Vita M. Golubovskaya

A small molecule inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride, targeting the Y397 site of Focal Adhesion Kinase decreases tumor growth

Focal adhesion kinase (FAK) is a nonreceptor kinase that is overexpressed in many types of tumors. We developed a novel cancer-therapy approach, targeting the main autophosphorylation site of FAK, Y397, by computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database. More than 140,000 small molecule compounds were docked into the N-terminal domain of the FAK crystal structure in 100 different orientations that identified 35 compounds. One compound, 14 (1,2,4,5-benzenetetraamine tetrahydrochloride), significantly decreased viability in most of the cells to the levels equal to or higher than control FAK inhibitor 1a (2-[5-chloro-2-[2-methoxy-4-(4-morpholinyl)phenylamino]pyrimidin-4-ylamino]-N-methylbenzamide, TAE226) from Novartis, Inc. Compound 14 specifically and directly blocked phosphorylation of Y397-FAK in a dose- and time-dependent manner. It increased cell detachment and inhibited cell adhesion in a dose-dependent manner. Furthermore, 14 effectively caused breast tumor regression in vivo. Thus, targeting the Y397 site of FAK with 14 inhibitor can be effectively used in cancer therapy.

A novel small molecule inhibitor of FAK decreases growth of human pancreatic cancer

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed in many types of tumors, including pancreatic cancer, and plays an important role in cell adhesion and survival signaling. Pancreatic cancer is a lethal disease and is very resistant to chemotherapy, and FAK has been shown recently to assist in tumor cell survival. Therefore, FAK is an excellent potential target for anti-cancer therapy. We identified a novel small molecule inhibitor (1,2,4,5-Benzenetetraamine tetrahydrochloride, that we called Y15) targeting the main autophosphorylation site of FAK and hypothesized that it would be an effective treatment strategy against human pancreatic cancer. Y15 specifically blocked phosphorylation of Y397-FAK and total phosphorylation of FAK. It directly inhibited FAK autophosphorylation in a dose- and time-dependent manner. Furthermore, Y15 increased pancreatic cancer cell detachment and inhibited cell adhesion in a dose-dependent manner. Y15 effectively caused human pancreatic tumor regression in vivo, when administered alone and its effects were synergistic with gemcitabine chemotherapy. This was accompanied by a decrease in Y397-phosphorylation of FAK in the tumors treated with Y15. Thus, targeting the Y397 site of FAK in pancreatic cancer with the small molecule inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride, is a potentially effective treatment strategy in this deadly disease.

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.

Focal adhesion kinase and P53 signaling in cancer cells

The progression of human cancer is characterized by a process of tumor cell motility, invasion, and metastasis to distant sites, requiring the cancer cells to be able to survive the apoptotic pressures of anchorage-independent conditions. One of the critical tyrosine kinases linked to these processes of tumor invasion and survival is the focal adhesion kinase (FAK). FAK was first isolated from human tumors, and FAK mRNA was found to be upregulated in invasive and metastatic human breast and colon cancer samples. Recently, the FAK promoter was cloned, and it has been found to contain p53-binding sites. p53 inhibits FAK transcription, and recent data show direct binding of FAK and p53 proteins in vitro and in vivo. The structure of FAK and p53, proteins interacting with FAK, and the role of FAK in tumorigenesis and FAK-p53-related therapy are reviewed. This review focuses on FAK signal transduction pathways, particularly on FAK and p53 signaling, revealing a new paradigm in cell biology, linking signaling from the extracellular matrix to the nucleus.

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.

FAK overexpression and p53 mutations are highly correlated in human breast cancer

Focal adhesion kinase (FAK) is overexpressed in a number of tumors, including breast cancer. Another marker of breast cancer tumorigenesis is the tumor suppressor gene p53 that is frequently mutated in breast cancer. In the present study, our aim was to find a correlation between FAK overexpression, p53 expression and mutation status in a population‐based series of invasive breast cancer tumors from the Carolina Breast Cancer Study. Immunohistochemical analyses of 622 breast cancer tumors revealed that expression of FAK and p53 were highly correlated (p = 0.0002) and FAK positive tumors were 1.8 times more likely to be p53 positive compared to FAK negative tumors [odds ratio (OR) = 1.8; 95% Confidence Interval (CI) 1.2–2.8, adjusted for age, race and stage at diagnosis]. Tumors positive for p53 expression showed higher intensity of FAK staining (p < 0.0001) and higher percent of FAK positive staining (p < 0.0005). From the same study, we evaluated 596 breast tumors for mutations in the p53 gene, using single strand conformational polymorphism and sequencing. Statistical analyses were performed to determine the correlation between p53 mutation status and FAK expression in these tumors. We found that FAK expression and p53 mutation were positively correlated (p < 0.0001) and FAK positive tumors were 2.5 times more likely to be p53 mutation positive compared to FAK negative tumors [adjusted OR = 2.5, 95% CI 1.6–3.9]. This is the first analysis demonstrating a high correlation between FAK expression and p53 mutations in a population‐based series of breast tumors.

p53 regulates FAK expression in human tumor cells.

Attenuation of the p53 protein is one of the most common abnormalities in human tumors. Another important marker of tumorigenesis is focal adhesion kinase (FAK), a 125‐kDa tyrosine kinase that is overexpressed at the mRNA and protein levels in a variety of human tumors. FAK is a critical regulator of adhesion, motility, metastasis, and survival signaling. We have characterized the FAK promoter and demonstrated that p53 can inhibit the FAK promoter activity in vitro. In the present study, we showed that p53 can bind the FAK promoter‐chromatin region in vivo by chromatin immunoprecipitation (ChIP) assay. Furthermore, we demonstrated down‐regulation of FAK mRNA and protein levels by adenoviral overexpression of p53. We introduced plasmids with different mutations in the DNA‐binding domain of p53 (R175H, p53 R248W and R273H) into HCTp53−/− cells and showed that these mutations of p53 did not bind FAK promoter and did not inhibit FAK promoter activity, unlike wild type p53. We analyzed primary breast and colon cancers for p53 mutations and FAK expression, and showed that FAK expression was increased in tumors containing mutations of p53 compared to tumors with wild type p53. In addition, tumor‐derived missense mutations in the DNA‐binding domain (R282, R249, and V173) also led to increased FAK promoter activity. Thus, the present data show that p53 can regulate FAK expression during tumorigenesis.

Focal Adhesion Kinase Versus p53: Apoptosis or Survival?

Focal adhesion kinase (FAK) is a tyrosine kinase that interacts with a multitude of signaling partners and helps cells to survive in the face of various proapoptotic signals. One of the most important interactions for FAK is with the tumor suppressor protein p53. p53 binds not only to the amino-terminal domain of FAK but also to the FAK promoter to inhibit its transcription. A study now reports the biological implications of the kinase-independent interaction of FAK with p53, which opens up future perspectives in cell signaling and cancer research. We focus on FAK and p53 signaling, which link signal transduction pathways from the extracellular matrix and cytoplasm to the nucleus, in human and mouse cells. FAK is proposed to be a critical scaffold protein that sequesters proapoptotic proteins, such as p53, to mediate cell survival.

Effect of focal adhesion kinase (FAK) downregulation with FAK antisense oligonucleotides and 5-fluorouracil on the viability of melanoma cell lines.

Inhibition of focal adhesion kinase (FAK), a non-receptor tyrosine kinase linked to tumour cell survival, causes cell rounding, loss of adhesion and apoptosis in human cancer cell lines. In this study, we tested antisense oligonucleotide inhibitors of FAK, in combination with 5-fluorouracil (5-FU), to increase its sensitivity in human melanoma cell lines. Antisense oligonucleotides directed to the 5′ mRNA sequence of FAK and missense control oligonucleotides were used. In BL melanoma cells, treatment with FAK antisense oligonucleotide was associated with a 2.5-fold increase in cell death compared with treatment with control oligonucleotide (33±2% vs. 13±3%, P<0.0001). 5-FU alone had no effect on BL cells (4.4% cell death, P=0.15). The addition of 5-FU after antisense oligonucleotide resulted in a significant synergistic increase in cell death equal to 69±2% compared with treatments with antisense oligonucleotide alone, 5-FU alone and control oligonucleotide (P<0.0001). Similar results were found in the C8161 melanoma cell line. In both cell lines, reduction in cell viability was accompanied by an increased loss of adhesion and increased apoptosis that was proportional to the decrease in viability. Treatment with antisense oligonucleotide plus 5-FU resulted in significantly decreased p125FAK expression in both C8161 and BL melanoma cell lines, demonstrated by Western blot analyses. These data show that the downregulation of FAK by antisense oligonucleotide combined with 5-FU chemotherapy results in a greater loss of adhesion and greater apoptosis in melanoma cells than treatment with either agent alone, suggesting that the combination may be a potential therapeutic agent for human melanoma in vivo.

TAE226‐Induced apoptosis in breast cancer cells with overexpressed Src or EGFR

Focal adhesion kinase, FAK is a 125 kDa nonreceptor tyrosine kinase that localizes to focal adhesions. FAK is overexpressed in human tumors and regulates cellular adhesion and survival signaling. We have shown previously that the dominant‐negative FAK, C‐terminal FAK‐CD, caused detachment and apoptosis in human breast cancer cells, and that overexpression of an activated form of Src tyrosine kinase or epidermal growth factor receptor, EGFR, suppressed FAK‐CD induced apoptotic effects in breast cancer cells. In the present study, we studied the effect of a novel FAK inhibitor, TAE226 (Novartis, Inc.), on the breast cancer cell lines. We used stable breast cancer cell lines overexpressing Src (MCF‐7‐Src and BT474‐Src) or overexpressing EGFR (BT474‐EGFR), and control breast cancer cell lines for the treatment with different doses of TAE226 drug. The detachment and apoptosis caused by TAE226 was analyzed and compared with the effect of the dominant‐negative adenoviral FAK‐CD. The TAE226 drug caused a dose‐dependent increase of detachment and apoptosis in both BT474 and MCF‐7‐Vector and Src cells and in BT474‐EGFR and BT474‐pcDNA3 cells. Additionally, TAE226 caused downregulation of Y397‐FAK, FAK and activation of PARP or caspase‐3 proteins. Both Src and EGFR‐overexpressing cells were not resistant to the TAE226 treatment compared to FAK‐CD treatment. In addition, normal breast MCF‐10A cell line was resistant to both TAE226 drug and to the Ad‐FAK‐CD inhibitor. Thus, inhibition of autophosphorylation activity of FAK with the TAE226 inhibitor at 10–20 µM is effective in causing apoptosis in breast cancer cells, resistant to the Ad‐FAK‐CD inhibitor that can be used effectively in therapy.