Abstract 231: Upregulation of HER3 (ErbB3) levels and function counteracts the antitumor effect of HER2 and PI3K inhibitors
Authors: Garrett Joan , Rinehart Cammie , Chakrabarty Anindita , Dave Bhuvanesh , Pao William , Manning Charles , Chang Jenny , Arteaga Carlos 
Published: June 2012

Abstract
We examined HER3 by immunohistochemistry in formalin-fixed tumor blocks of patients with HER2-overexpressing breast cancer treated with the HER2 tyrosine kinase inhibitor (TKI) lapatinib. HER3 levels increased 135% after 2 weeks of lapatinib therapy compared to pre-therapy levels (n=8; p=0.03). Treatment of mice bearing BT474 xenografts with lapatinib also increased levels of both HER3 mRNA and protein. Treatment with lapatinib of HER2-overexpressing BT474, SKBR3, and SUM225 cells also induced upregulation of HER3 RNA and protein. There was recovery of HER3 phosphorylation that correlated with recovery of P-Akt when P-HER2 was still inhibited. Inhibition of PI3K or Akt with small molecule inhibitors or with Akt1, Akt2, and Akt3 siRNA resulted in upregulation of HER3 mRNA/protein. Expression of myristoylated Akt reduced basal and lapatinib-induced HER3 mRNA and protein, suggesting that PI3K/Akt signaling downstream of HER2 represses HER3 expression.
We identified three putative FoxO3a sites in the ERRB3 promoter. The transcription factor FoxO3a is negatively regulated by AKT-induced phosphorylation. FoxO3a knockdown with siRNA reduced basal and lapatinib-induced increase in HER3 mRNA. We next conducted chromatin immunoprecipitation (ChIP) with FoxO antibodies. There was an increase in the HER3 PCR product using all three sets of primers specific to the FoxO binding sites upon treatment of BT474 cells with lapatinib, supporting direct FoxO protein binding to the HER3 promoter.
We hypothesized that sustained inhibition of HER3 and its output to PI3K/Akt is required for the optimal antitumor effect of HER2 inhibitors. Indeed, transfection with HER3 siRNA sensitized HER2+ cancer cells 3-fold to lapatinib-induced apoptosis. Similarly, the HER3 monoclonal antibody AMG-888 sensitized cells to lapatinib both in vitro and in vivo. Combined treatment with lapatinib and AMG-888 of mice bearing BT474 xenografts inhibited [18F]-FDG-PET uptake, P-HER3, and P-Akt and increased nuclear FoxO3a, all pharmacodynamic biomarkers of PI3K/Akt activity, more effectively than each inhibitor alone. The recovery of P-HER3 following lapatinib-induced inhibition of HER2 was not blocked by MET, FGFR2, EGFR, Src, and IGF-IR inhibitors, nor by pertuzumab. However, trastuzumab prevented recovery of P-HER3, suggesting that a ligand-independent HER2-HER3 interaction was involved in partial maintenance of P-HER3. Finally, treatment of mice bearing BT474 xenografts with lapatinib and trastuzumab eliminated 16/16 tumors within ≪3 weeks. Therefore, in HER2+ cancers, inhibition of HER2 with TKIs will inhibit PI3K/Akt and relieve suppression of HER3 expression. In turn, FoxO-induced upregulation of HER3 counteracts the full response to HER2 TKIs. These data suggest that current inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway in sustained fashion unless combined with HER3 antagonists.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 231. doi:10.1158/1538-7445.AM2011-231