantitative polymerase chain reaction in 90 patients with GC.Function assays were employed to assess the carcinogenic impact of RGS4,and the mechanism of its possible influence was detected by western blot analysis.A nude mouse xenograft model was established to study the effects of RGS4 on GC growth in vitro.RESULTS RGS4 was highly expressed in GC tissues compared with matched adjacent normal tissues.Elevated RGS4 expression was correlated with increased tumor-node-metastasis stage,increased tumor grade as well as poorer overall survival in patients with GC.Cell experiments demonstrated that RGS4 knockdown suppressed GC cell proliferation,migration and invasion.Similarly,xenograft experiments confirmed that RGS4 silencing significantly inhibited tumor growth.Moreover,RGS4 knockdown resulted in reduced phosphorylation levels of focal adhesion kinase,phosphatidyl-inositol-3-kinase,and protein kinase B,decreased vimentin and N-cadherin,and elevated E-cadherin.CONCLUSION High RGS4 expression in GC indicates a worse prognosis and RGS4 is a prognostic marker.RGS4 influences tumor progression via the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.

结直肠癌中FAK、FAK pY397、Akt、NF-κB表达及相关性研究

目的:检测黏着斑激酶(FAK)、磷酸化FAK(FAK pY397)、Akt和细胞核因子(NF-κB)在结直肠癌组织中的表达,并探讨其相关性。方法:采用免疫组织化学SP法,检测74例结直肠癌组织与10例正常结肠组织中FAK、FAK pY397、Akt和NF-κB的表达。结果:结直肠癌组织标本中FAK、FAK pY397、Akt和NF-κB的阳性率分别为82.43%、45.95%、52.70%和59.46%,除FAK pY397外,FAK、Akt、NF-κB均与正常结肠壁组织表达的差异显著(P<0.05)。FAK表达与分化程度、淋巴结转移有关(P<0.05),但不受患者性别、年龄、肿瘤的大小及位置、Dukes分期的影响。Akt阳性表达与分化程度显著相关(P<0.05),NF-κB表达与淋巴结转移显著有关(P<0.05),并且Akt、NF-κB表达与FAK表达呈显著正相关(P<0.05)。结论:FAK、Akt、NF-κB在结直肠癌组织中均高表达,且Akt、NF-κB表达与FAK有显著正相关性,提示生存信号传导通路FAK/Akt/NF-κB在结直肠癌的发生、演进中起重要作用。

FAK在肝细胞癌中的表达及临床病理意义

目的研究FAK在肝细胞癌组织中的表达情况以及其与临床病理特征的联系,并探讨其表达与肝细胞癌患者预后之间的关系。方法利用RT-PCR法检测了52例肝癌组织及对应的癌旁组织中FAK mRNA的表达水平。分析了FAK mRNA的表达水平与肝癌临床病理特征之间的关系。利用免疫组化的方法检测了FAK蛋白在30例肝癌组织及其癌旁组织和10例正常肝脏组织中的表达。Kaplan-Meier生存分析和Cox回归分析了FAK表达与肝癌手术治疗预后的关系。结果肝癌组织中FAK mRNA的表达水平显著高于其癌旁组织(0.48±0.12 vs.0.17±0.07;P<0.05)。FAK mRNA表达水平与肿瘤直径、组织病理分化程度、TNM分期、血管侵犯等临床病理特征显著相关(P<0.05)。Kaplan-Meier生存分析和COX回归模型分析发现FAK表达是影响肝细胞癌术后生存率的因素。30例肝癌组织细胞质中广泛表达FAK蛋白,主要定位于肝癌细胞质内,阳性率为60%(18/30),癌旁组织阳性率为26.3%(8/30),10例正常肝组织阳性表达率为20%(2/10)。结论 FAK在肝癌组织中高表达,这与肝癌的恶性临床病理特征相关,也是判断肝细胞癌手术治疗预后的指标,提示FAK可能成为肝癌潜在的分子标志物或治疗靶点。

FAK和Src在甲状腺乳头状癌中的表达及其在肿瘤侵袭转移中的临床意义及机制研究

目的:探讨黏着斑激酶(FAK)和Src在甲状腺乳头状癌侵袭转移中的临床意义。方法:利用免疫组化检测FAK和Src在100例甲状腺乳头状癌组织中的表达,并分析其与患者临床病理参数的关系。利用Western blot以及qRT-PCR检测FAK和Src在甲状腺癌细胞株中与上皮间质转化(EMT)的相关性。结果:FAK与甲状腺乳头状癌的年龄(P=0.000)、T分期(P=0.176)、N分期(P=0.000)、M分期(P=0.000)、临床分期(P=0.038)、局部复发(P=0.000)、淋巴结侵袭(P=0.014)以及与患者较低生存期(P<0.000 1)密切相关;Src与甲状腺乳头状癌的N分期(P=0.000)、M分期(P=0.002)、淋巴结侵袭(P=0.000)、包膜侵袭(P=0.029)以及患者的较低生存期(P<0.000 1)密切相关。并且高表达FAK与Src的细胞其N-cadherin、Vimentin的表达增高,而E-cadherin的表达降低。结论:FAK以及Src与甲状腺乳头状癌的侵袭转移密切相关,并可能通过EMT的方式促进其侵袭和转移。

低强度脉冲超声波对兔膝骨性关节炎软骨整合素-FAK-MAPKs信号通路蛋白表达的影响

目的:观察低强度脉冲超声波(low-intensity pulsed ultrasound,LIPUS)对兔膝骨性关节炎(osteoarthritis,OA)软骨中整合素-FAK-MAPKs力化学细胞信号转导通路相关蛋白表达的影响。方法:18只成年新西兰大白兔随机平均分成3组,分为正常对照组(normal control,NC),OA模型组(OA group,OA),OA模型照射组(O+L)。OA组接受右侧后肢前交叉韧带切断(ACLT)处理术后4周接受LIPUS假辐射,O+L组同样手术处理,术后4周接受LIPUS辐射,NC组仅切开关节囊。LIPUS作用6周后,处死实验动物,取右后肢,采用HE染色行改良Mankin评分比较各组胫骨平台关节表面病理学改变。同时,采用Western blot技术检测Ⅱ型胶原,MMP-13,整合素β1的蛋白表达水平以及FAK、MAPKs家族蛋白(p38、ERK1/2、JNK)磷酸化水平。结果:1组织学观察及Mankin评分:LIPUS干预后,OA软骨表层轻微不平整,染色轻度缺失,可见软骨细胞增殖;Mankin评分,NC组:4.67±0.57;OA组:10.57±2.55;O+L组:7.66±1.74。与NC组相比,OA组、O+L组Mankin评分明显增高,但OA组增高更为明显(P<0.05);与OA组相比,O+L组Mankin评分明显降低(P<0.05)。2Ⅱ型胶原、MMP-13含量:LIPUS干预后,II型胶原含量较NC组升高,MMP-13含量有明显下降。3整合素β1-FAK-MAPKs信号通路相关蛋白表达:LIPUS干预使整合素β1、磷酸化FAK表达增高;同时MAPKs信号通路相关蛋白ERK1/2、p38磷酸化水平明显下降,而JNK磷酸化水平无明显变化。结论:LIPUS可以减轻骨性关节炎软骨ECM损伤程度,与LIPUS产生机械应力使关节软骨中细胞表面应力受体之一整合素β1及其下游分子黏着斑激酶FAK磷酸化表达增高,进一步下游的磷酸化p38,ERK1/2表达下调有关。LIPUS的机械效应可经力化学转导途径作用于OA关节软骨,为治疗骨性关节炎提供一种新的思路。

反义FAK寡核苷酸抑制FAK-ERK1/2介导的平滑肌细胞迁移和粘附

目的 研究FAK ERK1 2信号通路在平滑肌细胞迁移和粘附中的作用及FAK反义寡核苷酸对其的调控作用。方法 通过纤粘连蛋白 (fibronectin ,FN)诱导平滑肌细胞迁移和粘附 ,以免疫沉淀和Westernblot方法检测粘着斑激酶 (focaladhesionkinase,FAK)和细胞外调控激酶 1 2 (extracellularregulationkinase ,ERK1 2 )及其磷酸化的表达量。将FAK反义寡核苷酸 (antisenseoligodeoxynucleotides,ODNs)经脂质体转染细胞 ,观察转染后反义ODN对FAK和ERK磷酸化、细胞粘附和迁移的影响。结果 FN在有效诱导平滑肌细胞迁移和粘附时FAK和ERK1 2也呈明显表达 ,FN2 0 μg·mL- 1 可使磷酸化处于较高表达量。脂质体可有效地介导ODNs转染。转染后FAK及ERK1 2磷酸化表达量明显减少。结论 由活化的FAK和ERK1 2介导的信号转导促进了细胞外基质诱导的SMCs迁移和增殖 ,反义FAKODNs可有效地对此进行抑制。

PTEN下调FAK磷酸化来抑制EGFR受体突变体引起的胶质瘤细胞侵袭

背景与目的:抑癌基因PTEN在胶质瘤中突变率达20%～40%,且PTEN缺失和表皮生长因子受体突变体EGFRvⅢ同时存在EGFR表达的胶质瘤组织中。PTEN通过直接与FAK作用从而降低其酪氨酸磷酸化来抑制肿瘤细胞侵袭。EGFRvⅢ表达,PTEN缺失都是肿瘤细胞侵袭性增加的原因之一。PTEN功能的丧失可能是EGFRvⅢ表达的肿瘤进一步发展成恶性侵袭性肿瘤的原因之一。本文将探讨在EGFRvⅢ表达和PTEN缺失的肿瘤细胞中转入PTEN以观察其是否能抑制EGFRvⅢ所引起的肿瘤细胞的侵袭。方法:将野生型PTEN及其突变体分别导入人胶质瘤细胞U87ΔEGFR中,利用Transwell-细胞侵袭实验观察细胞侵袭运动变化;免疫印迹实验检查FAK磷酸化变化;FAK表达载体来转染FAK磷酸化程度低的U87ΔEGFR-wtPTEN细胞,观察细胞侵袭运动变化。结果:PTEN和PTEN(G129E)能够抑制U87ΔEGFR细胞侵袭,并且下调FAK397位点磷酸化水平。U87ΔEGFR-wtPTEN细胞中FAK397位点磷酸化水平增加伴随着细胞侵袭能力的提高。结论:PTEN可能通过下调FAK397位点磷酸化水平来抑制EGFRvⅢ引起的胶质瘤细胞侵袭。

应用组织芯片技术检测KAI1、MRP-1、FAK蛋白在肺癌组织中的表达

背景与目的:肿瘤的发生发展、浸润转移与多基因改变密切相关。目前Kang-ai-1(KAI1)、移动相关蛋白(motility-relatedprotein-1,MRP-1)和局部粘着斑激酶(focaladhesionkinase,FAK)3种基因在肺癌中的共同作用研究较少。本研究旨在探讨这3种基因的蛋白产物在肺癌发生发展中的作用及其在肿瘤诊断和预后判断中的价值。方法:应用免疫组化SP方法检测包含240个点的肺癌组织芯片中KAI1、MRP-1和FAK蛋白的表达。结果:KAI1在肺癌原发灶中阳性率为25.9%,MRP-1为42.6%,与正常肺组织(100%)相比均显著下调;FAK蛋白在肺癌组织中阳性率为44.4%,与正常肺组织相比显著增高;KAI1、FAK两种蛋白在肺癌组织中的表达与患者的年龄、性别、肿瘤的大体类型及组织类型无关,而与肿瘤的分化程度、临床分期及是否伴有淋巴结转移密切相关,两种蛋白的表达呈显著负相关。MRP-1蛋白在肺癌组织中的表达与组织类型有关,小细胞肺癌与非小细胞肺癌相比差异显著,MRP-1与KAI1呈显著正相关,与FAK呈显著负相关。结论:KAI1、MRP-1和FAK的异常表达与肺癌的浸润转移有关,联合检测这3项指标对肺癌的发生发展有重要的预测作用。

大麻素受体1、FAK mRNA在小鼠肝纤维化形成过程中肝组织中的表达及相互关系

目的研究大麻素受体1(CB1)mRNA在肝纤维化形成过程中表达的变化,从基因转录水平探讨其与黏着斑激酶(FAK)的关系。方法采用10%四氯化碳腹腔注射制备肝纤维化模型,分别于造模第2、4、6、8周留取小鼠的肝组织及血清。通过肝组织病理对肝纤维化程度进行评分,荧光定量PCR检测CB1 mRNA和FAK mRNA水平,ELISA方法检测血清中转化生长因子(TGF)β1的含量。结果与正常对照组相比,各模型组小鼠肝组织中CB1 mRNA和FAK mRNA含量显著升高(P<0.05),而且随造模时间的延长,CB1 mRNA和FAK mRNA含量亦逐渐增高,各组之间差异有统计学意义(P<0.05)。CB1 mRNA的含量不但与肝纤维化评分呈显著正相关(r=0.747),而且与肝组织中FAK mRNA含量也呈显著正相关(r=0.907),P值均<0.01。各模型组小鼠血清中TGFβ1的水平随造模时间延长而不断升高,各组间差异具有统计学意义(P<0.05)。肝组织中CB1 mRNA的含量与血清TGFβ1水平呈显著正相关(r=0.542,P<0.01)。结论 CB1可能通过激活FAK,以磷脂酰肌醇-3-激酶(PI3K)通路诱导肝星状细胞(HSC)增殖并分泌大量TGFβ1,从而促进肝纤维化的形成。