Cancer Letters

Cancer Letters

Volume 380, Issue 1, 28 September 2016, Pages 20-30
Cancer Letters

Original Articles
The anti-HER3 antibody in combination with trastuzumab exerts synergistic antitumor activity in HER2-positive gastric cancer

https://doi.org/10.1016/j.canlet.2016.06.005Get rights and content

Highlights

  • Targeting both HER2 and HER3 with the combination of anti-HER3 antibody and trastuzumab may result in an improved treatment effects on HER2-positive GC.

  • The mechanism relies on inhibitory action of phosphorylation of HER3 as well as AKT and ERK signals.

  • A HER2-negative whereas NRG1-positive PDX model also benefited from combination treatment of anti-HER3 antibody and trastuzumab.

Abstract

The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited. HER3 seems to be the preferred dimerization partner with HER2 and is emerging as a key target for complete blockade of downstream pathways and better clinical response. In this study, we report that novel anti-HER3 antibodies (1A5–3D4) that can neutralize multiple modes of HER3 activation, combined with trastuzumab, exhibited synergistic inhibitory effect on the cell proliferation in HER2-positive GC cell lines. Follow-up studies revealed that the combination treatment significantly inhibited phosphorylation of HER3 as well as AKT and ERK signals. In vivo experiments further showed that the anti-tumor effect of trastuzumab was enhanced by its combination with 1A5–3D4 in NCI-N87 xenograft and patient derived xenografts (PDX). Particularly in an HER2-negative whereas neuregulin1 (a ligand of HER3) positive PDX, the combination was also superior to monotherapy. 1A5–3D4 in combination with trastuzumab exhibits a synergistic inhibitory effect on tumor activity, suggesting that targeting both HER2 and HER3 resulted in an improved treatment effects on HER2-positive GC.

Introduction

Gastric cancer (GC) is the third most common cause of cancer-related deaths worldwide, accounting for 723,000 deaths per year [1]. However, clinical approaches for treatment of GC are limited and the median survival for advanced (AGC) remains around 8 to 10 months [2]. Trastuzumab, a monoclonal antibody against human epidermal growth factor receptor 2 (HER2), is hitherto the only targeted biologic agent that has been demonstrated to confer overall survival benefit in the first-line treatment of HER2-positive AGC [3], and thus it is widely used in clinic. Nevertheless, the objective response rate of trastuzumab combined with chemotherapy was only 47%, and even initially responsive patients mostly suffered from progression within one year due to de novo or acquired resistance [3]. Thus, there is an urgent need to develop new therapeutic strategies that could overcome the limited efficacy of trastuzumab.

There are many proposed mechanisms underlying trastuzumab resistance [4]. Recent studies have shown that the activation of HER3 signaling plays a significant role in HER2-positive GC [5], [6]. HER3 is a kinase dead member of the HER family [7], whereas HER2 has no known ligand [8]. Although HER2 and HER3 are functionally incomplete on their own, their heterodimer formed in both ligand-dependent and ligand-independent manners [9] is the key activator of PI3K/AKT signaling in HER2-driven cancers [10], [11]. Prior studies have suggested that the compensatory resumed HER3 signaling is driven by high level of hetero-dimerization partner HER2 [12] or by PI3K/AKT driven inhibitory feedback loop in the presence of trastuzumab [13], [14]. Besides, elevated expression of the predominant ligand for HER3, neuregulin1 (NRG1), may drive the ligand-dependent HER3 signaling in GC and many other types of tumors [15], [16], [17]. Trastuzumab is able to block HER2–HER3 dimerization that occur in the absence of ligands by binding to domain IV of HER2 extracellular domain (ECD) [18], but it is not effective in blocking ligand-dependent HER2–HER3 dimerization, which is required to bind to domain II of HER2–ECD, resulting in unrestrained ligand-dependent HER3 signaling [9]. The inability of trastuzumab of fully inhibiting HER3 signaling and consequent PI3K/AKT signaling is considered as a reason for its limited therapeutic benefit [19].

In this regard, HER3 is emerging as a key target for complete blockade of the signaling pathway, in purpose of improving theefficacy of trastuzumab. While the rationale for HER3 blockage in GC is mainly rooted in previous experience with breast cancer [5], [20], [21]. Therapeutic targeting of HER3 in combination with trastuzumab need to be evaluated in GC [22]. Following this rationale, the HER3-targeted humanized monoclonal antibodies, 1A5 and 3D4, were developed. The two anti-HER3 antibodies bind to different epitopes of HER3, allowing them to simultaneously block both ligand-dependent and ligand-independent HER3 signaling. The aim of this study was to investigate the potential synergetic effects of a combination of anti-HER3 antibody and trastuzumab targeting HER2 and HER3 and its underlying mechanisms in the treatment of HER2-positive GC.

Section snippets

Reagents and antibodies

Trastuzumab (Herceptin) was purchased from Shanghai Roche Pharmaceutical Ltd., the anti-HER3 antibodies, 3D4 and 1A5, were produced by Beijing Cotimes Biotech Co.,Ltd (Patent Number: CN104011079A). NRG1 (rhNRG1-β1) was purchased from R&D Systems (396-HB-050). The HER2 (#4290), HER3 (#12708), p-HER3 (#2842), AKT (#4691), p-AKT (#4060), ERK (#4695), p-ERK (#4370), S6 (#2217), p-S6 (#4858) antibodies were purchased from Cell Signaling Technology. The anti-β-actin antibody (Lot#014M4759) was

Combination of 1A5–3D4 with trastuzumab synergistically inhibited cell proliferation and both PI3K/AKT and ERK pathways in HER2-positive GC cells

1A5 and 3D4 are two different antibodies designed to bind to sub-domains of HER3 without overlapping epitopes that allows them to simultaneously bind HER3. 1A5 inhibited HER3 phosphorylation irrespective with the levels of NRG1, whereas 3D4 can effectively prevent ligand-dependent phosphorylation of HER3 (Fig. S1A). Compared to 1A5 or 3D4 combined with trastuzumab, the combination of 1A5 and 3D4 (hereafter called 1A5–3D4) combined with trastuzumab showed more significantly synergistic effects

Discussion

Although clinical benefit has been well-established with trastuzumab, more potent targeted therapies for HER2 positive GC is necessary to overcome its limitation in efficacy. As the preferred dimerization partner for HER2, HER3 is a crucial activator with respect to the strength of interaction and downstream signaling. As HER3 lacks innate kinase activity, attention has been placed on targeting the HER3-ECD through antibodies, including antagonizing ligand binding to the ECD and blocking the

Conflict of interest

All the authors declare that they had no conflict of interest.

Acknowledgments

This study was supported by grants from National Natural Science Foundation of China (No. 81301915).

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    These authors contributed equally to this work.

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