FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

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

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

Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells

Focal adhesion kinase (pp125FAK) is present at sites of cell/extracellular matrix adhesion and has been implicated in the control of cell behaviour. In particular, as a key component of integrin-stimulated signal transduction pathways, pp125FAK is involved in cellular processes such as spreading, motility, growth and survival. In addition, a number of reports have indicated that pp125FAK may be up-regulated in human tumour cells of diverse origin, and consequently, a role has been proposed for pp125FAK in the development of invasive cancers. However, to date the mechanisms that lead to elevated pp125FAK expression in tumour cells have not been determined. Here we used in situ hybridization to confirm chromosome 8q as the genomic location of the human fak gene and report that elevation of pp125FAK protein in cell lines derived from invasive squamous cell carcinomas is accompanied by gains in copy number of the fak gene in all cases examined. In addition, we observed increased fak copy number in frozen sections of squamous cell carcinomas. Furthermore, increased dosage of the fak gene was also observed in many cell lines derived from human tumours of lung, breast and colon, including two cell lines Calu3 and HT29, in which fak was amplified. In addition, in an in vitro model for human colon cancer progression there was a copy number gain of the fak gene during conversion from adenoma to carcinoma, which was associated with increased pp125FAK protein expression. Thus, we show for the first time that many cell lines derived from invasive epithelial tumours have increased dosage of the fak gene, which may contribute to the elevated protein expression commonly observed. Although other genes near the fak locus are co-amplified or increased in copy number, including the proto-oncogene c-myc, the biological properties of pp125FAK in controlling the growth, survival and invasiveness of tumour cells, suggest that it may contribute to the selection pressure for maintaining increased dosage of the region of chromosome 8q that encodes these genes