FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo

Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. 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 hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas.     

A small molecule FAK kinase inhibitor,GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells

Focal adhesion kinase (FAK) hyperactivation is common in pancreatic ductal adenocarcinoma (PDAC). A small molecule, GSK2256098 (GlaxoSmithKline), has been developed to inhibit FAK activity through targeting the phosphorylation site of FAK, tyrosine (Y) 397. We sought to determine whether GSK2256098 inhibition of FAK Y397 phosphorylation attenuates PDAC-associated cell proliferation, motility and survival. Cultured PDAC cells were used as cellular models of GSK2256098-impaired abnormal growth. Western blot analysis, cell viability analysis, clonogenic survival, soft-agar and wound healing assays were performed. The responses of 6 PDAC cell lines in regards to FAK Y397 phosphorylation or activity to GSK2256098 treatments (0.1–10 μM) ranged from low (less than 20% inhibition) to high (more than 90% inhibition). The least and most sensitive cell lines (PANC-1 and L3.6P1) were selected for further analysis. GSK2256098 inhibition of FAK Y397 phosphorylation correlated with decreased levels of phosphorylated Akt and ERK in L3.6P1 cells. GSK2256098 decreased cell viability, anchorage-independent growth, and motility in a dose dependent manner. Current studies demonstrate that small molecule kinase inhibitors targeting FAK Y397 phosphorylation can inhibit PDAC cell growth. Assessments of FAK Y397 phosphorylation in biopsies may be used as a biomarker to select the subgroup of responsive patients and/or monitor the effects of GSK2256098 on FAK-modulated tumor growth during treatment.     

Inhibition of focal adhesion kinase decreases tumor growth in human neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously, we have shown that the MYCN oncogene, the primary adverse prognostic indicator in neuroblastoma, regulates the expression of FAK in neuroblastoma. In this study, we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN+/MYCN- neuroblastoma cell lines, we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment, decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 treatment than their MYCN negative counterparts. In addition, we have shown that treatment with Y15 in vivo leads to less tumor growth in nude mouse xenograft models, again with the greatest effects seen in MYCN+ tumor xenografts. The results of the current study suggest that FAK and phosphorylation at the Y397 site plays a role in neuroblastoma cell survival, and that the FAK Y397 phosphorylation site is a potential therapeutic target for this childhood tumor.     

FERM control of FAK function: Implications for cancer therapy

Integrins are transmembrane receptors that bind to extracellular matrix proteins and convey anchorage-dependent signals regulating normal cell proliferation. Integrin signals within the tumor micro-environment also impact cancer cell survival and invasion during tumor progression. These integrin-associated signaling events are transduced in part through the activation of non-receptor protein-tyrosine kinases. Focal adhesion kinase (FAK) is activated by β-subunit integrins in both normal and transformed cells. As genetic inactivation of β1 integrin or FAK yield early embryonic lethal phenotypes associated with decreased cell proliferation, and dominant-negative inhibition of FAK can cause increased cell apoptosis, there is a concern that FAK inhibition may have cytotoxic effects on cell growth or survival.However, FAK-specific small molecule inhibitors do not directly impact cell growth in culture, but yet show potent anti-tumor growth effects in vivo. Additionally, recent studies have shed new insight into the FAK kinase-independent regulation of cell proliferation and survival mediated by the FAK N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain.Herein, we review the role of the FAK FERM domain in both the intrinsic regulation of FAKkinase activity and how FERM-mediated nuclear localization of FAK promotes enhanced cell survival through the inhibition of tumor suppressor p53 activation during development and under conditions of cellular stress. As we find that FAK FERM-mediated regulation of p53 occurs in human carcinoma cells, elevated FAK expression in tumors may promote both kinase-dependent and –independent survival mechanisms. We discuss how the pharmacological inhibition of FAK kinase activity may impact tumor progression through combined effects of blocking both tumor- and stromal-associated signaling regulating neovascularization.     

Understanding the Roles of FAK in Cancer: Inhibitors,Genetic Models,and New Insights

Focal adhesion kinase (FAK) is a protein tyrosine kinase that regulates cellular adhesion, motility, proliferation and survival in various types of cells. Interestingly, FAK is activated and/or overexpressed in advanced cancers, and promotes cancer progression and metastasis. For this reason, FAK became a potential therapeutic target in cancer, and small molecule FAK inhibitors have been developed and are being tested in clinical phase trials. These inhibitors have demonstrated to be effective by inducing tumor cell apoptosis in addition to reducing metastasis and angiogenesis. Furthermore, several genetic FAK mouse models have made advancements in understanding the specific role of FAK both in tumors and in the tumor environment. In this review, we discuss FAK inhibitors as well as genetic mouse models to provide mechanistic insights into FAK signaling and its potential in cancer therapy.     

黏着斑激酶及其小分子抑制剂作为抗肿瘤药物的研究进展

抗黏着斑激酶是一种非受体型酪氨酸蛋白激酶,在许多肿瘤的发生和发展过程中均有过表达。研究表明,作为细胞内重要的骨架蛋白和调节多种细胞信号通路的关键分子,黏着斑激酶在肿瘤发生、发展、迁移和侵袭的各个阶段都起着重要作用。因此,以黏着斑激酶作为抗肿瘤靶点开发其抑制剂的研究受到广泛关注。综述黏着斑激酶的结构与功能、它与肿瘤的关联及其小分子抑制剂的研究与开发。     

MicroRNA-7 Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Targeting FAK Expression

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.     

A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells

Melanoma has the highest mortality rate of all skin cancers and a major cause of treatment failure is drug resistance. Tumors heterogeneity requires novel therapeutic strategies and new drugs targeting multiple pathways. One of the new approaches is targeting the scaffolding function of tumor related proteins such as focal adhesion kinase (FAK). FAK is overexpressed in most solid tumors and is involved in multiple protein-protein interactions critical for tumor cell survival, tumor neovascularization, progression and metastasis. In this study, we investigated the anticancer activity of the FAK scaffold inhibitor C4, targeted to the FAK-VEGFR-3 complex, against melanomas. We compared C4 inhibitory effects in BRAF mutant vs BRAF wild type melanomas. C4 effectively caused melanoma tumor regression in vivo, when administered alone and sensitized tumors to chemotherapy. The most dramatic effect of C4 was related to reduction of vasculature of both BRAF wild type and V600E mutant xenograft tumors. The in vivo effects of C4 were assessed in xenograft models using non-invasive multimodality imaging in conjunction with histologic and molecular biology methods. C4 inhibited cell viability, adhesion and motility of melanoma and endothelial cells, specifically blocked phosphorylation of VEGFR-3 and FAK and disrupted their complexes. Specificity of in vivo effects for C4 were confirmed by a decrease in tumor FAK and VEGFR-3 phosphorylation, reduction of vasculogenesis and reduced blood flow. Our collective observations provide evidence that a small molecule inhibitor targeted to the FAK protein-protein interaction site successfully inhibits melanoma growth through dual targeting of tumor and endothelial cells and is effective against both BRAF wild type and mutant melanomas.     

Focal adhesion kinase (FAK) binds RET kinase via its FERM domain, priming a direct and reciprocal RET-FAK transactivation mechanism

Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.     

Integrin-regulated FAK-Src signaling in normal and cancer cells

Integrins can alter cellular behavior through the recruitment and activation of signaling proteins such as non-receptor tyrosine kinases including focal adhesion kinase (FAK) and c-Src that form a dual kinase complex. The FAK-Src complex binds to and can phosphorylate various adaptor proteins such as p130Cas and paxillin. In normal cells, multiple integrin-regulated linkages exist to activate FAK or Src. Activated FAK-Src functions to promote cell motility, cell cycle progression and cell survival. Recent studies have found that the FAK-Src complex is activated in many tumor cells and generates signals leading to tumor growth and metastasis. As both FAK and Src catalytic activities are important in promoting VEGF-associated tumor angiogenesis and protease-associated tumor metastasis, support is growing that FAK and Src may be therapeutically relevant targets in the inhibition of tumor progression.     

High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.     

Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

Focal adhesion kinase (FAK) is a major signaling molecule which functions downstream of integrins or in conjunction with mitogenic signaling pathways. FAK is overexpressed and/or activated in many types of human tumors, in which it promotes cell adhesion, survival, migration and invasion. In addition to FAK''s ability to regulate signaling through its scaffolding activities, FAK encodes an intrinsic kinase activity. Although some FAK substrates have been identified, a more comprehensive analysis of substrates is lacking. In this study, we use a protein microarray to screen the human proteome for FAK substrates. We confirm that several of the proteins identified are bona fide in vitro FAK substrates, including several factors which are known to regulate the NFκB pathway. Finally, we identify a role for FAK''s kinase activity in both canonical and non-canonical NFκB signaling. Our screen therefore represents the first high throughput screen for FAK substrates and provides the basis for future in-depth analysis of the role of FAK''s kinase activity in the processes of tumorigenesis.     

The oncogenic and growth-suppressive functions of the integrin-linked kinase are distinguished by JNK1 expression in human cancer cells

While most reports detail an oncogenic function for the integrin-linked kinase (ILK) in human cancer, few describe a contradictory growth-suppressive function. We previously reported that ILK functions as either a tumor suppressor or an oncogene in rhabdomyosarcoma (RMS), in a manner linked to expression of the c-jun amino terminal kinase-1 (JNK1). However, studies in other tumors are lacking. With the advent of bioavailable small molecule inhibitors of ILK, defining both the function of ILK and biomarkers to predict its behaviour are of critical importance. Here, we studied the role of ILK in a panel of tumor cell lines. We demonstrate that ILK functions as either a growth-promoter or suppressor in numerous tumor cell lines. Further, cell lines in which ILK functioned as a growth suppressor displayed elevated JNK1 expression relative to cells in which ILK functioned as an oncogene. Comparison of endogenous JNK1 and JNK1β isoform expression levels to the cellular response to ILK overexpression demonstrated that JNK1β isoforms represent biomarkers differentiating the two functions of ILK. Moreover, RNAi and overexpression-based alteration of JNK1 expression levels was sufficient to switch the function of ILK in both transformed and untransformed cells. These results indicate widespread oncogenic and growth-suppressive functions for ILK in multiple human malignancies and suggest that JNK1 isoforms represent biomarkers for ILK neoplastic activity. These results provide a rationale for stratifying patients to receive ILK kinase inhibitors based on individualized tumor-specific ILK function.     

SD-208, a Novel Protein Kinase D Inhibitor,Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.     

A CK2-dependent mechanism for degradation of the PML tumor suppressor

The PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in human tumors through unknown posttranslational mechanisms. Casein kinase 2 (CK2) is oncogenic and frequently upregulated in human tumors. Here we show that CK2 regulates PML protein levels by promoting its ubiquitin-mediated degradation dependent on direct phosphorylation at Ser517. Consequently, PML mutants that are resistant to CK2 phosphorylation display increased tumor-suppressive functions. In a faithful mouse model of lung cancer, we demonstrate that Pml inactivation leads to increased tumorigenesis. Furthermore, CK2 pharmacological inhibition enhances the PML tumor-suppressive property in vivo. Importantly, we found an inverse correlation between CK2 kinase activity and PML protein levels in human lung cancer-derived cell lines and primary specimens. These data identify a key posttranslational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition.