Abstract
Pertuzumab (Perjeta) is an anti-HER2 monoclonal antibody that is used for treatment of HER2-positive breast cancers in combination with trastuzumab (Herceptin) and docetaxel and showed promising clinical outcomes. Pertuzumab is suggested to block heterodimerization of HER2 with EGFR and HER3 that abolishes canonical function of HER2. However, evidence on the exact mode of action of pertuzumab in homodimerization of HER2 are limited. In this study, we investigated the effect of pertuzumab and its combination with trastuzumab on HER2 homodimerization, phosphorylation and whole gene expression profile in Chinese hamster ovary (CHO) cells stably overexpressing human HER2 (CHO-K6). CHO-K6 cells were treated with pertuzumab, trastuzumab, and their combination, and then HER2 homodimerization and phosphorylation at seven pY sites were investigated. The effects of the monoclonal antibodies on whole gene expression and the expression of cell cycle stages, apoptosis, autophagy, and necrosis were studied by cDNA microarray. Results showed that pertuzumab had no significant effect on HER2 homodimerization, however, trastuzumab increased HER2 homodimerization. Interestingly, pertuzumab increased HER2 phosphorylation at Y1127, Y1139, and Y1196 residues, while trastuzumab increased HER2 phosphorylation at Y1196. More surprisingly, combination of pertuzumab and trastuzumab blocked the phosphorylation of Y1005 and Y1127 of HER2. Our results also showed that pertuzumab, but not trastuzumab, abrogated the effect of HER2 overexpression on cell cycle in particular G1/S transition, G2/M transition, and M phase, whereas trastuzumab abolished the inhibitory effect of HER2 on apoptosis. Our findings confirm that pertuzumab is unable to inhibit HER2 homodimerization but induces HER2 phosphorylation at some pY sites that abolishes HER2 effects on cell cycle progress. These data suggest that the clinical effects of pertuzumab may mostly through the inhibition of HER2 heterodimers, rather than HER2 homodimers and that pertuzumab binding to HER2 may inhibit non-canonical HER2 activation and function in non-HER-mediated and dimerization-independent pathway(s).
Highlights
HER2 (ErbB2/Neu) is a 185 kDa transmembrane receptor belongs to the tyrosine kinase epidermal growth factor receptor family including other receptors EGFR (HER1/ErbB1), HER3 (ErbB3) and HER4 (ErbB4) [1,2,3] Dimerization of HER receptors leads to activation of their intracellular tyrosine kinase domains leading to the phosphorylation of both receptors [3]
To study the effect of the pertuzumab on cell death pathways we investigated the expression of selected marker genes for apoptosis, autophagy and necrosis in the cells treated with pertuzumab, trastuzumab, or their combination
Pertuzumab induces phosphorylation of HER2 at Y1127, Y1139, and Y1196 phospho-sites independent of HER2 homodimerization
Summary
HER2 (ErbB2/Neu) is a 185 kDa transmembrane receptor belongs to the tyrosine kinase epidermal growth factor receptor family including other receptors EGFR (HER1/ErbB1), HER3 (ErbB3) and HER4. (ErbB4) [1,2,3] Dimerization of HER receptors leads to activation of their intracellular tyrosine kinase domains leading to the phosphorylation of both receptors [3]. HER2 kinase tyrosine kinase domain tak which promotes cell growth, division, and motility [3]. Activation of HER2 domain heterodimerization with either. HER2 as an oncogene and amplification causes overe is encoded by the ERBB2 gene which is known as an oncogene known and amplification causes overexpression of HER2 mostly due to gene amplificatio of HER2 receptor in the cells. HER2 in tumor thanofthat normal cells in 15–30 is overexpressed more than 10 times in tumor cells than that10intimes normal cells incells all in breast ovarian cancers [8,9], and of all lung adenoca cancers [2,5,6,7], 2–66% of all ovarian cancers [8,9], and 4–35% of all lung adenocarcinoma [10,11]
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