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FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

ABSTRACT: 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 ?M 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.

SUBMITTER: Heffler M

PROVIDER: S-EPMC3625516 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

Heffler Melissa M Golubovskaya Vita M VM Conroy Jeffrey J Liu Song S Wang Dan D Cance William G WG Dunn Kelli B KB

Anti-cancer agents in medicinal chemistry 20130501 4

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 inhibi ...[more]

PMID: 22934709

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Project description:Purpose: 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. Methods: We treated SW620 colon cancer cells with Y15 to inhibit FAK. We used two strategies to inhibit HAS: (1) cells were transfected with siRNA (HAS3 inhibited); a scrambled sequence was used as a control (HAS3 scrambled), and (2) cells were treated with the HAS inhibitor 4-methylumbelliferone (4-MU). To determine the effect on viability, MTT assays were performed on transfected cells treated with Y15, and wild type cells treated with Y15 alone, 4-MU alone or Y15+4-MU. Treated and untreated cells were submitted to the gene microarray facility for expression profiling. RT-PCR was done to confirm the results. Results: HAS and FAK inhibition affected cell viability. Y15 and 4-MU decreased viability in a dose-dependent manner; viability was further inhibited by treatment with Y15+4-MU in combination (p<0.05). HAS-inhibited cells treated with as little as 2 M of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05), suggesting 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). Of 29 genes that were common to both groups, 9 were down-regulated (CBS, DHRS3, EEPD1, ESPN, FAM46C, GRTP1, IL20RA, INHBE, SCNN1A) and 4 were up-regulated (ANXA1, MALL, RGS2, SNAI2). RT-PCR confirmed these findings. Among the genes affected by FAK or HAS3 inhibition were FOX genes (apoptosis, cell cycle regulation), ANXA1 (apoptosis, proliferation), IL8 (cell cycle regulation, adhesion, proliferation), RGS2 (cell cycle regulation), CEACAM6 (adhesion), SNAI2 (transcription regulation), and SFRP5 (apoptosis). Several genes were specific to either FAK or HAS3 inhibition and several were common to both. Gene expression profiles of samples isolated from human colorectal cancer cells (SW620). A comparison of gene expression between untreated cells and cells treated with 4mcM of Y15. A second comparison between cells transfected with siRNA to HAS3 (HAS3-silenced) and cells transfected with a scrambled control sequence (sc). Two replicates each.

Project description:PURPOSE:Proinflammatory cytokines interferon gamma (IFN-?), tumor necrosis factor alpha (TNF-?), and interleukin-1 beta (IL-1?) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-?, TNF-?, and IL-1? have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. METHODS:ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-?, TNF-?, and/or IL-1?, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. RESULTS:Proinflammatory cytokines (IFN-? + TNF-? + IL-1?) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). CONCLUSIONS:RPE cells exposed to proinflammatory cytokines IFN-?, TNF-?, and IL-1? showed decreased expression of key genes involved in the visual cycle, epithelial morphology, and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology.

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FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

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FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

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