Abstract 531: HER4 cleavage and nuclear localization mediate Trastuzumab resistance in HER2-positive breast cancer.
Authors: Mohd Nafi Siti Norasikin , Gijsen Merel , Roxanis Ioannis , Kramer-Marek Gabriela , Capala Jacek , Kong Anthony 
Published: August 2014

Abstract
Background. Trastuzumab is used in HER2 positive breast cancer patients and has been shown to increase survival in these patients. However, drug resistance imposes a crucial limitation to the successful treatment of HER2 positive breast cancer. Multiple mechanisms have been proposed but the role of HER4 in relation to Trastuzumab treatment and resistance remains unclear. This study aims to investigate the role of HER4 in relation to Trastuzumab treatment and resistance in HER2 positive breast cancer. Methods. The effects of Trastuzumab on the protein and mRNA levels of HER4 were studied in both naïve and Trastuzumab resistant cells using western blot and RT-PCR. HER4 localisation after Trastuzumab treatment was determined by confocal microscopy and nuclear fractionation. A gamma-secretase inhibitor (GSI) and a pan-HER kinase inhibitor, Neratinib, were used to inhibit HER4 cleavage during Trastuzumab treatment. The role of HER4 was further validated using siRNA experiments. HER4 level and its localisation were also assessed by IHC in BT474 xenograft samples treated with Trastuzumab or control. To understand the prognostic role of HER4, TMAs of a cohort of HER2 positive breast cancer patients were stained for HER4 expression using IHC and the levels were correlated with patients’ outcome. Results. The basal level of HER4 in HER2 positive SKBR3 and BT474 is comparable to HER2 negative MCF7 cells. In SKBR3 and BT474, Trastuzumab treatment upregulated HER4 mRNA and protein expression in a dose responsive manner. Moreover, HER480kDa was also increased and an enhanced nuclear localisation was observed, indicating that Trastuzumab promotes HER4 cleavage. Resistant cells appear to have more nuclear HER4 and Trastuzumab withdrawal causes a decrease in nuclear intensity. HER4 downregulation via siRNA reduces cell viability in Trastuzumab sensitive and resistant cells. In SKBR3, GSI decreased HER4 nuclear staining and decreased Trastuzumab induced HER4 nuclear translocation. In addition, GSI also enhanced response to Trastuzumab in decreasing cell viability. Neratinib also blocks HER480kDa formation induced by Trastuzumab, and combination treatment of Trastuzumab and Neratinib had an additive inhibition effect on cell viability. There was a significant upregulation of nuclear HER4 in a Trastuzumab treated BT474 xenograft model compared to the control. Nuclear HER4 was also prognostic in a cohort of HER2 positive breast cancer patients. Conclusion. This study shows that Trastuzumab upregulates HER4 in vitro and in vivo. Nuclear HER4 seems to mediate Trastuzumab resistance in HER2 positive breast cancer cells. HER4 knockdown as well as drug treatments that decrease nuclear HER4 may enhance sensitivity to Trastuzumab. We believe that the prognostic and predictive values of nuclear HER4 in relation to Trastuzumab resistance need to be further validated in HER2 positive breast cancer patients.
Citation Format: Siti Norasikin Mohd Nafi, Merel Gijsen, Ioannis Roxanis, Gabriela Kramer-Marek, Jacek Capala, Anthony Kong. HER4 cleavage and nuclear localization mediate Trastuzumab resistance in HER2-positive breast 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 531. doi:10.1158/1538-7445.AM2013-531