Abstract 5631: Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer .
Authors: Khelwatty Said Abdullah , Essapen Sharadah , Seddon Alan M. , Fan Zhen , Modjtahedi Helmout 
Published: August 2014

Abstract
The aberrant expression and activation of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (also called HER or erbB family) have been implicated in a wide range of cancers. These receptors have emerged as important therapeutic targets in human cancers. To date, of the two classes of HER inhibitors (i.e. monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs), cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients develop resistance to the EGFR inhibitors and consequently the duration of response to these treatments is often short. In this study on colorectal cancer cells, we evaluated whether there was any association between the expression levels of the HER family members, putative colorectal cancer stem cell (CSC) markers CD133 and CD44, and ABC transporter and the development of resistance to anti-EGFR mAb ICR62 or afatinib, an irreversible erbB blocker. A wide range of techniques were employed in this study including FACS analysis, Western blot analysis and SRB assay on the EGFR over-expressing human colorectal tumour cell line DiFi, which is highly sensitive to treatment with mAb ICR62 (IC50=1.2 nM) and afatinib (IC50=10 nM). Continuous exposure of DiFi cells to increasing doses of mAb ICR62 or afatinib for 6-10 months resulted in the emergence of ICR62 variant DiFi cells (DiFi62) and afatinib-variant DiFi cells (DiFiAf) with IC50 values of 209 nM and 35 nM respectively. Interestingly, we found that ICR62 resistant DiFi cells (DiFi62) also become less sensitive to treatment with 5FU, oxaloplatin and irinotecan, but remained highly sensitive to treatment with afatinib (IC50=8 nM). In contrast to DiFi62 cells, afatinib-resistant DiFiAf cells were less sensitive to treatment with 5FU and irinotecan, but like parental DiFi cells remained highly sensitive to treatment with ICR62 and oxaliplatin. Of the HER family members, there was only upregulation of EGFR in both DiFi62 and DiFiAf cells, but no changes in the expression levels of other members of the HER family or CD133. In contrast, the expression of CD44, the other putative colorectal CSC marker, was found to be downregulated by around 38% in DiFiAf cells. Of the ABC transporters, there was an increase in the expression of p-glycoprotein in both DiFi62 and DiFiAf cells and an increased expression of both MRP2 and ABCG2 in DiFi62, but not in DiFiAf cells. Taken together, our results suggest that colorectal tumour cells which acquire resistance to anti-EGFR mAb ICR62 remain sensitive to treatment with the irreversible erbB blocker afatinib and vice versa, suggesting that mechanisms underlying the antitumor activity of ICR62 and afatinib are different in these cells. In addition, upregulation of the EGFR upon treatment with these inhibitors may contribute to resistance.
Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Alan M. Seddon, Zhen Fan, Helmout Modjtahedi. Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5631. doi:10.1158/1538-7445.AM2013-5631