The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. with lesser expression of COX-2 experienced a significantly higher rate of grade 2 to 3 3 skin reactions, which GW 9662 were a biomarker of response after cetuximab treatment [19]. A case statement showed a partial response of colorectal malignancy to the combination of cetuximab and celecoxib [20]. Furthermore, analysis of tissue samples from Rabbit Polyclonal to Actin-pan 130 participants in the IMC-0144 trial of cetuximab in patients with metastatic colorectal malignancy showed that polymorphisms in predicted progression-free survival independently of K-mutation status [21]. However, a phase II trial to explore the clinical and biological effects of combined blockade of the EGFR and COX-2 pathways using cetuximab and celecoxib was terminated early owing to lack of sufficient clinical activity and lack of laboratory evidence that this drugs were actually blocking EGFR and COX-2 activity [10]. Therefore, whether dual blockade of EGFR and COX-2 pathways represents a rational approach to benefit colorectal cancer patients remains elusive. Here, we report findings from our study to identify differences in global gene expression between DiFi human colorectal malignancy cells; DiFi5, a DiFi subline with acquired resistance to cetuximab; and DiFi-AG, a DiFi subline with acquired resistance to an EGFR tyrosine kinase inhibitor (TKI). Our study independently identified as the gene with the greatest difference in expression between cetuximab-resistant DiFi5 cells and cetuximab-sensitive DiFi and DiFi-AG cells. We next performed several functional studies using both genetic and pharmacological approaches to validate COX-2 upregulation as a major mechanism conferring resistance to cetuximab. Our results provide important mechanistic data supporting dual targeting of EGFR and COX-2 as a rational approach for treating metastatic colorectal malignancy. RESULTS Characterization of EGFR inhibition-resistant DiFi sublines and identification of genes differentially expressed between cetuximab-sensitive DiFi cells and cetuximab-resistant DiFi subline cells DiFi human colorectal malignancy cells exhibit unusually high sensitivity to EGFR inhibition: the cells readily undergo apoptosis after treatment with EGFR-blocking monoclonal antibodies or EGFR TKIs [22C27]. We previously reported generation and characterization of DiFi5, a cetuximab-resistant DiFi subline, through chronic exposure of parental DiFi cells to cetuximab with stepwise increase in concentrations in culture medium [27]. We later adopted a similar approach to generate a DiFi subline with acquired resistance to the EGFR TKI AG1478. This subline, termed DiFi-AG, exhibited strong resistance to AG1478 up to 10 M (Physique ?(Physique1A,1A, right panel). However, DiFi-AG cells remained considerably sensitive to cetuximab (Physique ?(Physique1A,1A, left panel). In contrast, DiFi5 cells are resistant to both cetuximab and AG1478 (Physique ?(Figure1A).1A). This interesting obtaining indicates that different mechanisms underlie development of resistance to EGFR inhibitors with different mechanisms of action (i.e., cetuximab versus AG1478). The differences between DiFi5 and DiFi-AG cells in response to cetuximab and AG1478 suggested that these cell lines could be used to identify pathways uniquely associated with response to cetuximab. Open in a separate window Physique 1 Characterization of EGFR inhibition-resistant DiFi sublines and identification of genes differentially expressed between cetuximab-sensitive and cetuximab-resistant DiFi cells(A) DiFi, DiFi5, and DiFi-AG cells were cultured in 0.5% fetal bovine serum containing the indicated concentrations of cetuximab or AG1478 for 5 days and then subjected to MTT assays. The data shown are the optical density (OD) values of treated cell groups at the end of treatment, expressed as a percentage of the OD value of the corresponding untreated or vehicle-treated cells. The color matched *symbols show GW 9662 0.05 for comparison of the values of GW 9662 DiFi5 or DiFi-AG with corresponding values of DiFi cells. (B) Results from Affymetrix HG-U133A microarray gene expression analysis. Total linkage analysis of gene expression classified DiFi5 cells in a cluster unique from DiFi and DiFi-AG cells. The waterfall graph shows results from one of two impartial analyses for the 62 genes with fold switch greater GW 9662 than 2 between the two clusters. experienced the highest level of fold change. Additional information is usually offered in the GEO database (access number “type”:”entrez-geo”,”attrs”:”text”:”GSE71210″,”term_id”:”71210″GSE71210). (C) Total RNA isolated from your indicated cell lines was subjected to RT-PCR amplification using a pair of COX-2-specific primers. The RT-PCR products were analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide and visualized with UV light. (D) Cell lysates from your indicated cell lines were subjected to Western blot analysis using a COX-2-specific antibody. The level of -actin was used as.
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