RESUMO
The selective oestrogen receptor (ER) degrader (SERD), fulvestrant, is limited in its use for the treatment of breast cancer (BC) by its poor oral bioavailability. Comparison of the orally bioavailable investigational SERD elacestrant, versus fulvestrant, demonstrates both drugs impact tumour growth of ER+ patient-derived xenograft models harbouring several ESR1 mutations but that elacestrant is active after acquired resistance to fulvestrant. In cell line models of endocrine sensitive and resistant breast cancer both drugs impact the ER-cistrome, ER-interactome and transcription of oestrogen-regulated genes similarly, confirming the anti-oestrogenic activity of elacestrant. The addition of elacestrant to CDK4/6 inhibitors enhances the antiproliferative effect compared to monotherapy. Furthermore, elacestrant inhibits the growth of palbociclib-resistant cells. Lastly, resistance to elacestrant involves Type-I and Type-II receptor tyrosine kinases which are amenable to therapeutic targeting. Our data support the wider clinical testing of elacestrant.
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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.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Sequenciamento do Exoma/métodos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/uso terapêutico , Piridinas/uso terapêutico , Quinase 1 Polo-LikeRESUMO
Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Receptores de Estrogênio/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fulvestranto/administração & dosagem , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Interferência de RNA , Receptores de Estrogênio/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Tamoxifeno/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
After publication of the original article [1], we were notified that an author's surname has been erroneously spelled. Elisabetta Maragoni's family name should be replaced with Marangoni.
RESUMO
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.
RESUMO
BACKGROUND: Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS: The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS: Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS: Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.
Assuntos
Inibidores da Aromatase/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Inibidores da Aromatase/efeitos adversos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase 4/genética , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Receptor ErbB-2/genéticaRESUMO
BACKGROUND: Endocrine therapy reduces breast cancer mortality by 40%, but resistance remains a major clinical problem. In this study, we sought to investigate the impact of aromatase inhibitor (AI) therapy on gene expression and identify gene modules representing key biological pathways that relate to early AI therapy resistance. METHODS: Global gene expression was measured on pairs of core-cut biopsies taken at baseline and at surgery from 254 patients with ER-positive primary breast cancer randomised to receive 2-week presurgical AI (n = 198) or no presurgical treatment (control n = 56) from the POETIC trial. Data from the AI group was adjusted to eliminate artefactual process-related changes identified in the control group. The response was assessed by changes in the proliferation marker, Ki67. RESULTS: High baseline ESR1 expression associated with better AI response in HER2+ tumours but not HER2- tumours. In HER2- tumours, baseline expression of 48 genes associated with poor antiproliferative response (p < 0.005) including PERP and YWHAQ, the two most significant, and the transcription co-regulators (SAP130, HDAC4, and NCOA7) which were among the top 16 most significant. Baseline gene signature scores measuring cell proliferation, growth factor signalling (ERBB2-GS, RET/GDNF-GS, and IGF-1-GS), and immune activity (STAT1-GS) were significantly higher in poor AI responders. Two weeks of AI caused downregulation of genes involved in cell proliferation and ER signalling, as expected. Signature scores of E2F activation and TP53 dysfunction after 2-week AI were associated with poor AI response in both HER2- and HER2+ patients. CONCLUSIONS: There is a high degree of heterogeneity in adaptive mechanisms after as little as 2-week AI therapy; however, all appear to converge on cell cycle regulation. Our data support the evaluation of whether an E2F signatures after short-term exposure to AI may identify those patients most likely to benefit from the early addition of CDK4/6 inhibitors. TRIAL REGISTRATION: ISRCTN, ISRCTN63882543, registered on 18 December 2007.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptores de Estrogênio/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Período Perioperatório , Pós-Menopausa , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Transdução de Sinais/genéticaRESUMO
Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/administração & dosagem , Receptor alfa de Estrogênio/antagonistas & inibidores , Indazóis/administração & dosagem , Mutação , Administração Oral , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Indazóis/química , Indazóis/farmacologia , Células MCF-7 , Camundongos , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Resistance to endocrine therapy remains a major clinical problem in the treatment of oestrogen-receptor positive (ER+) breast cancer. Studies show androgen-receptor (AR) remains present in 80-90% of metastatic breast cancers providing support for blockade of AR-signalling. However, clinical studies with abiraterone, which blocks cytochrome P450 17A1 (CYP17A1) showed limited benefit. METHODS: In order to address this, we assessed the impact of abiraterone on cell-viability, cell-death, ER-mediated transactivation and recruitment to target promoters. together with ligand-binding assays in a panel of ER+ breast cancer cell lines that were either oestrogen-dependent, modelling endocrine-sensitive disease, or oestrogen-independent modelling relapse on an aromatase inhibitor. The latter, harboured wild-type (wt) or naturally occurring ESR1 mutations. RESULTS: Similar to oestrogen, abiraterone showed paradoxical impact on proliferation by stimulating cell growth or death, depending on whether the cells are hormone-dependent or have undergone prolonged oestrogen-deprivation, respectively. Abiraterone increased ER-turnover, induced ER-mediated transactivation and ER-degradation via the proteasome. CONCLUSIONS: Our study confirms the oestrogenic activity of abiraterone and highlights its differential impact on cells dependent on oestrogen for their proliferation vs. those that are ligand-independent and harbour wt or mutant ESR1. These properties could impact the clinical efficacy of abiraterone in breast cancer.
Assuntos
Androstenos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/genética , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Apoptose/efeitos dos fármacos , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Mutação , Metástase Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Receptores Androgênicos/genética , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/genética , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Estradiol/farmacologia , Estrogênios/metabolismo , Everolimo/farmacologia , Feminino , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
To investigate the impact of sampling methodology on gene expression data from primary estrogen receptor-positive (ER+) breast cancer biopsies, global gene expression was measured in core-cut biopsies at baseline and surgery from patients randomly assigned to receive either two weeks of presurgical aromatase inhibitor (AI; n = 157) or no presurgical treatment (n = 56). Those genes most markedly altered in the AI group (eg, FOS, DUSP1, RGS1, FOSB) were similarly altered in the no treatment group; some widely investigated genes that were apparently unaffected in the AI group (eg, MYC) were counter-altered in the control group, masking actual AI-dependent changes. In the absence of a control group, these artefactual changes would likely lead to the most affected genes being the erroneous focus of research. The findings are likely relevant to all archival collections of ER+ breast cancer.
RESUMO
Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/análogos & derivados , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/genética , Linhagem Celular Tumoral , Estradiol/uso terapêutico , Feminino , Fulvestranto , Humanos , Células MCF-7 , Mutação , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêuticoRESUMO
BACKGROUND: Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting. METHODS: To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed. RESULTS: The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30-50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy. CONCLUSION: Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.
Assuntos
Antineoplásicos Hormonais/farmacologia , Vias Biossintéticas , Neoplasias da Mama/metabolismo , Colesterol/biossíntese , Resistencia a Medicamentos Antineoplásicos , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ésteres do Colesterol/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fenótipo , Prognóstico , Proteoma , Proteômica/métodos , Interferência de RNA , Transcriptoma , Resultado do TratamentoRESUMO
Despite the effectiveness of endocrine therapies in estrogen receptor positive (ER+) breast cancer, approximately 40% of patients relapse. Previously, we identified the Focal-adhesion kinase canonical pathway as a major contributor of resistance to estrogen deprivation and cellular-sarcoma kinase (c-src) as a dominant gene in this pathway. Dasatinib, a pan-src inhibitor, has recently been used in clinical trials to treat ER+ patients but has shown mixed success. In the following study, using isogenic cell line models, we provide a potential explanation for these findings and suggest a sub-group that may benefit. A panel of isogenic cell lines modelling resistance to aromatase inhibitors (LTED) and tamoxifen (TAMR) were assessed for response to dasatinib ± endocrine therapy. Dasatinib caused a dose-dependent decrease in proliferation in MCF7-TAMR cells and resensitized them to tamoxifen and fulvestrant but not in HCC1428-TAMR. In contrast, in estrogen-deprived conditions, dasatinib increased the proliferation rate of parental-MCF7 cells and had no effect on MCF7-LTED or HCC1428-LTED. Treatment with dasatinib caused a decrease in src-phosphorylation and inhibition of downstream pathways, including AKT and ERK1/2 in all cell lines tested, but only the MCF7-TAMR showed a concomitant decrease in markers of cell cycle progression. Inhibition of src also caused a significant decrease in cell migration in both MCF7-LTED and MCF7-TAMR cells. Finally, we showed that, in MCF7-TAMR cells, in contrast to tamoxifen sensitive cell lines, ER is expressed throughout the cell rather than being restricted to the nucleus and that treatment with dasatinib resulted in nuclear shuttling of ER, which was associated with an increase in ER-mediated transcription. These data suggest that src has differential effects in endocrine-resistant cell lines, particularly in tamoxifen resistant models, with low ER genomic activity, providing further evidence of the importance of patient selection for clinical trials testing dasatinib utility in ER+ breast cancer.
Assuntos
Antineoplásicos Hormonais/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/genética , Quinases da Família src/antagonistas & inibidores , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Humanos , Células MCF-7 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologia , Ativação Transcricional , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Aromatase inhibitors (AI) have become the first-line endocrine treatment of choice for postmenopausal estrogen receptor-positive (ER(+)) breast cancer patients, but resistance remains a major challenge. Metabolic reprogramming is a hallmark of cancer and may contribute to drug resistance. Here, we investigated the link between altered breast cancer metabolism and AI resistance using AI-resistant and sensitive breast cancer cells, patient tumor samples, and AI-sensitive human xenografts. We found that long-term estrogen deprivation (LTED), a model of AI resistance, was associated with increased glycolysis dependency. Targeting the glycolysis-priming enzyme hexokinase-2 (HK2) in combination with the AI, letrozole, synergistically reduced cell viability in AI-sensitive models. Conversely, MCF7-LTED cells, which displayed a high degree of metabolic plasticity, switched to oxidative phosphorylation when glycolysis was impaired. This effect was ER dependent as breast cancer cells with undetectable levels of ER failed to exhibit metabolic plasticity. MCF7-LTED cells were also more motile than their parental counterparts and assumed amoeboid-like invasive abilities upon glycolysis inhibition with 2-deoxyglucose (2-DG). Mechanistic investigations further revealed an important role for miR-155 in metabolic reprogramming. Suppression of miR-155 resulted in sensitization of MCF7-LTED cells to metformin treatment and impairment of 2-DG-induced motility. Notably, high baseline miR-155 expression correlated with poor response to AI therapy in a cohort of ER(+) breast cancers treated with neoadjuvant anastrozole. These findings suggest that miR-155 represents a biomarker potentially capable of identifying the subset of breast cancers most likely to adapt to and relapse on AI therapy.
Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Estrogênios/genética , MicroRNAs/genética , Receptores de Estrogênio/genética , Anastrozol , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Glicólise/efeitos dos fármacos , Glicólise/genética , Hexoquinase/genética , Humanos , Letrozol , Células MCF-7 , Camundongos , Camundongos Nus , Terapia Neoadjuvante/métodos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Triazóis/farmacologiaRESUMO
Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.
Assuntos
Antineoplásicos/administração & dosagem , Inibidores da Aromatase/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Reação em Cadeia da Polimerase MultiplexRESUMO
PI3K/AKT/mTOR signaling plays an important role in breast cancer. Its interaction with estrogen receptor (ER) signaling becomes more complex and interdependent 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 breast cancer treatment. AZD5363, a novel pan-AKT kinase catalytic inhibitor, was examined in a panel of ER(+) breast cancer 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 < 500 nmol/L) except HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the lines (GI50 â¼ 100 nmol/L). AZD5363 resensitized 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 IGFI 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 tumor progression after cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a potential therapy for breast cancer 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.
Assuntos
Antineoplásicos Hormonais/farmacologia , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Sinergismo Farmacológico , Estradiol/farmacologia , Feminino , Fulvestranto , Perfilação da Expressão Gênica , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ativação Transcricional , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. METHODS: Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n=84), gene expression profiling (n=47), matched pre- and post-AI aCGH (n=19 pairs) and Ki67-based AI-response analysis (n=39). RESULTS: Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). CONCLUSIONS: These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Animais , Antineoplásicos Hormonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores da Aromatase/farmacologia , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Colina Quinase/genética , Colina Quinase/metabolismo , Aberrações Cromossômicas , Análise por Conglomerados , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Humanos , Camundongos , Terapia Neoadjuvante , Estadiamento de Neoplasias , Prognóstico , Receptores de Estrogênio/metabolismo , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
INTRODUCTION: Endocrine therapies target oestrogenic stimulation of breast cancer (BC) growth, but resistance remains problematic. Our aims in this study were (1) to identify genes most strongly associated with resistance to endocrine therapy by intersecting global gene transcription data from patients treated presurgically with the aromatase inhibitor anastrazole with those from MCF7 cells adapted to long-term oestrogen deprivation (LTED) (2) to assess the clinical value of selected genes in public clinical data sets and (3) to determine the impact of targeting these genes with novel agents. METHODS: Gene expression and Ki67 data were available from 69 postmenopausal women with oestrogen receptor-positive (ER+) early BC, at baseline and 2 weeks after anastrazole treatment, and from cell lines adapted to LTED. The functional consequences of target genes on proliferation, ER-mediated transcription and downstream cell signalling were assessed. RESULTS: By intersecting genes predictive of a poor change in Ki67 with those upregulated in LTED cells, we identified 32 genes strongly correlated with poor antiproliferative response that were associated with inflammation and/or immunity. In a panel of LTED cell lines, C-X-C chemokine receptor type 7 (CXCR7) and CXCR4 were upregulated compared to their wild types (wt), and CXCR7, but not CXCR4, was associated with reduced relapse-free survival in patients with ER+ BC. The CXCR4 small interfering RNA variant (siCXCR4) had no specific effect on the proliferation of wt-SUM44, wt-MCF7 and their LTED derivatives. In contrast, siCXCR7, as well as CCX733, a CXCR7 antagonist, specifically suppressed the proliferation of MCF7-LTED cells. siCXCR7 suppressed proteins associated with G1/S transition and inhibited ER transactivation in MCF7-LTED, but not wt-MCF7, by impeding association between ER and proline-, glutamic acid- and leucine-rich protein 1, an ER coactivator. CONCLUSIONS: These data highlight CXCR7 as a potential therapeutic target warranting clinical investigation in endocrine-resistant BC.
Assuntos
Antineoplásicos Hormonais/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/metabolismo , Receptores CXCR/metabolismo , Receptores de Estrogênio/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Apoptose , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Fosforilação , Pós-Menopausa , Processamento de Proteína Pós-Traducional , Receptores CXCR/genética , Ativação TranscricionalRESUMO
PURPOSE: Endocrine therapies include aromatase inhibitors and the selective estrogen receptor (ER) downregulator fulvestrant. This study aimed to determine whether the reported efficacy of fulvestrant over anastrozole, and high- over low-dose fulvestrant, reflect distinct transcriptional responses. EXPERIMENTAL DESIGN: Global gene expression profiles from ERα-positive breast carcinomas before and during presurgical treatment with fulvestrant (n = 22) or anastrozole (n = 81), and corresponding in vitro models, were compared. Transcripts responding differently to fulvestrant and estrogen deprivation were identified and integrated using Gene Ontology, pathway and network analyses to evaluate their potential significance. RESULTS: The overall transcriptional response to fulvestrant and estrogen deprivation was correlated (r = 0.61 in presurgical studies, r = 0.87 in vitro), involving downregulation of estrogen-regulated and proliferation-associated genes. The transcriptional response to fulvestrant was of greater magnitude than estrogen deprivation (slope = 0.62 in presurgical studies, slope = 0.63 in vitro). Comparative analyses identified 28 genes and 40 Gene Ontology categories affected differentially by fulvestrant. Seventeen fulvestrant-specific genes, including CAV1/2, SNAI2, and NRP1, associated with ERα, androgen receptor (AR), and TP53, in a network regulating cell cycle, death, survival, and tumor morphology. Eighteen genes responding differently to fulvestrant specifically predicted antiproliferative response to fulvestrant, but not anastrozole. Transcriptional effects of low-dose fulvestrant correlated with high-dose treatment, but were of lower magnitude (ratio = 0.29). CONCLUSIONS: The transcriptional response to fulvestrant has much in common with estrogen deprivation, but is stronger with distinctions potentially attributable to arrest of estrogen-independent ERα activity and involvement of AR signaling. Genes responding differently to fulvestrant may have predictive utility. These data are consistent with the clinical efficacy of fulvestrant versus anastrozole and higher dosing regimens.