05-537 Sigma-AldrichAnti-FAK Antibody, clone 4.47

In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.

Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. Vascular endothelial growth factor receptor-3 (VEGFR-3), another tyrosine kinase, has also been found to be important in the development of many human tumors including neuroblastoma. Recent reports have found that FAK and VEGFR-3 interact, and we have previously shown that both of these kinases interact in neuroblastoma. We have hypothesized that interruption of the FAK-VEGFR-3 interaction would lead to decreased neuroblastoma cell survival. In the current study, we examined the effects of a small molecule, chloropyramine hydrochloride (C4), designed to disrupt the FAK-VEGFR-3 interaction, upon cellular attachment, migration, and survival in two human neuroblastoma cell lines. We also utilized a murine xenograft model to study the impact of C4 upon tumor growth. In these studies, we showed that disruption of the FAK-VEGFR-3 interaction led to decreased cellular attachment, migration, and survival in vitro. In addition, treatment of murine xenografts with chloropyramine hydrochloride decreased neuroblastoma xenograft growth. Further, this molecule acted synergistically with standard chemotherapy to further decrease neuroblastoma xenograft growth. The findings from this current study help to further our understanding of the regulation of neuroblastoma tumorigenesis, and may provide novel therapeutic strategies and targets for neuroblastoma and other solid tumors of childhood.

Aberrant activation of Wnt/β-catenin signaling plays an unequivocal role in colorectal cancer, but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge. New insights into the regulation of this pathway could reveal new therapeutic point of intervention, therefore are greatly needed. Here we report a novel FAK/PYK2/GSK3β(Y216)/β-catenin regulation axis: FAK and PYK2, elevated in adenomas in APC(min/+) mice and in human colorectal cancer tissues, functioned redundantly to promote the Wnt/β-catenin pathway by phosphorylating GSK3β(Y216) to reinforce pathway output-β-catenin accumulation and intestinal tumorigenesis. We previously showed that Wnt-induced β-catenin accumulation requires Wnt-induced GSK3β/β-TrCP interaction; the current study revealed that phosphorylation of GSK3β(Y216) was a molecular determinant of GSK3β recruitment of β-TrCP. Pharmacological inhibition of FAK/PYK2 suppressed adenoma formation in APC(min/+) mice accompanied with reduced intestinal levels of phospho-GSK3β(Y216) and β-catenin, indicating that FAK/PYK2/GSK3β(Y216) axis is critical for the activation of Wnt/β-catenin signaling in APC driven intestinal tumorigenesis.

Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development.

Ovarian clear cell carcinoma (OCCC) displays a higher resistance to first line chemotherapy, requiring the development of new therapeutics. We previously identified a frequent chromosomal gain at 8q24 that harbors the focal-adhesion kinase (FAK) gene; the potential of this gene as a therapeutic target remains to be evaluated in OCCCs. We first examined the dependence of OCCCs on FAK and the PI3K/AKT signaling pathway. FAK was overexpressed in 20% of 67 OCCC samples, and this overexpression was correlated with its copy number gain. FAK copy number gains and mutations in PIK3CA accounted for about 40% of OCCC samples, suggesting that the FAK/PI3K/AKT axis is an attractive candidate for targeted therapeutics. We, therefore, treated ovarian cancer cell lines, including OCCC subtypes, with the FAK inhibitors PF-562,271 (PF271), and PF-573,228 (PF228). Ovarian cancer cells were more sensitive to PF271 than PF228. We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271. We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis. RMGI and OVISE cells were more sensitive to ABT-737 than OVMANA and SKOV3 cells, which have PIK3CA mutations. Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway. Therefore, PF271/ABT-737 treatment led to the inhibition of the anti-apoptotic proteins MCL1 and BCL-XL/BCL-2. We suggest that pharmacological inhibition of BCL-XL and FAK/PYK2 can be a potential therapeutic strategy for the treatment of OCCC.

Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and nonosseous renal Ewing sarcoma. Children with advanced, metastatic, or relapsed disease have a poor disease-free survival rate. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading to the hypothesis that FAK contributes to pediatric kidney tumors and would affect cellular survival. In the current study, FAK was present and phosphorylated in pediatric kidney tumor specimens. Moreover, the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines were examined using a number of parallel approaches to block FAK, including RNA interference and small-molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Furthermore, small-molecule inhibition of FAK led to decreased SK-NEP-1 xenograft growth in vivo. These data deepen the knowledge of the tumorigenic process in pediatric renal tumors, and provide desperately needed therapeutic strategies and targets for these rare, but difficult to treat, malignancies.This study provides a fundamental understanding of tumorigenesis in difficult to treat renal tumors and provides an impetus for new avenues of research and potential for novel, targeted therapies.

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.

Focal adhesion Kinase (FAK) is a nonreceptor protein tyrosine kinase that is overexpressed in tumors and plays a significant role in tumor survival and metastasis. The purpose of the study is to perform correlation of FAK expression with patient prognostic factors using tissue microarrays (TMA) samples.We analyzed FAK expression by immunohistochemical staining in 196 breast primary tumor samples from stage II-IV patients and in 117 metastatic tissues matched to the primary tumors using TMA that were stained with FAK monoclonal antibody.High FAK expression in primary tumors was associated with a younger age of patients (p = 0.033), lymphovascular invasion (p = 0.001) and with the triple-negative phenotype (p = 0.033). FAK expression in 117 metastatic tissues positively correlated with FAK expression in matched primary tumors by Spearman correlation analysis. In addition, a strong positive correlation was observed between high FAK expression and shorter overall survival and progression free survival in patients with metastatic tumors.The data demonstrate a high potential for FAK as a therapeutic target, especially in triple-negative breast cancer patients with high FAK expression.

Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

Neuroblastoma, the most common extracranial solid tumor of childhood, is responsible for over 15 % of pediatric cancer deaths. We have shown that neuroblastoma cell lines overexpress focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase that controls a number of tumorigenic pathways. In this study, we hypothesized that inhibition of FAK would result in decreased cellular migration and invasion in neuroblastoma cell lines, and decrease metastasis in a murine model. We utilized non-isogenic and isogenic MYCN human neuroblastoma cell lines and parallel methods of FAK inhibition. Cell viability, migration, and invasion assays were employed to assess the effects of FAK inhibition in vitro. A nude mouse model was utilized to determine the effects of FAK inhibition on in vivo liver metastasis. FAK knockdown with siRNA resulted in decreased invasion and migration in neuroblastoma cell lines, and the effects of siRNA-induced FAK inhibition were more pronounced in MYCN amplified cell lines. In addition, abrogation of FAK with a small molecule inhibitors resulted in decreased cell survival, migration and invasion in neuroblastoma cell lines, again most pronounced in cell lines with MYCN amplification. Finally, small molecule FAK inhibition in a nude mouse model resulted in a significant decrease in metastatic tumor burden in SK-N-BE(2) injected animals. We believe that FAK plays an important role in maintaining and propagating the metastatic phenotype of neuroblastoma cells, and this driver role is exaggerated in cell lines that overexpress MYCN. FAK inhibition warrants further investigation as a potential therapeutic target in the treatment of aggressive neuroblastoma.