Abstract

G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported. We overexpressed S310F as well as G309A, G309E and S310Y HER2 mutants and tested their reactivity to trastuzumab and pertuzumab. All mutants reacted to trastuzumab, but S310F mutant did not react to pertuzumab along with S310Y or G309E mutants. Thereafter, we tested the effects of trastuzumab and pertuzumab on 5637 cell line expressing both wild-type HER2 and S310F mutant. The ligand-independent HER2 homodimerization blocking antibody, trastuzumab, did not inhibit the activation of the HER2 receptor, suggesting that the S310F HER2 mutant did not form homodimers or heterodimers with wild-type HER2. Because 5637 cells overexpressed the EGFR, the effects of cetuximab and gefitinib were determined, and both inhibited the activation of HER2 and significantly reduced cell growth. Because pertuzumab did not inhibit the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is expected to occur on the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule interaction analyses using TIRF microscopy were performed, which showed that S310F mutant successfully formed complexes with EGFR. In conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab.

Highlights

  • Human epidermal growth factor receptor 2 (HER2)/ErbB2/Neu is a member of the human epidermal growth factor receptor (EGFR, HER) family of homologous transmembrane receptor tyrosine kinases

  • The extracellular domain of four mutants and wild-type HER2 was prepared as a fusion protein of human Cκ through cloning into a mammalian expression vector, transfection and purification using an affinity column reactive to human Cκ from the culture supernatant

  • D769H and V777L mutations increased the phosphorylation of both HER2 and EGFR [27,35]

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Summary

Introduction

HER2/ErbB2/Neu is a member of the human epidermal growth factor receptor (EGFR, HER) family of homologous transmembrane receptor tyrosine kinases. There are 11 types of ligands reactive to HER family receptors, including those generated by alternative splicing [1]. The roles for these receptors and their ligands have been well described in many types of human cancers, including breast, colon, pancreatic, ovarian, brain, and lung cancers [2]. Human epidermal growth factor receptor 2 (HER2) has no known ligand but forms heterodimers with other EGFR family members and activates downstream signaling through the phosphoinositide 3-kinase/AKT and MAPK pathways [4]

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