Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance

Chemotherapy resistance in solid tumors is broad and encompasses diverse unrelated drugs. Three-dimensional multicellular spheroids (MCSs) are a good model for studying in vitro drug resistance. In the current study, we investigated the role of focal adhesion kinase (FAK) in 5-fluorouracil (5-FU) chemoresistance in colon carcinoma MCS culture cells. The expression of FAK was inhibited significantly by specific small hairpin RNA targeting FAK. The suppression of FAK expression did not affect the growth of spheroid cells. However, silencing of FAK combined with 5-FU treatment significantly decreased the 50% inhibitory concentration (IC₅₀) of 5-FU and markedly increased the population of apoptosis cells, which was associated with the reduction of the levels of Akt and nuclear factor–kappa B (NF-κB). Moreover, knockdown of FAK could inhibit tumor growth and increase the sensitivity of the tumor to 5-FU in the nude mouse xenograft. These results indicate that while not affecting cellular proliferation in the absence of 5-FU, RNA interference targeting FAK potentiated 5-FU-induced cytotoxicity in vitro and in vivo , and partially reversed multicellular resistance, which may contribute to its chemosensitizing effect through efficiently suppressing Akt/NF-κB activity. ( Cancer Sci 2009; 100: 1708–1713)     

黏着斑激酶活化与肿瘤进程研究进展

细胞质非受体型酪氨酸激酶黏着斑激酶（focaladhesionkinase，FAK），作为信号转导的关键分子，传递由胞外基质到细胞质的信号，在许多高度恶化肿瘤中表达升高，并促进细胞形态学变化、形成伪足小体和侵袭伪足，进而导致入侵表型的出现。因此，研究FAK的结构、功能以及在细胞运动、细胞存活、肿瘤发生过程中的作用意义重大。本文就FAK的活化以及在肿瘤发生过程中的作用作一综述。     

Role of expression of focal adhesion kinase in progression of hepatocellular carcinoma.

PURPOSE: Although hepatocellular carcinoma (HCC) is the most common cancer of the human liver, the mechanisms that regulate HCC development and progression remain unclear. The aim of this study was to investigate whether focal adhesion kinase (FAK) is involved in the progression of human HCC. EXPERIMENTAL DESIGN: Western blot analysis for FAK was performed on three HCC cell lines. We reviewed 64 consecutive patients who had undergone initial liver resection for HCC without preoperative treatment. Immunohistochemistry analysis for FAK was performed on paraffin-embedded tissues. FAK expression was confirmed by Western blot analysis in several clinical samples. We investigated the correlation between FAK expression and clinical outcome. RESULTS: FAK proteins were detected in all HCC cell lines. Hepatocytes in the normal liver and chronic hepatitis with or without cirrhosis were negative for immunohistochemical staining for FAK expression. Cytoplasmic FAK expression was observed in 18 of 64 patients (28.1%), and this positive staining was correlated with gender (P < 0.05), a lower level of serum albumin (P < 0.05), and portal venous invasion (P < 0.01). Positive staining for FAK was associated with significantly poorer survival (P < 0.05). In multivariate analysis, FAK overexpression was an independent factor in determining the prognosis of patients. CONCLUSIONS: These data suggest that FAK plays an important role in promoting tumor progression, especially vascular invasion, in HCC. FAK could play an important role in HCC progression and would be a novel target for HCC therapeutics as well as a prognostic marker.     

Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression

Introduction  

Rho signaling regulates key cellular processes including proliferation, survival, and migration, and it has been implicated in the development of many types of cancer including breast cancer. P190B Rho GTPase activating protein (RhoGAP) functions as a major inhibitor of the Rho GTPases. P190B is required for mammary gland morphogenesis, and overexpression of p190B in the mammary gland induces hyperplastic lesions. Hence, we hypothesized that p190B may play a pivotal role in mammary tumorigenesis.     

Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase.

Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN). Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal-shaped cells to fusiform-shaped ones with pseudopodia. MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not. A small GTPase Rho and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y-27632, an inhibitor of ROCK. Y-27632 also suppressed the formation of fusiform-shaped pseudopodia-carrying MM1 cells that was induced by stimulation with the combination of LPA and FN. LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. The inhibitory effect of Y-27632 on LPA-induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles. Y-27632 suppressed LPA-induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y-27632 inhibits cellular migration and morphological change, at least in part, through this regulation.     

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.     

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin-focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3β pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src-miR-542-3p-ILK-FAK circuit plays a crucial role in controlling tumor progression.     

Expression of focal adhesion kinase in small-cell lung carcinoma

BACKGROUND:

The focal adhesion kinase (FAK) is a non‐receptor tyrosine kinase linked to tumor growth, invasion, and metastasis. FAK is overexpressed and associated with prognosis in many cancers, but its prognostic value in small‐cell lung carcinoma (SCLC) is unknown and was the focus of this study.

METHODS:

Total FAK expression was analyzed via immunohistochemistry in tissue microarrays consisting of formalin‐fixed, paraffin‐embedded SCLC specimens from 85 patients. FAK staining scores were tested for correlations with pathological characteristics and clinical outcomes. Phospho‐paxillin was also tested in 35 of the 85 specimens to evaluate whether FAK expression was associated with downstream signaling.

RESULTS:

Specific FAK expression was localized to the cytoplasm of 78/85 (92%) SCLCs. FAK expression was scored low in 11 (13%), moderate in 17 (20%), and high in 50 (59%) SCLCs. FAK staining scores treated as continuous variables did not correlate with SCLC disease stage, response to therapy, recurrence/progression‐free survival, or overall survival. Moreover, total FAK expression did not correlate with phospho‐paxillin Tyr¹¹⁸ expression.

CONCLUSIONS:

Total FAK is strongly expressed in a majority of SCLC tumors. However, the expression evaluated via immunohistochemistry is not a prognostic factor in patients with SCLC. Cancer 2012;. © 2011 American Cancer Society.     

Migration of renal carcinoma cells is dependent on protein kinase Cdelta via beta1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesion were assessed through chemotaxis analysis and adhesion to an endothelial monolayer, respectively. Cell proliferation was analysed by a BrdU incorporation assay. The expression and activity of beta1 integrins and FAK were analysed by Western blot analysis. GF109203X reduced cell migration to 69%, the activity of beta1 integrins to 63% and FAK expression to 82% compared to untreated cells. Rottlerin reduced cell migration in a concentration-dependent manner to 36%, cell proliferation to 81%, expression and activity of beta1 integrins to 72 and 79%, and expression and activity of FAK to 56 and 76% of untreated cells, respectively. RO31-8220 also reduced the expression and activity of beta1 integrins as well as the expression of FAK to 84, 66 and 66% of untreated cells, respectively. GO6976 reduced the expression of FAK to 60% of untreated cells. Cell migration was only slightly reduced by GO6976 to 84% of untreated cells, and cell adhesion remained uninfluenced. These findings show a critical role of PKCdelta in the regulation of tumor cell migration, which seems to be caused by affecting the expression and activity of beta1 integrins and FAK. These results can provide a basis for new strategies in preventing metastases of renal cell carcinoma.     

Deguelin inhibits vasculogenic function of endothelial progenitor cells in tumor progression and metastasis via suppression of focal adhesion

Deguelin is a nature-derived chemopreventive drug. Endothelial progenitor cells (EPCs) are bone-marrow (BM)-derived key components to induce new blood vessels in early tumorigenesis and metastasis. Here we determined whether deguelin inhibits EPC function in vitro and in vivo at doses not affecting cancer cell apoptosis. Deguelin significantly reduced the number of EPC colony forming units of BM-derived c-kit+/sca-1+ mononuclear cells (MNCs), proliferation, migration, and adhesion to endothelial cell monolayers, and suppressed incorporation of EPC into tube-like vessel networks when co-cultured with endothelial cells. Deguelin caused cell cycle arrest at G1 without induction of apoptosis in EPC. In a mouse tumor xenograft model, tumor growth, lung metastasis and tumor-induced circulating EPCs were supressed by deguelin treatment (2 mg/kg). In mice tranplanted with GFP-expressing BM-MNCs, deguelin reduced the co-localization of CD31 and GFP, suggesting suppression of BM-derived EPC incoporation into tumor vessels. Interestingly, focal adhesion kinase (FAK)-integrin-linked kinase (ILK) activation and actin polymerization were repressed by deguelin. Decreased number of focal adhesions and a depolarized morphology was found in deguelin-treated EPCs. Taken together, our results suggest that the deguelin inhibits tumorigenesis and metastasis via EPC suppression and that suppression of focal adhesion by FAK-integrin-ILK-dependent actin remodeling is a key underlying molecular mechanism.     

The adhesion molecule L1 (CD171) promotes melanoma progression

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.     

Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model

Expression of focal adhesion kinase (FAK) is elevated in malignant breast cancer, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. Here, we have inhibited FAK activity or expression in murine 4T1 breast carcinoma cells via dominant-negative focal adhesion kinase-related non-kinase (FRNK) or anti-FAK short hairpin RNA (shRNA) expression, respectively. Neither FRNK nor FAK shRNA ( approximately 80% reduced FAK levels) affected 4T1 proliferation in culture, whereas reduced FAK activity or expression blocked 4T1 cell invasion through Matrigel and resulted in 2-3-fold lower urokinase plasminogen activator (uPA) expression. Control 4T1 cells implanted into mammary fat pads of BALB/c mice exhibited spontaneous metastasis to the lungs, to the peritoneal cavity, and resulted in 90% lethality within 21 days. Whereas FAK shRNA-expressing 4T1 cells formed tumors in mice with low levels of apoptosis, when mammary-injected, these cells did not exhibit lung metastasis after 21 days and caused only 40% lethality up to 60 days. Transient re-expression of wild-type but not kinase-dead FAK in 4T1 FAK shRNA cells promoted uPA production and mammary to lung metastasis within 7 days. In fact, stable human uPA overexpression in 4T1 FAK shRNA cells promoted Matrigel invasion and lung metastasis equal to 4T1 controls. Conversely, treatment with plasminogen activator inhibitor-1 or neutralizing antibody to uPA blocked Matrigel invasion of 4T1 control cells. These studies provide the first direct proof that FAK catalytic activity can facilitate metastatic breast cancer progression by regulating uPA expression.     

黏着斑激酶与口腔鳞状细胞癌关系的研究进展

黏着斑激酶（focal adhesion kinase,FAK）是一种位于细胞内的非受体型酪氨酸激酶,介导多条信号通路,在肿瘤生长、发展及转移过程中起至关重要的作用。口腔肿瘤中 95%是来源于口腔黏膜的鳞状细胞癌,深入了解 FAK 与口腔鳞状细胞癌的关系,有助于更好地预防和治疗口腔鳞状细胞癌,本文就 FAK 与口腔鳞状细胞癌关系的研究进展作一综述。     

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.     

Transforming growth factor beta regulates cell-cell adhesion through extracellular matrix remodeling and activation of focal adhesion kinase in human colon carcinoma Moser cells

Transforming growth factor (TGF) beta is a potent regulator of cell-matrix and cell-cell adhesions (collectively termed cellular adhesions). Cellular adhesions play crucial roles in controlling the differentiation of epithelial cells and in maintaining the integrity of the epithelium. Loss of TGF beta-responsiveness is thought to be an important early initiating event in the malignant progression of epithelial cancer. In the TGFbeta-responsive human colon adenocarcinoma Moser cells, TGFbeta promotes cellular adhesions and suppresses their malignant phenotype. TGFbeta promotes cell-matrix adhesion by inducing the synthesis of extracellular matrix (ECM) adhesion molecules and the expression of integrin receptors for these molecules (termed ECM remodeling). TGFbeta promotes cell-cell adhesion through the induction of E-cadherin expression, an epithelial associated homotypic cell-cell adhesion molecule, which also functions as a tumor suppressor in colon cancer. How TGFbeta regulates E-cadherin expression is not known. In this study, we showed that the induction of E-cadherin by TGFbeta was mediated through the activation of focal adhesion kinase (FAK), a major signaling molecule in focal adhesion contacts and that the activation of FAK was due to ECM remodeling and increased cell-matrix interactions. Thus, TGFbeta regulates cell-cell adhesion through its ability to remodel the ECM and to activate FAK through ECM remodeling.     

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。     

Chromobox 4 (CBX4) promotes tumor progression and stemness via activating CDC20 in gastric cancer

BackgroundThe Chromobox homolog 4 (CBX4) has been found to be overexpressed in multiple malignancies. However, the associations between CBX4 and gastric cancer (GC) have remained unclear. This study aimed to determine the biological roles of CBX4 in GC and identify effective therapeutic targets.MethodsThe 3-(4,5-dimethylthiazol-2-yl) (MTT) assays were used to screen CBX family members. Differential analysis was utilized to evaluate the CBX4 levels. Kaplan-Meier analysis was used to perform prognostic analysis. Western blotting assay, quantitative polymerase chain reaction (qPCR) assay and immunohistochemistry (IHC) were used to assess CBX4 expressions. Colony formation assay, Cell Counting Kit-8 (CCK-8) assay, and Transwell assay were used to assess progression features of cells. The tail vein injection model was utilized to determine the metastatic efficacy of GC cells. Tumor sphere formation assay was used to assess tumor stemness maintenance ability. Chromatin immunoprecipitation (ChIP)-qPCR assay was used to evaluate the associations between CBX4 and CDC20. A subcutaneous tumor model was used to assess the in vivo growth ability of GC.ResultsThe MTT assay revealed that only CBX4 inhibition could lead to notable restriction of GC growth, as compared to others. Differential analysis suggested that CBX4 was upregulated in tumor samples relative to normal tissues. Less favorable overall survival (OS) outcomes were noticed in GC patients with high CBX4 in comparison to those with low CBX4. High CBX4 could notably enhance cell proliferation capacity, migration ability, and in vivo metastatic efficacy. Gene set enrichment analysis (GSEA) indicated the relationships between CBX4 and GC stemness, and CBX4 overexpression could remarkably elevate self-renewal ability of GC cells. In addition, CBX4 could mainly promote CDC20 messenger RNA (mRNA) levels, and targeting CBX4 suppressed the relative CDC20 levels. The ChIP-qPCR assay further demonstrated that CBX4 coordinated with H3K4me3 to bind at the CDC20 promoter region. Additionally, CBX4 depended on CDC20 to drive GC growth. Lastly, downregulated CBX4 could notably inhibit the growth of GC in vivo.ConclusionsThis study highlights the oncogenic roles of CBX4 in GC. CBX4 activates CDC20 to maintain stemness features of GC, thereby creating therapeutic vulnerabilities in the treatment of GC.     

FRNK抑制人乳腺癌细胞体外增殖及机制研究

目的：研究黏着斑相关非激酶（focal adhesion kinase related non—kinase，FRNK）对人乳腺癌MCF-7细胞增殖的抑制作用及相关机制。方法：通过RT—PCR方法克隆目的基因FRNK，构建pcDNA3．1-FRNK重组质粒；经脂质体分别介导重组质粒（pcDNA3．1-FRNK）、阳性对照质粒（pcDNA3．1-GFP）和空质粒（pcDNA3．1）转染MCF-7细胞，以正常MCF-7细胞作为空白对照；通过检测转染后细胞FRNK蛋白的表达，并结合转染pcDNA3．1-GFP后细胞表达荧光的多寡来评估转染效率；采用MTT法研究转染后12、24、48和72h各时间点细胞的增殖情况；转染质粒48h后，采用流式细胞术分析MCF-7细胞周期，Westernblot法检测细胞核内NF-κBp65的表达。结果：pcDNA3．1-FRNK质粒可经脂质体介导高效转染MCF-7细胞，促进细胞FRNK的表达，FRNK表达量在转染后48h达高峰；转染pcDNA3．1-FRNK后，MCF-7细胞增殖趋缓，且这种抑制增殖呈一定的时间依赖性；转染FRNK基因后，S＋G2／M期的细胞比例较正常细胞显著下降（P〈0．05）；转染pcDNA3．1-FRNK后MCF-7细胞核内NF-teBp65蛋白表达减少。结论：FRNK可抑制MCF-7细胞增殖，其抑制作用与下调NF-κBp65核易位相关。