Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Abstract

Background: Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER⁺) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility.

Methods: A panel of ER⁺ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1 ^wt or ESR1 ^Y537S , modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies.

Results: Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001.

Conclusions: Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.

Keywords: Breast cancer; Endocrine resistance; Everomilus; Neratinib; Oestrogen receptor.

Fig. 1

Simplified schematic diagram of the pathways described in this study. a Growth factor signalling (IGFR and ERBB) leads to activation of PI3K and phosphorylation of AKT. AKT inhibits TCS1/2, resulting in upregulation of mTORC1. In parallel, mTORC1 can also be upregulated by the RAS-RAF-MEK-ERK signalling pathway. ERK phosphorylates and inactivates TCS2 also leading to mTORC1 activation. S6 K1 activity increases as a result of mTORC1 activation. S6 K1 suppresses mTORC2 and IRS1. ER is also a target of S6 K1 leading to phosphorylation of serine 167. b Inhibition of mTORC1 with everolimus suppresses S6 K1 removing the negative feedback loop causing a rise in IRS1 and AKT activity via loss of suppression on mTORC2. Increased AKT activity suppresses TCS1/2 and increases expression of growth factor receptors (ERBB2/3) enhancing RAS-RAF-ERK signalling. c The dual blockade of ERBBs (neratinib) and mTORC1 signalling (everolimus) may suppress the two feedback loops described in b. Yellow shows normal mTORC signalling cascade; blue represents activated proteins; red represents inhibited proteins; dotted lines show loss of normal feedback loops

Fig. 2

Anti-proliferative effect of a RAD001 and b neratinib in endocrine-resistant and -sensitive BC cell lines. Cells were treated in the absence or presence of exogenous E2 (0.01 nM) and doubling concentrations of RAD001 or neratinib. Treatments were performed at day 1 and day 3 after seeding. After 6 days of treatment, cell viability was analysed using a cell titer-glo assay. Data are expressed as fold-change relative to dextran charcoal (DCC) control. Error bars represent mean ± SEM

Fig. 3

Anti-proliferative effect of RAD001 (RAD), neratinib (Ner), or their combination in endocrine-resistant and -sensitive BC cell lines. Cell lines were treated with vehicle or sub-optimal concentrations for each drug alone or in combination, both in the absence and presence of 0.01 nM exogenous E2. After 6 days of treatment, cell viability was analysed using cell titer-glo and data expressed as fold-change relative to vehicle control. Error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Concentrations used in dextran charcoal (DCC): wt-MCF7 (0.75 nM RAD001, 2000 nM neratinib); MCF7-LTED (0.75 nM RAD001, 500 nM neratinib); wt-SUM44 (0.75 nM RAD001, 2000 nM neratinib); SUM44-LTED (0.4 nM RAD001, 500 nM neratinib); wt-HCC1428 (12.5 nM RAD001, 1200 nM neratinib); HCC1428-LTED (3 nM RAD001, 250 nM neratinib). Concentrations used in E2: wt-MCF7 (1.5 nM RAD001, 200 nM neratinib); MCF7-LTED (1.5 nM RAD001, 300 nM neratinib); wt-SUM44 (0.37 nM RAD001, 450 nM neratinib); SUM44-LTED (0.37 nM RAD001, 250 nM neratinib); wt-HCC1428 (1.5 nM RAD001, 500 nM neratinib); HCC1428-LTED (3 nM RAD001, 250 nM neratinib)

Fig. 4

Effect of RAD001 (RAD), neratinib (Ner), or their combination with endocrine agents on cell signalling pathways governing cell cycle. Endocrine-resistant and -sensitive BC cell lines were treated for 24 h with the drug combinations indicated. Whole-cell extracts were assessed for expression on S6 kinase, ERK1/2, AKT, and ERBB signalling together with markers of cell cycle and apoptosis by immunoblotting. IC₅₀ values were used for RAD001 and neratinib together with standard concentrations of oestradiol (E2; 0.01 nM), 4-hydroxytamoxifen (4-OHT; 10 nM), and fulvestrant (ICI; 1 nM) (with exception of HCC1428-LTED where 10 nM was used). ERBB pathways are highlighted in pink, ERK1/2 in blue, mTORC1/AKT in green, and cell cycle in orange. wt-MCF7 (2 nM RAD001, 500 nM neratinib); MCF7-LTED (4 nM RAD001, 750 nM neratinib); wt-SUM44 (3 nM RAD001, 700 nM neratinib); SUM44-LTED (3 nM RAD001, 700 nM neratinib); wt-HCC1428 (3 nM RAD001, 1000 nM neratinib); HCC1428-LTED (10 nM RAD001, 500 nM neratinib)

Fig. 5

Effect of RAD001 (RAD), neratinib (Ner), or their combination on oestrogen receptor (ER)-mediated transactivation and recruitment of the ER basal transcription machinery. a Cell lines were co-transfected with EREIItkLuc and pCH110 and treated for 24 h with RAD001 and neratinib in the absence of E2 (DCC). Luciferase activity was normalized by β-galactosidase from triplicate wells and fold-changes expressed relative to the DCC control. b ChIP analysis to determine the effect of neratinib, RAD001, or the combination on recruitment of ER to the TFF1 promoter in wt-HCC1428 and HCC1428-LTED. Error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Concentration used for transactivation assay and ChIP: wt-MCF7 (2 nM RAD001, 500 nM neratinib); MCF7-LTED (4 nM RAD001, 650 nM neratinib); wt-SUM44 (3 nM RAD001, 700 nM neratinib); SUM44-LTED (3 nM RAD001, 700 nM neratinib); wt-HCC1428 (3 nM RAD001, 700 nM neratinib); HCC1428-LTED (10 nM RAD001, 300 nM neratinib). ns not significant

Fig. 6

Effect of RAD001 (RAD) and neratinib (Ner) alone or in combination with endocrine therapy in vivo. a Long-term study assessing the relative mean changes in tumour volume over 41 days of treatment and b effect of drug regimes on animal weight. Error bars represent mean ± SEM (n = 7–9 animals per group). RAD001, 2 mg/kg; neratinib, 40 mg/kg; fulvestrant (ICI), 5 mg/kg. c–f GSEA enrichment plots for 198 genes known to be induced by sustained activation of ERK in response to EGF activity. Plots show the profile of the running Enrichment Score and positions of GeneSet Members on the Rank Ordered List for rank gene lists generated from the comparison of c neratinib vs. vehicle (Veh), d RAD001 vs. vehicle, e RAD001 + neratinib vs. RAD001, and f RAD001 + neratinib + ICI vs. RAD001 + neratinib