Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated.

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.

Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth.

BACKGROUND: Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis. METHODS: We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site. RESULTS: By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil. CONCLUSIONS: Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches.

A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field.

Down-regulation of ALDH1A3, CD44 or MDR1 sensitizes resistant cancer cells to FAK autophosphorylation inhibitor Y15.

PURPOSE: Focal adhesion kinase is an important survival signal in cancer. Recently, we demonstrated that the autophosphorylation inhibitor of FAK, Y15, effectively inhibited cancer cell growth. We detected many cancer cell lines sensitive to Y15 and also detected several cell lines such as colon cancer Lovo-1 and thyroid K1 more resistant to Y15. We sought to determine the main players responsible for the resistance. METHODS: To reveal the signaling pathways responsible for the increased resistance of these cancer cells to the inhibitor of FAK, we performed a microarray gene profile study in both sensitive and resistant cells treated with Y15 inhibitor to compare with the more sensitive cells. RESULTS: Among unique genes up-regulated by Y15 in Lovo-1 and K1 resistant cells, a stem cell marker-ALDH1A3-was detected to be up-regulated >twofold. The resistant Lovo-1 and thyroid K1 cells overexpressed ALDH1A3 and CD44 versus sensitive cells. Treatment with ALDH1A3 siRNAs or ALDH inhibitor, DEAB sensitized resistant Lovo-1 and K1 cells to Y15 inhibitor, decreased viability and caused G1 cell cycle arrest more effectively than each agent alone. In addition, down-regulation of CD44 that was overexpressed in resistant Lovo-1 cells with CD44 siRNA effectively decreased the viability of cells in combination with Y15. In addition, down-regulation of overexpressed MDR1 with specific inhibitor, PSC-833, also sensitized resistant colon cancer cells to Y15. CONCLUSIONS: This report clearly demonstrates the mechanism of resistance to FAK autophosphorylation inhibitor and the mechanism to overcome it that is important for developing FAK-targeted therapy approaches.

Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells.

MiR-138 and MiR-135 directly target focal adhesion kinase, inhibit cell invasion, and increase sensitivity to chemotherapy in cancer cells.

Focal Adhesion Kinase is a 125 kDa non-receptor kinase and overexpressed in many types of tumors. Recently, short noncoding RNAs, called microRNAs have been discovered as regulators of gene expression mainly through binding to the untranslated region (UTR) of mRNA. In this report we show that MiR-138 and MiR-135 down-regulated FAK expression in cancer cells. MiR-138 and MiR-135 inhibited FAK protein expression in different cancer cell lines. The computer analysis of 3'FAK-untranslated region (FAKUTR) identified one conserved MiR-138 binding site (CACCAGCA) at positions 3514-3521 and one conserved MiR-135 (AAGCCAU) binding site at positions 4278-4284 in the FAK-UTR. By a dual-luciferase assay we demonstrate that MiR-138 and MiR-135 directly bound the FAK untranslated region using FAK-UTR-Target (FAK-UTR) luciferase plasmid and inhibited its luciferase activity. The sitedirected mutagenesis of the MiR-138 and MiR-135 binding sites in the FAK-UTR abrogated MiR-138 and MiR-135-directed inhibition of FAK-UTR. Real-time PCR demonstrated that cells transfected with MiR-138 and MiR-135 expressed decreased FAK mRNA levels. Moreover, stable expression of MiR-138 and MiR-135 in 293 and HeLa cells decreased cell invasion and increased sensitivity to 5- fluorouracil (5-FU), FAK inhibitor, Y15, and doxorubicin. In addition, MiR-138 significantly decreased 293 xenograft tumor growth in vivo. This is the first report on regulation of FAK expression by MiR-135 and MiR138 that affected invasion, drug sensitivity, and tumor growth in cancer cells, which is important to the development of FAK-targeted therapeutics and understanding their novel regulations and functions.

The microarray gene profiling analysis of glioblastoma cancer cells reveals genes affected by FAK inhibitor Y15 and combination of Y15 and temozolomide.

Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and detected 8087 and 6555 genes, which were significantly either up- or down-regulated by Y15-treatment in DBTRG and U87 cells, respectively (p<0.05). Moreover, DBTRG and U87 cells treated with Y15 changed expression of 1332 and 462 genes more than 1.5 fold, p<0.05, respectively and had 237 common genes affected by Y15. The common genes up-regulated by Y15 included GADD45A, HSPA6 (heat-shock 70); DUSP1, DUSP 5 (dual-phosphatase 5); CDKN1A (p21) and common down-regulated genes included kinesins, such as KIF11, 14, 20A, 20B; topoisomerase II, TOP2A; cyclin F; cell cycle protein: BUB1; PARP1, POLA1. In addition, we detected genes affected by temozolomide and by combination of Y15 and temozolomide treatment in U87 cells. Among genes up-regulated by Y15 and temozolomide more significantly than by each agent alone were: COX7B; interferon, gamma-inducible transcript: IFI16; DDIT4; GADD45G and down-regulated: KIF3A, AKT1; ABL; JAK1, GLI3 and ALDH1A3. Thus, microarray gene expression analysis can be effective in establishing genes affected in response to FAK inhibitor alone and in response to combination of Y15 with temozolomide that is important for glioblastoma therapy.

Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex.

Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53+/+ and p53-/- cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53+/+ cells but not in p53-/- cells. Among up-regulated genes in HCT p53+/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53+/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

Mitoxantrone targets the ATP-binding site of FAK, binds the FAK kinase domain and decreases FAK, Pyk-2, c-Src, and IGF-1R in vitro kinase activities.

Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors and plays a key role in cell adhesion, spreading, motility, proliferation, invasion, angiogenesis, and survival. Recently, FAK has been proposed as a target for cancer therapy, and we performed computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database to target the ATP-binding site of FAK, K454. More than 140,000 small molecule compounds were docked into the crystal structure of the kinase domain of FAK in 100 different orientations using DOCK5.1 that identified small molecule compounds, targeting the K454 site, called A-compounds. To find the therapeutic efficacy of these compounds, we examined the effect of twenty small molecule compounds on cell viability by MTT assays in different cancer cell lines. One compound, A18 (1,4-bis(diethylamino)-5,8- dihydroxy anthraquinon) was a mitoxantrone derivative and significantly decreased viability in most of the cells comparable to the to the level of FAK kinase inhibitors TAE-226 (Novartis, Inc) and PF-573,228 (Pfizer). The A18 compound specifically blocked autophosphorylation of FAK like TAE-226 and PF-228. ForteBio Octet Binding assay demonstrated that mitoxantrone (1,4-dihydroxy- 5,8-bis[2-(2-hydroxyethylamino) ethylamino] anthracene-9,10-dione directly binds the FAK-kinase domain. In addition, mitoxantrone significantly decreased the viability of breast cancer cells in a dose-dependent manner and inhibited the kinase activity of FAK and Y56/577 FAK phosphorylation at 10-20 µM. Mitoxantrone did not affect phosphorylation of EGFR, but decreased Pyk-2, c-Src, and IGF-1R kinase activities. The data demonstrate that mitoxantrone decreases cancer viability, binds FAK-Kinase domain, inhibits its kinase activity, and also inhibits in vitro kinase activities of Pyk-2 and IGF-1R. Thus, this novel function of the mitoxantrone drug can be critical for future development of anti-cancer agents and FAK-targeted therapy research.

A small-molecule inhibitor, 5'-O-tritylthymidine, targets FAK and Mdm-2 interaction, and blocks breast and colon tumorigenesis in vivo.

Focal Adhesion Kinase (FAK) is overexpressed in many types of tumors and plays an important role in survival. We developed a novel approach, targeting FAK-protein interactions by computer modeling and screening of NCI small molecule drug database. In this report we targeted FAK and Mdm-2 protein interaction to decrease tumor growth. By macromolecular modeling we found a model of FAK and Mdm-2 interaction and performed screening of > 200,000 small molecule compounds from NCI database with drug-like characteristics, targeting the FAK-Mdm-2 interaction. We identified 5';-O-Tritylthymidine, called M13 compound that significantly decreased viability in different cancer cells. M13 was docked into the pocket of FAK and Mdm-2 interaction and was directly bound to the FAK-N terminal domain by ForteBio Octet assay. In addition, M13 compound affected FAK and Mdm-2 levels and decreased complex of FAK and Mdm-2 proteins in breast and colon cancer cells. M13 re-activated p53 activity inhibited by FAK with Mdm-2 promoter. M13 decreased viability, clonogenicity, increased detachment and apoptosis in a dose-dependent manner in BT474 breast and in HCT116 colon cancer cells in vitro. M13 decreased FAK, activated p53 and caspase-8 in both cell lines. In addition, M13 decreased breast and colon tumor growth in vivo. M13 activated p53 and decreased FAK in tumor samples consistent with decreased tumor growth. The data demonstrate a novel approach for targeting FAK and Mdm-2 protein interaction, provide a model of FAK and Mdm-2 interaction, identify M13 compound targeting this interaction and decreasing tumor growth that is critical for future targeted therapeutics.

Pharmacologic blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide.

Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.