Direct estrogen receptor (ER) / HER family crosstalk mediating sensitivity to lumretuzumab and pertuzumab in ER+ breast cancer

Abstract

Bidirectional cross talk between members of the human epidermal growth factor family of receptors (HER) and the estrogen receptor (ER) is believed to underlie resistance mechanisms that develop in response to treatment with anti-HER agents and endocrine therapy. We investigated the interaction between HER2, HER3 and the ER in vitro using human embryonic kidney cells transfected with human HER2, HER3, and ERα. We also investigated the additive efficacy of combination regimens consisting of anti-HER3 (lumretuzumab), anti-HER2 (pertuzumab), and endocrine (fulvestrant) therapy in vivo. Our data show that both HER2 and HER3 can directly complex with the ER and can mediate phosphorylation of the ER. Phosphorylation of the ER was only observed in cells that expressed both HER2 and ERα or in heregulin-stimulated cells that expressed both HER3 and ERα. Using a mouse xenograft model of ER+/HER2-low (HER2 immunohistochemistry 1+ or 2+ without gene amplification) human breast cancer we show that the combination of lumretuzumab and pertuzumab is highly efficacious and induces long-lasting tumor regression in vivo and adding endocrine therapy (fulvestrant) to this combination further improved efficacy. In addition, a prolonged clinical response was observed with the combination of lumretuzumab and pertuzumab in a patient with ER+/HER2-low breast cancer who had failed endocrine therapy. These preclinical data confirm that direct cross talk exists between HER2/HER3 and ER which may explain the resistance mechanisms to endocrine therapy and monoclonal antibodies that target HER2 and HER3. Our data also indicate that the triplet of anti-HER2, anti-HER3, and endocrine therapy might be an efficacious combination for treating patients with ER+/HER2-low breast cancer, which is an area of significant unmet medical need.

Fig 1. Lumretuzumab and pertuzumab both potently inhibit HER2/HER3 signaling.

MC7-7 cells (3 × 10⁵ per six-well plate) were stimulated with or without HRG (5 ng/mL for 10 minutes) in vitro and treated with pertuzumab, trastuzumab, or lumretuzumab each at 10 μmol/L or vehicle control (PBS) for 1 hour, after which cell lysates were examined by Western blotting. Downregulation of pHER3, pMAPK, and pSHC was greater with pertuzumab than trastuzumab (A). Lumretuzumab monotherapy effectively inhibited HER2/HER3 signaling (B). All experiments were carried out in triplicate.

Fig 2. Lumretuzumab plus pertuzumab induces long-lasting tumor remission and inhibits HER2/HER3 signaling in estrogen-dependent breast cancer.

(A) Outbred athymic (nu/nu) female mice (n = 10 per treatment group) bearing ER+/HER2-low/HER3+ human breast cancer xenografts (HBCx-19) were treated with single-agent lumretuzumab (10 mg/kg i.p.), single-agent pertuzumab (15 mg/kg i.p. with a two-fold loading dose), the combination of both antibodies, or with vehicle only (control). All treatments were given weekly beginning on Day 26 when median tumor size was 100–150 mm³ for 6 weeks (until Day 61). (B) Longer term follow-up of the mice depicted in Fig 1A showed that remissions in mice treated with lumretuzumab plus pertuzumab were long-lasting after the final dose of combination therapy on Day 61 (black arrow). Inhibition of MAPK (C) and AKT (D) in tumors harvested from mice on Day 94 (Day 40 for vehicle controls) was greatest with combination therapy.

Fig 3. HER2 and HER3 both interact and co-immunoprecipitate with the ERα.

(A–C) HEK 293 cells transfected with human HER2 and ERα (alone or together) and stimulated with (+) or without (-) HRG were either immunoprecipitated with an anti-HER2 antibody and blotted against ERα (A) or vice versa (B). Results show that HER2 and ERα co-precipitated in cells transfected with cDNAs encoding both proteins, indicating that HER2 and ERα can form a complex. Western blotting of total cell lysate revealed that the ERα was phosphorylated only in cells that expressed both HER2 and ERα (C). The experiments were also conducted in HEK 293 cells transfected with human HER3 and ERα (alone or together; D–F). Again, HER3 and ERα co-precipitated (D&E) and ERα was only phosphorylated in HRG-treated cells expressing both HER3 and ERα (F). Data represent the results of 2–3 independent experiments.

Fig 4. Adding anti-estrogen therapy further improves the efficacy of lumretuzumab and pertuzumab.

HBCx-19 ER+/HER2-low/HER3+ breast cancer xenograft-bearing mice (n = 10 per treatment group) were treated with lumretuzumab (3 mg/kg i.p.), pertuzumab (3 mg/kg i.p.), and fulvestrant (50 mg/kg i.m.) either as single agents or in combination. Lower (sub-optimal) doses of lumretuzumab and pertuzumab were used to discriminate the additional contribution of fulvestrant. All treatments were given weekly beginning on Day 26 when median tumor size was 100–150 mm³ for 6 weeks (until Day 57).

Fig 5. Tumor regression in a breast cancer patient treated with lumretuzumab plus pertuzumab and paclitaxel.

This 37-year-old patient received 23 cycles of lumretuzumab and pertuzumab. The black arrow indicates the discontinuation of paclitaxel treatment after 18 weeks. Circles indicate the timing of tumor assessments. Tumors were assessed according to RECIST version 1.1.