ABSTRACT
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.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Aniline Compounds/pharmacology , Colonic Neoplasms/drug therapy , Hyaluronan Receptors/metabolism , Protein Kinase Inhibitors/pharmacology , Thyroid Neoplasms/drug therapy , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Cell Line, Tumor , Colonic Neoplasms/metabolism , Down-Regulation , Drug Resistance, Neoplasm , Female , Focal Adhesion Kinase 1/antagonists & inhibitors , Focal Adhesion Kinase 1/metabolism , Humans , Mice , Mice, Nude , Phosphorylation , Thyroid Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Focal adhesion kinase (FAK) increasingly has been implicated in cancer growth and progression. 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) is a small molecule FAK inhibitor that blocks the Y397 autophosphorylation site. FAK inhibitor, Y15 decreased Y397 FAK in different colon cancer cells lines in a dose-dependent manner. In addition, Y15 decreased phosphorylated Src in SW480 and SW620 cells. Y15 decreased cell viability, increased detachment, and increased apoptosis in SW480 and SW620 cells in vitro. Combination of FAK inhibitor Y15 and Src inhibitor PP2 decreased colon cancer cell viability more effectively than each agent alone. In addition, when combined with 5-FU, oxaliplatin or 5-FU and oxaliplatin, colon cancer viability was decreased further, demonstrating that dual and triple therapy synergistically inhibits cell viability. In vivo, Y15 decreased subcutaneous SW620 tumor growth by 28%. Combination of oral Y15 with 5-FU/or oxaliplatin decreased tumor growth by 48% more effectively than each inhibitor alone. Finally, tumors treated with Y15 expressed less Y397 phosphorylation, Src phosphorylation and had greater apoptosis than controls. Thus, the small molecule FAK inhibitor, Y15, inhibits cell growth in vitro and in vivo and enhances the efficacy of chemotherapy, demonstrating that it can be an effective therapeutic inhibitor for treating colon cancer.
Subject(s)
Colonic Neoplasms/drug therapy , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Female , Focal Adhesion Protein-Tyrosine Kinases/metabolism , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Phenylenediamines/pharmacology , Phosphorylation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2 µM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p < 0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p < 0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heatshock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways.
Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Glucuronosyltransferase/antagonists & inhibitors , Hymecromone/pharmacology , Aniline Compounds/chemistry , Antineoplastic Agents/chemistry , Cell Survival/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Focal Adhesion Protein-Tyrosine Kinases/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Hyaluronan Synthases , Hymecromone/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Hyaluronan (HA) and hyaluronan synthases (HAS) have been implicated in cancer growth and progression. We previously have shown that HAS3 and HA mediate tumor growth in SW620 colon cancer cells, but the mechanism remains poorly understood. In addition, the effect of HAS3 inhibition on tumor growth with other cells lines has not been explored. We therefore hypothesized that inhibition of HAS3 in highly tumorigenic HCT116 colon cancer cells would decrease tumor growth and that the underlying mechanism would involve altering proliferation and/or apoptosis. HAS3 expression was inhibited by transfection with siRNA; a scrambled sequence served as a control. Stable transfectants were injected into the flanks of nude mice and tumor growth followed for 30 days. Proliferation and apoptosis were then assessed in the harvested tumors. Results were compared using the Students' t-test and ANOVA where appropriate. siRNA transfection decreased HAS3 expression, protein production, and pericellular HA retention, and decreased in vivo tumor growth. Proliferation was unaffected in the HCT116 tumors, but increased slightly in the SW620 tumors. In contrast, HAS3 inhibition significantly increased apoptosis in all tumors. HAS3 inhibition decreases subcutaneous tumor growth by colon cancer cells and significantly increases apoptosis with less effect on proliferation. These data show that HAS3 and HA mediate colon cancer growth by inhibiting apoptosis.
Subject(s)
Apoptosis , Biomarkers, Tumor/metabolism , Colonic Neoplasms/genetics , Glucuronosyltransferase/antagonists & inhibitors , Hyaluronic Acid/metabolism , RNA, Small Interfering/genetics , Animals , Biomarkers, Tumor/genetics , Caspase 3/genetics , Caspase 3/metabolism , Cell Proliferation , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , HCT116 Cells , Humans , Hyaluronan Synthases , Injections, Subcutaneous , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Nude , RNA, Small Interfering/metabolism , Transfection , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
Despite advances in medical and surgical therapy, cancer kills more than half a million people in the United States annually, and the majority of these patients succumb to metastatic disease. The traditional approach to treating systemic disease has been the use of cytotoxic chemotherapy. However, chemotherapy is rarely curative and toxicity is often dose limiting. In addition, the effects of chemotherapy are nonspecific, targeting both malignant and normal tissues. As a result, recent efforts increasingly have focused on developing agents that target specific molecules in tumor cells in order to both improve efficacy and limit toxicity. This review summarizes the history and current use of targeted molecular therapy for cancer, with a special emphasis on recently developed inhibitors of Focal Adhesion Kinase (FAK).
