Focal Adhesion Kinase mRNA Levels Research Articles

Abstract 5805: Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase

Abstract Triple-negative breast cancer (TNBC) exhibits an aggressive behavior associated with poor prognosis due to the absence of established molecular targets. Focal adhesion kinase (FAK) is a major determinant and participates in the acquisition of migration and invasion, as well as the maintenance of breast cancer stem cell (BCSC)-like traits in TNBC. We sought to investigate the effect of ebastine, a second-generation antihistamine on apoptosis, FAK activation, BCSC subpopulations and metastasis in TNBC in vitro and in vivo. TCGA dataset analysis revealed that mRNA levels of FAK were highly expressed in TNBC compared to other breast cancer subtypes. We found that ebastine binds to the tyrosine kinase domain of FAK, which blocks phosphorylation at the Y397 and Y576/577 residues and subsequent inactivation of SRC. Ebastine-induced apoptosis was associated with attenuation of JAK2/STAT3 and MEK/ERK signaling in TNBC cells. Kinetic analysis revealed a concentration-dependent impairment of cell migration in the presence of ebastine in MDA-MB-231 and 4T1 cells in vitro. Ebastine targets BCSC-like cell populations as evidenced by a sharp decline in the expression of the BCSC markers ALDH1, CD44 and CD49f and suppression of mammosphere-forming capacity. Ebastine administration led to a significant reduction in the growth ebastine of BCSC-enriched 4T1 mammospheres orthotopically injected into the mammary glands of Balb/c mice. Ebastine administration significantly impeded angiogenesis and distant metastasis while reducing MMP-2/-9 levels in circulating blood in vivo. Importantly, biochemical analysis in mice serum showed that ebastine had no effect on liver and kidney function. Our findings suggest that EBA may be an effective therapeutic repositioning candidate for the simultaneous targeting of multiple survival signaling pathways for the treatment of molecularly heterogeneous TNBC. Further investigation of ebastine as an anti-metastatic agent for the treatment of TNBC is warranted. Citation Format: Juyeon Seo, Minsu Park, Dongmi Ko, Seongjae Kim, Soeun Park, Kee Dal Nam, Yong Koo Kang, So Ra Seuk, Jaeyoun Park, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5805.

IL-1β enhances cell adhesion through laminin 5 and β4 integrin in gingival epithelial cells.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6β4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1β induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin β3, β4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1β (1 ng/mL), and IL-1β, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1β. After IL-1β stimulation, colocalization of laminin 5 and β4 integrin was increased after 1 h, colocalization of β4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of β4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1β treatment (3 h) delayed wound healing. These results suggest that IL-1β enhances cell adhesion by altering localization of epithelial adhesion molecules.

Abstract 1455: FAK overexpression upregulates glucose metabolism in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive brain cancer, afflicting nearly 1 in 6 adult patients with brain tumors. Currently, no effective curative treatment for this CNS neoplasm exists. The high prevalence and dismal prognosis of GBM increases the need to identify novel therapeutic targets. Previous research has suggested that increased expression of focal adhesion kinase (FAK) may play a role in tumor metabolism. Our preliminary data using datamining approach of the TCGA and Oncomine™ databases showed that mRNA levels of FAK correlate with expression levels of the glucose transporter GLUT1. Subsequent mass-spectrometric and immunoprecipitation experiments provided evidence that FAK binds to GLUT1, while immunohistochemistry and immunocytochemistry experiments confirmed their interaction. Western blot analysis using shRNA to silence FAK demonstrated a significant decrease in GLUT1 expression. Cells treated with the same FAK shRNA showed decreased metabolites from anaerobic glycolysis, instead relying on oxygen-dependent metabolism utilizing oxidative phosphorylation for their energy supply. Collectively, we propose that FAK is a viable target in modulating glucose uptake and metabolism in GBM. Citation Format: Kiran Kumar Velpula, Michael Pajor, Maheedhara R. Guda, Collin M. Labak, Simon Park, Andrew J. Tsung. FAK overexpression upregulates glucose metabolism in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1455.

Expression and Clinical Significance of Focal Adhesion Kinase in Patients with Acute Leukemia

To explore the expression of focal adhesion kinase (FAK) in patients with acute leukemia (AL) and its clinical significance. The FAK mRNA levels in leukemic cells of 50 patients with AL and bone marrow mononuclear cells(BMMNC) of 10 healthy controls were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The bone marrow cell chromosomes were prepared using direct and short term culture, and karyotyping was performed by R-banding technique. The expression positive rate of FAK mRNA in leukemic cells of 50 patients with AL was 66.0%, which was significantly higher than that in normal controls (20.0%) (P<0.05). The expression positive rates and levels of FAK mRNA in both ALL and ANLL cells were statistically significantly higher than those in normal controls (P<0.05), but the difference between ALL and ANLL cells was not significant (P>0.05). All the expression- positive rates and levels of FAK mRNA in newly diagnosed AL patients and the relapsed AL patients were significantly higher than those in normal controls (P<0.05), but the difference between the newly diagnosed AL patients and the relapsed AL patients was not significant (P>0.05). Morecover, the expression plsitive rate and levels of FAK mRNA in the remitted patients were lower than those in newly diagnosed and relapsed patients (P<0.05), but were no statistically significantly different from normal controls (P>0.05). The expression- positive rate and level of FAK mRNA in the AL patients with abnormal karyotype were significantly higher than those in the AL patients without abnormal karyotype (P<0.05). FAK mRNA over-express in the leukemia patients, which may be related with the outome of disease, possibly providing novel targeting sites for the treatment of leukemia.

FAK regulates E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 melanoma cells.

Focal adhesion kinase (FAK) is involved in tumor cell migration and metastasis. However, the underlying mechanism remains unclear. Here, we present a signaling pathway involved in the regulation of melanoma cell migration by FAK. We found that the interference of FAK expression suppressed B16F10 cell migration/metastasis, and altered the expressions of genes involved in melanoma migration/metastasis. The down-regulation of FAK inhibited the expression of p-SrcY416, p-ERK1/2, Stat3 and p-Stat3Y705, while promoted the expression of PPARγ, miR-125b and E-cadherin. Then we found that FAK inhibited E-cadherin expression via p-SrcY416/p-ERK1/2/ p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 cell. Moreover, miR-125b inhibited B16F10 cell migration. Furthermore, we repeated the key data with human melanoma cell line A375. The results obtained from A375 cells fell in line with those from B16F10 cells. Using Oncomine database, we found that the mRNA levels of FAK, Src, ERK1/2 and Stat3 increased, while the mRNA levels of PPARγ, C21orf34 (miR-125b host gene) and E-cadherin decreased in human metastatic melanoma. The data from human breast cancer confirmed those from metastatic melanoma.Taken together, our study suggests that down-regulation of FAK promotes E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway. Our findings provide a novel explanation regarding how FAK promotes melanoma cell migration, suggesting that FAK might be a potential target for melanoma therapy.

MicroRNA-543 suppresses endometrial cancer oncogenicity via targeting FAK and TWIST1 expression

Focal adhesion kinase (FAK) is a critical mediator of extracellular matrix signaling, cell survival, proliferation and motility. Twist homolog 1 (TWIST1) is a transcription factor and serves as a powerful oncogene. Increased FAK and TWIST1 expression are observed in a variety of solid human tumors and correlate with metastasis and poor survival. Here, we identify miR-543 as a direct regulator of FAK and TWIST1 expression in endometrial cancer. Forced expression of miR-543 in endometrial cancer cell lines decreases both endogenous FAK and TWIST1 mRNA and protein levels. Forced expression of miR-543 in aggressive endometrial cancer cell lines also impairs tumor cell monolayer proliferation, anchorage-independent growth, migration and invasion. Endogenous miR-543 expression is decreased in malignant versus normal endometrium tissue and the levels of miR-543 inversely correlate with mRNA levels of FAK and TWIST1. miR-543 expression is decreased in endometrial cancer and serves as a tumor suppressor by targeting FAK and TWIST1 expression.

Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression.

Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the peri-ovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer.

Biological behavior of fibroblast-like cells cultured on anodized-hydrothermally treated titanium with a nanotopographic surface structure

PurposeThe interface between the transmucosal portion of endosseous implants surface and the connective tissue is characterized by fibroblast-rich barrier tissue, which is important for the long-term stability and maintenance of the implant. This study investigated the effect of cell adhesion on focal adhesion kinase (FAK) protein and on gene expression over a 72-h culture period. Fibroblast-like cells were cultured on anodized-hydrothermally treated commercially pure titanium with nanotopographic structure (SA-treated c.p.Ti) surfaces. MethodsMurine fibroblast-like NIH/3T3 cells were cultured for 10–72h on c.p.Ti, anodic oxide (AO) c.p.Ti, and SA-treated c.p.Ti disks. Cell morphology was analyzed using scanning electron microscopy (SEM). Cytoskeletal structure and FAK protein localization were analyzed using confocal laser scanning microscopy (CLSM). FAK mRNA levels were analyzed using real-time quantitative RT-PCR. ResultsSEM and CLSM showed increased NIH/3T3 cell adhesion with time, and actin filaments oriented parallel with the filopodium-like extensions on all disks. Filopodium-like extensions were bound tightly to the nanotopographic structure surface of cultures on SA-treated c.p.Ti, and especially at 72h. FAK protein was localized along cellular extensions on SA-treated c.p.Ti and the expression of FAK mRNA was significantly higher on these disks than on c.p.Ti and AO c.p.Ti after 72h (P<0.05). ConclusionsNIH/3T3 fibroblast-like cells have the capacity to adhere to SA-treated c.p.Ti as a transmucosal portion of implant surface material and express focal adhesion molecules, which may play a key role in the maintenance of a mucosal tissue barrier

The Role of Focal Adhesion Kinase in the TGF-β-Induced Myofibroblast Transdifferentiation of Human Tenon's Fibroblasts

PurposeTo investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-β-induced myofibroblast transdifferentiation of human Tenon's fibroblasts.MethodsPrimary cultured human Tenon's fibroblasts were exposed to TGF-β1 for up to 48 hours. The mRNA levels of FAK, α smooth muscle actin (αSMA), and β-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, αSMA, and β-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNAFAK) molecules were delivered into the cells, the expressions of αSMA proteins were determined by Western immunoblots.ResultsIn human Tenon's fibroblasts, TGF-β1 significantly increased the mRNA and protein expressions of αSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-β1-induced expression of αSMA was attenuated.ConclusionsOur data suggest that FAK may be associated with the TGF-β1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.

Expression of paxillin and FAK mRNA and the related clinical significance in esophageal carcinoma

The objective of the present study was to investigate the expression of paxillin and focal adhesion kinase (FAK) mRNA in esophageal carcinoma tissues, and their relationship with clinicopathological parameters, as well as to analyze the correlation of paxillin and FAK mRNA levels in esophageal carcinoma. By using reverse transcription polymerase chain reaction (RT-PCR), the mRNA expression levels of paxillin and FAK were detected in 121 samples of esophageal carcinoma, 43 samples of atypical hyperplasia and 56 samples of normal esophageal mucosa. The results showed that the positive rates of paxillin and FAK mRNA expression in esophageal carcinoma were 87.6 and 80.17%, respectively, which were significantly higher (P<0.05) than those in atypical hyperplasia (44.19 and 39.53%) and normal esophageal mucosa (5.36 and 12.5%). Notably, paxillin and FAK mRNA expression levels were significantly correlated with the differentiation degree and depth of invasion of esophageal carcinoma and with lymph node metastasis (P<0.05). In addition, paxillin and FAK mRNA expression levels in esophageal carcinoma were positively correlated (r=0.4804, P=0.000). In conclusion, the combined detection of paxillin and FAK mRNA expression is expected to provide a theoretical basis for the molecular diagnosis of esophageal carcinoma.

Expression and prognostic significance of focal adhesion kinase in hepatocellular carcinoma

To examine the expressions of focal adhesion kinase (FAK) and its clinical significance in hepatocellular carcinoma (HCC). We determined the expression levels of FAK on both steady-state mRNA and protein levels in 50 HCC samples by quantitative real-time PCR and immunohistochemistry, respectively. The correlations between FAK expression and various clinicopathological parameters were analyzed. The expression of FAK on the mRNA level was consistent with that on the protein level. FAK mRNA levels in tumor tissues were significantly higher than in the paratumor tissues (0.229 +/- 0.027 vs. 0.163 +/- 0.019; P < 0.001), but lower than in the macroscopic cancer emboli (0.506 +/- 0.155 vs. 0.377 +/- 0.176; P < 0.05). Compared within the tumor tissues, FAK expressions were significantly higher in those with cancer emboli than those without (0.343 +/- 0.05 vs. 0.165 +/- 0.025; P = 0.003). Univariate and multivariate analyses revealed that FAK expression was an independent prognostic factor for disease-free survival and overall survival. Both 3-year disease-free survival rates and overall survival rates in FAK-negative group were significantly higher than in positive group (52 vs. 20% and 72 vs. 29%, P < 0.001). Our results suggest that FAK expression is up-regulated in HCC and its expression is an independent prognostic factor for HCC.

Small GTPase Ras and Rho Expression in Rat Osteoblasts during Spaceflight

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1alpha,25 dihydroxyvitamin D(3). The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-beta, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-gamma (PLC-gamma) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-gamma1 and -gamma2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein-coupled receptors are transmitted through PLC-beta and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-beta were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein-coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts.

Up-regulation of focal adhesion kinase in non-small cell lung cancer

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase linked to the integrin and growth factor receptor-signalling pathways that regulates a number of the biological processes involved in neoplastic transformation, invasion and metastases, such as cell adhesion, migration and apoptosis. Its up-regulation might play a role in the tumourigenesis of invasive tumours, but its involvement in human lung cancer tissues has not yet been determined. We immunohistochemically compared FAK expression and localisation in 60 formalin-fixed and paraffin-embedded non-small cell lung cancer (NSCLC) tissues with that in the surrounding non-neoplastic tissue and in a further five microscopically normal lungs. FAK mRNA levels were quantitatively determined by real-time RT-PCR in frozen tissue specimens of all of the tumours and 21 matched non-neoplastic lung parenchymas, and protein expression in 16 homogenates of the matched neoplastic/non-neoplastic specimens was evaluated by Western blotting. The three different techniques showed that FAK is weakly expressed in non-neoplastic lung parenchyma and up-regulated in NSCLCs. Moreover, Western blotting and real-time RT-PCR indicated a statistically significant correlation between FAK up-regulation and higher disease stages (I+II versus III+IV, p=0.019 and 0.028, respectively). Our results provide evidence that FAK is up-regulated in NSCLCs, and suggest its potential involvement in lung cancer progression.

Overexpression of Focal Adhesion Kinase in Head and Neck Squamous Cell Carcinoma Is Independent of fak Gene Copy Number

The development of human malignancies can involve the aberrant regulation of intracellular signal transduction pathways that regulate cell-extracellular matrix interactions. In the current study, we aimed to evaluate focal adhesion kinase (FAK) at both genetic and protein expression levels in head and neck squamous cell carcinomas (HNSCC) and to explore the prognostic significance of FAK. A total of 211 tissue specimens, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, were analyzed using immunohistochemistry. The fak gene dosage was determined in 33 tumors. Correlations among DNA, protein, and clinicopathologic variables were analyzed. FAK protein was overexpressed in HNSCCs compared with corresponding normal mucosa. High expression levels were found in 62% of the samples. Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions. All lymph node metastases examined showed FAK overexpression, with significant correlation with the expression in matched primary tumor. DNA copy number ratios for fak were higher in 39% of the tumors compared with normal mucosa. However, elevated FAK expression did not correlate with gains on DNA level, and not all cases with an amplification of the fak gene displayed protein overexpression. Similar data were obtained in five HNSCC-derived cell lines, in which FAK mRNA levels were precisely correlated with FAK protein levels. FAK protein overexpression in tumors correlated with nodal metastases. These findings suggest an involvement of FAK in the onset and progression of HNSCC and provide an insight into a mechanism of FAK activation alternative to gene amplification.