Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling

The Focal Adhesion Kinase Suppresses Transformation-associated, Anchorage-independent Apoptosis in Human Breast Cancer Cells: INVOLVEMENT OF DEATH RECEPTOR-RELATED SIGNALING PATHWAYS

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway

FAK overexpression and p53 mutations are highly correlated in human breast cancer

Focal Adhesion Kinase (FAK) is overexpressed in a number of tumors, including breast cancer. Another marker of breast cancer tumorigenesis is the tumor suppressor gene p53 that is frequently mutated in breast cancer. In the present study, our aim was to find a correlation between FAK overexpression, p53 expression and mutation status in a population-based series of invasive breast cancer tumors from the Carolina Breast Cancer Study. Immunohistochemical analyses of 622 breast cancer tumors revealed that expression of FAK and p53 were highly correlated (P = 0.0002) and FAK positive tumors were 1.8 times more likely to be p53 positive compared to FAK negative tumors [odds ratio (OR) = 1.8; 95% Confidence Interval (CI) 1.2 – 2.8, adjusted for age, race and stage at diagnosis]. Tumors positive for p53 expression showed higher intensity of FAK staining (P<0.0001) and higher percent of FAK positive staining (P<0.0005). From the same study, we evaluated 596 breast tumors for mutations in the p53 gene, using SSCP (single strand conformational polymorphism) and sequencing. Statistical analyses were performed to determine the correlation between p53 mutation status and FAK expression in these tumors. We found that FAK expression and p53 mutation were positively correlated (P<0.0001) and FAK positive tumors were 2.5 times more likely to be p53 mutation positive compared to FAK negative tumors [adjusted OR = 2.5, 95% CI 1.6–3.9]. This is the first analysis demonstrating a high correlation between FAK expression and p53 mutations in a population-based series of breast tumors

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation <it>in vitro</it>, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD <it>in vivo</it>, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.</p> <p>Results</p> <p>Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability <it>in vitro </it>and inhibited tumorigenesis <it>in vivo</it>. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.</p> <p>Conclusion</p> <p>Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis <it>in vivo </it>and reveal specific expression of genes affected by silencing of FAK.</p

Predicting Breast Cancer Response to Neoadjuvant Chemotherapy Using Pretreatment Diffuse Optical Spectroscopic-Texture Analysis

Purpose: Diffuse optical spectroscopy (DOS) has been demonstrated capable of monitoring response to neoadjuvant chemotherapy (NAC) in locally advanced breast cancer (LABC) patients. In this study, we evaluate texture features of pre-treatment DOS functional maps for predicting LABC response to NAC. Methods: LABC patients (n = 37) underwent DOS-breast imaging before starting neoadjuvant chemotherapy. Breast-tissue parametric maps were constructed and texture analyses were performed based on grey level co-occurrence matrices (GLCM) for feature extraction. Ground-truth labels as responders (R) or non-responders (NR) were assigned to patients based on Miller-Payne pathological response criteria. The capability of DOS-textural features computed on volumetric tumour data before the start of treatment (i.e. “pre-treatment”) to predict patient responses to NAC was evaluated using a leave-one-out validation scheme at subject level. Data were analysed using a logistic regression, naïve Bayes, and k-nearest neighbour (k-NN) classifiers. Results: Data indicated that textural characteristics of pre-treatment DOS parametric maps can differentiate between treatment response outcomes. The HbO2-homogeneity resulted in the highest accuracy amongst univariate parameters in predicting response to chemotherapy: sensitivity (%Sn) and specificity (%Sp) were 86.5 and 89.0%, respectively and accuracy was 87.8%. The highest predictors using multivariate (binary) combination features were the Hb-Contrast + HbO2-Homogeneity which resulted in a %Sn/%Sp = 78.0/81.0% and an accuracy of 79.5%. Conclusions: This study demonstrated that pre-treatment tumour DOS-texture features can predict breast cancer response to NAC and potentially guide treatments

TAE226-induced apoptosis in breast cancer cells with overexpressed Src or EGFR.

Focal adhesion kinase, FAK is a 125 kDa nonreceptor tyrosine kinase that localizes to focal adhesions. FAK is overexpressed in human tumors and regulates cellular adhesion and survival signaling. We have shown previously that the dominant-negative FAK, C-terminal FAK-CD, caused detachment and apoptosis in human breast cancer cells, and that overexpression of an activated form of Src tyrosine kinase or epidermal growth factor receptor, EGFR, suppressed FAK-CD induced apoptotic effects in breast cancer cells. In the present study, we studied the effect of a novel FAK inhibitor, TAE226 (Novartis, Inc.), on the breast cancer cell lines. We used stable breast cancer cell lines overexpressing Src (MCF-7-Src and BT474-Src) or overexpressing EGFR (BT474-EGFR), and control breast cancer cell lines for the treatment with different doses of TAE226 drug. The detachment and apoptosis caused by TAE226 was analyzed and compared with the effect of the dominant-negative adenoviral FAK-CD. The TAE226 drug caused a dose-dependent increase of detachment and apoptosis in both BT474 and MCF-7-Vector and Src cells and in BT474-EGFR and BT474-pcDNA3 cells. Additionally, TAE226 caused downregulation of Y397-FAK, FAK and activation of PARP or caspase-3 proteins. Both Src and EGFR-overexpressing cells were not resistant to the TAE226 treatment compared to FAK-CD treatment. In addition, normal breast MCF-10A cell line was resistant to both TAE226 drug and to the Ad-FAK-CD inhibitor. Thus, inhibition of autophosphorylation activity of FAK with the TAE226 inhibitor at 10-20 microM is effective in causing apoptosis in breast cancer cells, resistant to the Ad-FAK-CD inhibitor that can be used effectively in therapy

Focal adhesion kinase and cancer

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that resides at the sites of integrin clustering, known as focal adhesions. The FAK protein has a molecular mass of 125kDa and is encoded by the FAK gene located on human chromosome 8q24. Structurally, FAK consists of an amino-terminal regulatory FERM domain, a central catalytic kinase domain, two proline-rich motifs, and a carboxy-terminal focal adhesion targeting domain. FAK has been shown to be an important mediator of cell growth, cell proliferation, cell survival and cell migration, all of which are often dysfunctional in cancer cells. Our lab was the first to isolate FAK from primary human tissue and link it to the process of tumorigenesis. We analyzed FAK mRNA expression in normal, invasive and metastatic human tissues and demonstrated through Northern blot analysis that normal tissues had very low levels of FAK mRNA while primary and metastatic tumors significantly overexpressed FAK. We also demonstrated and confirmed FAK overexpression in colorectal carcinoma and liver metastases with real-time PCR. In this review we summarized immunohistochemical data of FAK expression and role in different cancer types tumors and discussed FAK inhibition therapy approaches