Identification and validation of COX-2 as a co-target for overcoming cetuximab resistance in colorectal cancer cells

Cetuximab, an epidermal growth factor receptor (EGFR)-blocking antibody, was approved for treatment of metastatic colorectal cancer over a decade ago; however, patients' responses to cetuximab vary substantially due to intrinsic and acquired resistance to cetuximab. Here, we report our findings using Affymetrix HG-U133A array to examine changes in global gene expression between DiFi, a human colorectal cancer cell line that is highly sensitive to cetuximab, and two other cell lines: DiFi5, a DiFi subline with acquired resistance to cetuximab, and DiFi-AG, a DiFi subline with acquired resistance to the EGFR tyrosine kinase inhibitor AG1478 but sensitivity to cetuximab. We identified prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2 (COX-2), as the gene with the greatest difference between the cetuximab-resistant DiFi5 cells and the cetuximab-sensitive DiFi cells and DiFi-AG cells. Reverse transcription polymerase chain reaction and Western blotting validated upregulation of COX-2 in DiFi5 but not in DiFi or DiFi-AG cells. We developed COX-2 knockdown stable clones from DiFi5 cells and demonstrated that genetic knockdown of COX-2 partially re-sensitized DiFi5 cells to cetuximab. We further confirmed that cetuximab in combination with a COX-2 inhibitor led to cell death via apoptosis or autophagy not only in DiFi5 cells but also in another colorectal cancer cell line naturally resistant to cetuximab. Our findings support further evaluation of the strategy of combining cetuximab and a COX-2 inhibitor for treatment of metastatic colorectal cancer.