Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer.
ABSTRACT: BACKGROUND:Endocrine therapies are still the main strategy for the treatment of oestrogen receptor-positive (ER+) breast cancers (BC), but resistance remains problematic. Cross-talk between ER and PI3K/AKT/mTORC has been associated with ligand-independent transcription of ER. We have previously reported the anti-proliferative effects of the combination of everolimus (an mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape via AKT signalling can lead to resistance; therefore, the use of dual mTORC1/2 inhibitors has met with significant interest. METHODS:To address this, we tested the effect of vistusertib, a dual mTORC1 and mTORC2 inhibitor, in a panel of endocrine-resistant and endocrine-sensitive ER+ BC cell lines, with varying PTEN, PIK3CA and ESR1 mutation status. End-points included proliferation, cell signalling, cell cycle and effect on ER-mediated transcription. Two patient-derived xenografts (PDX) modelling endocrine resistance were used to assess the efficacy of vistusertib, fulvestrant or the combination on tumour progression, and biomarker studies were conducted using immunohistochemistry and RNA-seq technologies. RESULTS:Vistusertib caused a dose-dependent decrease in proliferation of all the cell lines tested and reduced abundance of mTORC1, mTORC2 and cell cycle markers, but caused an increase in abundance of EGFR, IGF1R and ERBB3 in a context-dependent manner. ER-mediated transcription showed minimal effect of vistusertib. Combined therapy of vistusertib with fulvestrant showed synergy in two ER+ PDX models of resistance to endocrine therapy and delayed tumour progression after cessation of therapy. CONCLUSIONS:These data support the notion that models of acquired endocrine resistance may have a different sensitivity to mTOR inhibitor/endocrine therapy combinations.
Project description:INTRODUCTION:Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER?+?endocrine resistant BC cells. METHODS:In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS:RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ?1 ?M), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P?<0.001) growth inhibitor of TamR (IC50 18?nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P?<0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P?<0.05). Co-treating with AZD8055 alongside tamoxifen (P?<0.01) or oestrogen deprivation (P?<0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS:This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.
Project description: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.
Project description:Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n?=?68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.
Project description:Breast cancers (BCs) typically express estrogen receptors (ERs) but frequently exhibit de novo or acquired resistance to hormonal therapies. Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors. In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts. Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens. Importantly, in PDX tumors with acquired tamoxifen resistance, NOTCH4 inhibition reduced BCSC activity. Thus, we establish that BCSC and NOTCH4 activities predict both de novo and acquired tamoxifen resistance and that combining endocrine therapy with targeting JAG1-NOTCH4 overcomes resistance in human breast cancers.
Project description:Breast cancers (BCs) with expression of estrogen receptor-alpha (ER?) occur in more than 70% of newly-diagnosed patients in the U.S. Endocrine therapy with antiestrogens or aromatase inhibitors is an important intervention for BCs that express ER?, and it remains one of the most effective targeted treatment strategies. However, a substantial proportion of patients with localized disease, and essentially all patients with metastatic BC, become resistant to current endocrine therapies. ER? is present in most resistant BCs, and in many of these its activity continues to regulate BC growth. Fulvestrant represents one class of ER? antagonists termed selective ER downregulators (SERDs). Treatment with fulvestrant causes ER? down-regulation, an event that helps overcome several resistance mechanisms. Unfortunately, full antitumor efficacy of fulvestrant is limited by its poor bioavailability in clinic. We have designed and tested a new generation of steroid-like SERDs. Using ER?-positive BC cells in vitro, we find that these compounds suppress ER? protein levels with efficacy similar to fulvestrant. Moreover, these new SERDs markedly inhibit ER?-positive BC cell transcription and proliferation in vitro even in the presence of estradiol-17?. In vivo, the SERD termed JD128 significantly inhibited tumor growth in MCF-7 xenograft models in a dose-dependent manner (P?<?0.001). Further, our findings indicate that these SERDs also interact with ER-positive immune cells in the tumor microenvironment such as myeloid-derived suppressor cells (MDSC), tumor infiltrating lymphocytes and other selected immune cell subpopulations. SERD-induced inhibition of MDSCs and concurrent actions on CD8+ and CD4?+?T-cells promotes interaction of immune checkpoint inhibitors with BC cells in preclinical models, thereby leading to enhanced tumor killing even among highly aggressive BCs such as triple-negative BC that lack ER? expression. Since monotherapy with immune checkpoint inhibitors has not been effective for most BCs, combination therapies with SERDs that enhance immune recognition may increase immunotherapy responses in BC and improve patient survival. Hence, ER? antagonists that also promote ER downregulation may potentially benefit patients who are unresponsive to current endocrine therapies.
Project description:Fulvestrant is the first selective estrogen receptor (ER) downregulator available in clinical practice. It is a pure antiestrogen with no agonistic effects, leading to degradation of ER alpha, with activity in tamoxifen-resistant breast cancer (BC) models. Pharmacokinetic and pharmacodynamic studies and several postmarketing clinical trials led to the definition of the optimal dose at 500 mg intramuscularly on days 1, 15, and 29 and then every 28 days. Targeting ER alpha, fulvestrant is a cornerstone of treatment in luminal BCs, whose growth is largely driven by the ER pathway. In endocrine therapy-naïve patients with hormone receptor-positive, HER2- advanced BC (ABC), fulvestrant yielded significantly longer progression-free survival compared to anastrozole in the Phase III FALCON study. Due to its mechanism of action and pharmacokinetic properties, fulvestrant is an ideal backbone for combination therapies. Preclinical studies have shown synergism with drugs acting on signaling pathways involved in the development of endocrine resistance, among which the cyclin D/cyclin-dependent kinase 4-6/retinoblastoma pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin pathway, contributing to overcoming or delaying endocrine resistance. In the Phase III PALOMA-3 trial, a combination of the cyclin-dependent kinase 4/6 inhibitor palbociclib with fulvestrant significantly improved progression-free survival over fulvestrant alone in women with hormone receptor positive, HER2- ABC progressing during prior endocrine therapy. This led to approval of the combination in this clinical setting. Similar results were obtained with abemaciclib and ribociclib. Combination with pan-PI3K inhibitors, though showing some efficacy, was hampered by the toxicity of these agents, and studies in combinations with more selective inhibitors of the ?-catalytic subunit of PI3K are ongoing. Fulvestrant has shown partial activity also in patients with tumors harboring mutations of the ESR1 gene. It is thus a key drug in the treatment of ABC, whose role in combination with new targeted agents is still evolving.
Project description:<h4>Background</h4>Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer.<h4>Methods</h4>We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer.<h4>Results</h4>We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model.<h4>Conclusions</h4>We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.
Project description:A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.
Project description:Here we characterise the response of models of ER-positive breast cancer to treatment with the small molecule MDM2 inhibitor NVP-CGM097, a dihydroisoquinolinone derivative currently evaluated in a phase I clinical trial. We show that NVP-CGM097 reduces tumour cell viability of in vitro and in vivo models of endocrine sensitive, endocrine resistant and palbociclib (CDK4/6 inhibitor) resistant p53 wildtype (p53wt) ER-positive breast cancer. NVP-CGM097 synergises with both fulvestrant and palbociclib in models of therapy resistance. Importantly, we identify the key mechanisms of the synergistic interactions between NVP-CGM097 and endocrine therapy, which occurs through the inhibition of E2F Targets and G2M Checkpoint signalling and induction of senescence, rather than depending upon upregulation of p53 dependent apoptotic pathways. Moreover, we find these same pathways are synergistically targeted during the combination treatment of ER positive breast cancer models with NVP-CGM097 and palbociclib. This indicates the genuine potential of MDM2 inhibition as therapy in advanced ER-positive breast cancer as combination endocrine therapy and CDK4/6 inhibitor treatment becomes embedded as standard of care. Overall design: Experimental Design: In vitro and in vivo models were treated with NVP-CGM097 alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in treatment naïve and therapy resistant ER positive cell lines. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant breast cancer. Results: We demonstrate that NVP-CGM097 inhibits tumour cells in multiple clinically relevant models of ER-positive breast cancer. Activation of p53 by NVP-CGM097 leads to cell cycle arrest and increased apoptosis in p53-wildtype breast cancer cell lines, and reduces tumour growth in a PDX model. NVP-CGM097 in combination with fulvestrant or palbociclib do not enhance apoptosis, but instead yields profound downregulation of cell cycle-related transcriptional programmes. Cell lines resistant to fulvestrant or palbociclib become senescent upon treatment with combination therapy, and a fulvestrant resistant PDX model reduces tumour growth with combined NVP-CGM097 and fulvestrant treatment.
Project description:Phosphoinositide-3-kinase/protein-kinaseB/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling plays an important role in breast cancer (BC). Its interaction with estrogen receptor (ER) signalling becomes more complex and inter-dependent with acquired endocrine resistance. Targeting mTOR combined with endocrine therapy has shown clinical utility, however, a negative feedback-loop exists downstream of PI3K/AKT/mTOR. Direct blockade of AKT together with endocrine therapy may improve BC treatment. AZD5363, a novel pan-AKT kinase catalytic inhibitor, was examined in a panel of ER+ BC cell lines (MCF7, HCC1428, T47D, ZR75.1) adapted to long-term-estrogen-deprivation (LTED) or tamoxifen (TamR). AZD5363 caused a dose-dependent decrease in proliferation in all cell lines tested (GI50<500nM) except HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the lines (GI50~100nM). AZD5363 re-sensitised TamR cells to tamoxifen and acted synergistically with fulvestrant. AZD5363 decreased p-AKT/mTOR targets leading to a reduction in ERα-mediated transcription in a context specific manner and concomitant decrease in recruitment of ER and CREB-binding protein (CBP) to estrogen-response-elements located on the TFF1, PGR and GREB1 promoters. Furthermore, AZD5363 reduced expression of cell-cycle-regulatory proteins. Global gene expression highlighted ERBB2-ERBB3, ERK5 and IGF1 signaling pathways driven by MYC as potential feedback-loops. Combined treatment with AZD5363 and fulvestrant showed synergy in an ER+ patient derived xenograft and delayed tumour progression post-cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a potential therapy for BC that is sensitive or resistant to E-deprivation or tamoxifen and that activated AKT is a determinant of response, supporting the need for clinical evaluation. Cell lines were treated in biological triplicates in the absence of estrogen with or without AZD5363 for 24hours in order to identify gene changes associated with perturbation of AKT signalling