Inhibition of FAK and VEGFR-3 binding decreases tumorigenicity in neuroblastoma.

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.

Targeting the C-terminal focal adhesion kinase scaffold in pancreatic cancer.

Preliminary studies in our laboratory have demonstrated the importance of both the NH2 and COOH terminus scaffolding functions of focal adhesion kinase (FAK). Here, we describe a new small molecule inhibitor, C10, that targets the FAK C-terminus scaffold. C10 showed marked selectivity for cells overexpressing VEGFR3 when tested in isogenic cell lines, MCF7 and MCF7-VEGFR3. C10 preferentially inhibited pancreatic tumor growth in vivo in cells with high FAK-Y925 and VEGFR3 expression. Treatment with C10 led to a significant inhibition in endothelial cell proliferation and tumor endothelial and lymphatic vessel density and decrease in interstitial fluid pressure. These results highlight the underlying importance of targeting the FAK scaffold to treat human cancers.

Design, synthesis, and biological evaluation of novel FAK scaffold inhibitors targeting the FAK-VEGFR3 protein-protein interaction.

Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK-VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK-VEGFR3 protein-protein interaction inhibitors.

Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

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.

Inhibition of focal adhesion kinase and src increases detachment and apoptosis in human neuroblastoma cell lines.

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that is overexpressed in a number of human tumors including neuroblastoma, and regulates both cellular adhesion and survival. We have studied the effects of FAK inhibition upon neuroblastoma using adenovirus-containing FAK-CD (AdFAK-CD). Utilizing an isogenic MYCN+/MYCN- neuroblastoma cell line, we found that the MYCN+ cells are more sensitive to FAK inhibition with AdFAK-CD than their MYCN negative counterparts. In addition, we have shown that phosphorylation of Src is increased in the untreated isogenic MYCN- neuroblastoma cells, and that the decreased sensitivity of the MYCN- neuroblastoma cells to FAK inhibition with AdFAK-CD is abrogated by the addition of the Src family kinase inhibitor, PP2. The results of the current study suggest that both FAK and Src play a role in protecting neuroblastoma cells from apoptosis, and that dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor.

Small molecule chloropyramine hydrochloride (C4) targets the binding site of focal adhesion kinase and vascular endothelial growth factor receptor 3 and suppresses breast cancer growth in vivo.

FAK is a tyrosine kinase that functions as a key orchestrator of signals leading to invasion and metastasis. Since FAK interacts directly with a number of critical proteins involved in survival signaling in tumor cells, we hypothesized that targeting a key protein-protein interface with druglike small molecules was a feasible strategy for inhibiting tumor growth. In this study, we targeted the protein-protein interface between FAK and VEGFR-3 and identified compound C4 (chloropyramine hydrochloride) as a drug capable of (1) inhibiting the biochemical function of VEGFR-3 and FAK, (2) inhibiting proliferation of a diverse set of cancer cell types in vitro, and (3) reducing tumor growth in vivo. Chloropyramine hydrochloride reduced tumor growth as a single agent, while concomitant administration with doxorubicin had a pronounced synergistic effect. Our data demonstrate that the FAK-VEGFR-3 interaction can be targeted by small druglike molecules and this interaction can provide the basis for highly specific novel cancer therapeutics.

Vascular endothelial growth factor receptor-3 promotes breast cancer cell proliferation, motility and survival in vitro and tumor formation in vivo.

Vascular endothelial growth factor receptor-3 is a receptor tyrosine kinase that is overexpressed in some human carcinomas, but its role in tumorigenesis has not been fully elucidated. We examined VEGFR-3 expression in normal, nonneoplastic and early stage malignant breast tissues and have shown that VEGFR-3 upregulation in breast cancer preceded tumor cell invasion, suggesting that VEGFR-3 may function as a survival signal. We characterized the biological effects of VEGFR-3 over-expression in human breast cancer cells based on two approaches: gain of function by overexpressing VEGFR-3 in MCF-7 breast cancer cells and loss of function by RNAi-mediated silencing of VEGFR-3 in MCF-7-VEGFR-3 and BT474 cells. VEGFR-3 overexpression increased cellular proliferation by 40% when MCF7-VEGFR-3 cells were compared to parental MCF7 cells, and proliferation was reduced by more than 40% when endogenous VEGFR-3 was downregulated in BT474 cells. VEGFR-3 overexpression promoted a three-fold increase in motility and invasion and both motility and invasion were inhibited by downregulation of VEGFR-3. Furthermore, VEGFR-3 overexpression promoted cellular survival under stress conditions induced by staurosporine treatment and led to anchorage-independent growth. VEGFR-3 overexpression dramatically increased tumor formation in both hormone-dependent and independent xenograft models. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger than control xenografts. Finally, downregulation of VEGFR-3 expression in both xenograft model cell lines led to a significant reduction of tumor growth. For the first time, we have demonstrated that VEGFR-3 overexpression promotes breast cancer cell proliferation, motility, survival, anchorage-independent growth and tumorogenicity in the absence of ligand expression.

Crystallization of the focal adhesion kinase targeting (FAT) domain in a primitive orthorhombic space group.

X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 A resolution and reduced to the primitive orthorhombic lattice. A single molecule was predicted to be present in the asymmetric unit based on the Matthews coefficient. The data were phased using molecular-replacement methods using an existing model of the FAK FAT domain. All structures of human focal adhesion kinase FAT domains solved to date have been solved in a C-centered orthorhombic space group.

Focal adhesion kinase expression in human neuroblastoma: immunohistochemical and real-time PCR analyses.

PURPOSE: The focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase important in signaling between cells and their extracellular matrix. Studies have shown that FAK expression is up-regulated in several human tumors and is related to tumor progression. We recently found an increase in p125(FAK) expression in human neuroblastoma cells lines and wished to determine its expression in human neuroblastoma specimens and evaluate for a possible correlation between p125(FAK) expression and known prognostic factors for neuroblastoma. We hypothesized that p125(FAK) expression would be up-regulated in advanced human neuroblastomas. EXPERIMENTAL DESIGN: Using immunohistochemical techniques with monoclonal antibody 4.47 specific for p125(FAK) expression, we analyzed 70 formalin-fixed, paraffin-embedded human neuroblastoma specimens for p125(FAK) staining. In addition, real-time PCR was used to determine the abundance of FAK mRNA in 17 matched human neuroblastoma mRNA specimens. RESULTS: FAK staining was present in 51 of the 70 tumor specimens (73%). Immunohistochemical staining of p125(FAK) in the ganglion-type tumor cells correlated with advanced International Neuroblastoma Staging System tumor stages and FAK mRNA abundance. In addition, p125(FAK) staining was significantly increased in stage IV tumors with amplification of the N-MYC oncogene. CONCLUSIONS: These novel findings provide evidence that FAK is expressed by advanced-stage neuroblastoma and provide a rationale for targeting FAK in the treatment of this tumor.

FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells.

Pancreatic cancer is a lethal disease accounting for the fourth leading cause of cancer death in USA. Focal adhesion kinase (FAK) and the insulin-like growth factor-I receptor (IGF-1R) are tyrosine kinases that activate common pathways, leading to increased proliferation and cell survival. Sparse information is available regarding their contribution to the malignant behavior of pancreatic cancer. We analyzed the relationship between FAK and IGF-1R in human pancreatic cancer cells, determined which downstream signaling pathways are altered following kinase inhibition or downregulation and studied whether dual kinase inhibition represents a potential novel treatment strategy in this deadly disease. Using immunoprecipitation and confocal microscopy, we show for the first time that FAK and IGF-1R physically interact in pancreatic cancer cells and that inhibition of tyrosine phosphorylation of either kinase disrupts their interaction. Decreasing phosphorylation of either FAK or IGF-1R alone resulted in little inhibition of cell viability or increased apoptosis. However, dual inhibition of FAK, using either a dominant-negative construct (FAK-CD) or small interfering RNA, and IGF-1R, using a specific small molecule tyrosine kinase inhibitor (AEW-541) or stable expression of a truncated, mutated IGF-1R, led to a synergistic decrease in cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) and increase in cell detachment and apoptosis compared with inhibition of either pathway alone. Dual kinase inhibition with FAK-CD and AEW-541 resulted in a marked increase in apoptosis when FAK was displaced from the focal adhesions. Inhibition of both tyrosine kinase activities via a novel single small molecular inhibitor (TAE 226), at low doses specific for FAK and IGF-1R, resulted in significant inhibition of cell viability, decrease in phosphorylation of ERK and Akt and increase in apoptosis accompanied by cleavage of Poly (ADP-ribose) polymerase (PARP) and activation of caspase-3 in pancreatic cancer cells. Thus, simultaneous inhibition of both tyrosine kinases represents a potential novel therapeutic approach in human pancreatic adenocarcinoma.

TAE226 inhibits human neuroblastoma cell survival.

PURPOSE: Neuroblastoma is one of the most devastating pediatric solid tumors and is unresponsive to many interventions. TAE226 is a novel small molecule FAK inhibitor. We investigated the effects of TAE226 on neuroblastoma cells in vitro. MATERIALS AND METHODS: Human neuroblastoma cell lines were treated with varying concentrations of TAE226. Following treatment, cell viability, cell cycle, and apoptosis were evaluated. RESULTS: Treatment of human neuroblastoma cell lines with TAE226 resulted in a concentration dependent decrease in FAK phosphorylation, decrease in cellular viability, cell cycle arrest, and an increase in apoptosis. CONCLUSIONS: Targeting FAK provides potential therapeutic options for the treatment of neuroblastoma and deserves further investigation.

The 7-amino-acid site in the proline-rich region of the N-terminal domain of p53 is involved in the interaction with FAK and is critical for p53 functioning.

It is known that p53 alterations are commonly found in tumour cells. Another marker of tumorigenesis is FAK (focal adhesion kinase), a non-receptor kinase that is overexpressed in many types of tumours. Previously we determined that the N-terminal domain of FAK physically interacted with the N-terminal domain of p53. In the present study, using phage display, sitedirected mutagenesis, pulldown and immunoprecipitation assays we localized the site of FAK binding to a 7-amino-acid region(amino acids 65-71) in the N-terminal proline-rich domain of human p53. Mutation of the binding site in p53 reversed the suppressive effect of FAK on p53-mediated transactivation ofp21, BAX (Bcl-2-associated X protein) and Mdm2 (murine double minute 2) promoters. In addition, to functionally test this p53 site, we conjugated p53 peptides [wild-type (containing the wild-type binding site) and mutant (with a mutated 7-aminoacid binding site)] to a TAT peptide sequence to penetrate the cells, and demonstrated that the wild-type p53 peptide disrupted binding of FAK and p53 proteins and significantly inhibited cell viability of HCT116 p53+/+ cells compared with the control mutant peptide and HCT116 p53-/- cells. Furthermore, the TAT-p53 peptide decreased the viability of MCF-7 cells, whereas the mutant peptide did not cause this effect. Normal fibroblast p53+/+ and p53-/- MEF (murine embryonic fibroblast) cells and breast MCF10A cells were not sensitive to p53 peptide. Thus, for the first time, we have identified the binding site of the p53 andFAK interaction and have demonstrated that mutating this site and targeting the site with peptides affects p53 functioning and viability in the cells.

Shb gene knockdown increases the susceptibility of SVR endothelial tumor cells to apoptotic stimuli in vitro and in vivo.

The Shb adapter protein is an Src homology 2-domain containing signaling intermediate operating downstream of several tyrosine kinase receptors, including vascular endothelial growth factor receptor-2. Shb is multifunctional and apoptosis is one response that Shb regulates. Inhibition of angiogenesis can be used in cancer therapy, and one way to achieve this is by inducing endothelial cell apoptosis. The angiosarcoma cell line SVR is of endothelial origin and can be used as a tool for studying in vivo inhibition of angiogenesis, and we thus employed an Shb-knockdown strategy using an inducible lentiviral system to reduce Shb levels in SVR cells and to study their responses. Shb knockdown increases the susceptibility of SVR cells to the apoptotic agents, cisplatin and staurosporine. Simultaneously, Shb knockdown causes reduced focal adhesion kinase (FAK) activation, monitored as phosphorylation of the regulatory residues tyrosines 576/577. No detectable effects on Akt or extracellular signal-regulated kinase activity were noted. The altered FAK activity coincided with an elongated cell phenotype that was particularly noticeable in the presence of staurosporine. In order to relate the effects of Shb knockdown to in vivo tumorigenicity, cells were exposed to the angiogenesis inhibitor honokiol, and again the cells with reduced Shb content exhibited increased apoptosis. Tumor growth in vivo was strongly reduced in the Shb-knockdown cells upon honokiol treatment. It is concluded that Shb regulates apoptosis and cell shape in tumor endothelial cells via FAK, and that Shb is a potential target for inhibition of angiogenesis.

N-MYC regulates focal adhesion kinase expression in human neuroblastoma.

N-MYC is a transcription factor that plays an important role in cellular survival in neuroblastoma, and amplification of the N-MYC oncogene is the primary adverse prognostic indicator for neuroblastoma. Focal adhesion kinase (FAK) is a survival factor that has been shown to be overexpressed in many types of human cancers. In this study, we investigated the role of N-MYC regulation of FAK expression in neuroblastoma. We first found a correlation between N-MYC and FAK expression in neuroblastoma. Real time quantitative PCR demonstrated an increase in FAK mRNA abundance in the N-MYC-amplified IMR-32 compared with the nonamplified SK-N-AS neuroblastoma cell lines. FAK protein expression also correlated positively with N-MYC expression in the N-MYC-amplified IMR-32 versus nonamplified SK-N-AS neuroblastoma cell lines. The same results were seen with the isogenic N-MYC(+) (Tet(-)) and N-MYC(-) (Tet(+)) neuroblastoma cell lines. Promoter-reporter assays showed that activity of the FAK promoter was increased in the N-MYC-amplified IMR-32 cell line, in the N-MYC-transfected SK-N-AS nonamplified cell line, and in the isogenic N-MYC(+) (Tet(-)) neuroblastoma cell lines compared with the nonamplified and N-MYC-nonexpressing cell lines. We also identified two N-MYC binding sites in the FAK promoter sequence and showed binding of N-MYC transcription factor to the FAK promoter through electrophoretic mobility shift, chromatin immunoprecipitation, and dual luciferase assays. Finally down-regulation of FAK expression in N-MYC-inducible neuroblastoma cell lines with FAK small interfering RNA or a dominant-negative FAK inhibitor (AdFAK-CD) significantly decreased viability and increased apoptosis in the N-MYC(+) (Tet(-)) cells compared with the isogenic N-MYC(-) (Tet(+)) cells, demonstrating the biological significance of FAK overexpression in the N-MYC-expressing cell lines. This is the first report linking N-MYC and FAK in neuroblastoma, and it clearly demonstrates that N-MYC induces FAK expression. The results indicate that N-MYC regulation of FAK expression can control cellular functions in isogenic N-MYC(-/+) (Tet(+/-)) neuroblastoma cell lines.

Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells.

Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in human cancer and play an important role in survival signaling. In addition to its involvement with cell survival, VEGFR-3 is a primary factor in lymphatic angiogenesis. Because FAK function is regulated by its COOH terminus (FAK-CD), we used FAK-CD as a target to identify binding partners. We isolated a peptide from a phage library that bound to FAK-CD, specifically the focal adhesion targeting domain of FAK and was homologous to VEGFR-3, suggesting these two tyrosine kinases physically interact. We have also shown that VEGFR-3 is overexpressed in human breast tumors and cancer cell lines. For the first time, we have shown the physical association of FAK and VEGFR-3. The association between the NH(2) terminus of VEGFR-3, containing the peptide identified by phage display, and the COOH terminus of FAK was detected by in vitro and in vivo binding studies. We then coupled a 12-amino-acid VEGFR-3 peptide, AV3, to a TAT cellular penetration sequence and showed that AV3 and not control-scrambled peptide caused specific displacement of FAK from the focal adhesions and affected colocalization of FAK and VEGFR-3. In addition, AV3 peptide decreased proliferation and caused cell detachment and apoptosis in breast cancer cell lines but not in normal breast cells. Thus, the FAK/VEGFR-3 interaction may have a potential use to develop novel molecular therapeutics to target the signaling between FAK and VEGFR-3 in human tumors.