RESUMO
The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease. SIGNIFICANCE: Targeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinases , Resistencia a Medicamentos Antineoplásicos/genética , Receptores de Estrogênio/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Recidiva , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quinase 9 Dependente de Ciclina/genéticaRESUMO
Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Feminino , Humanos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteoma/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/uso terapêuticoRESUMO
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.
RESUMO
Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.
Assuntos
Proteína de Ligação a CREB/fisiologia , Carcinogênese/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Proteína de Ligação a CREB/genética , Proliferação de Células/genética , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Genômica/métodos , Células HCT116 , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
Assuntos
Antineoplásicos/farmacologia , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Furanos/farmacologia , Macroautofagia , Fosfatidilcolinas/biossíntese , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Células CHO , Linhagem Celular Tumoral , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Macroautofagia/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Seleção de Pacientes , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Mutations in the ligand-binding domain (LBD) of the ESR1 gene result in resistance to estrogen deprivation therapy (EDT) in breast cancer. Their detection might enable optimization of therapy strategies. However, the predictive utility of the primary tumor (PT) is limited, and obtaining serial biopsies of metastatic lesions is challenging. To underline their application as a liquid biopsy, single circulating tumor cells (CTCs) were analyzed with a next-generation sequencing approach for the ESR1 coding region. CTCs from 46 metastatic luminal breast cancer patients were enriched using CellSearch system and isolated by micromanipulation. Their genomic DNA was amplified and the ESR1 gene was sequenced. Furthermore, tissue samples from corresponding PTs and/or metastatic lesions were investigated. ESR1 mutations were detected in 12 patients-exclusively in patients treated with EDT (P = 0.048). In seven cases mutations were located in the hotspot regions in the LBD. Six novel mutations were identified. ESR1 mutations were absent in PT tissue samples and were detected only in metastases obtained after CTC characterization. Single-cell CTC analysis for ESR1 mutations could be of clinical value to identify patients who progress under EDT and therefore benefit from an early switch to an alternative endocrine therapy or other treatment regimens. Furthermore, our data indicate that mutations outside the LBD's hotspot regions might also contribute to resistance to EDT.
Assuntos
Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Mutação , Células Neoplásicas Circulantes , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Pessoa de Meia-Idade , Análise de Célula ÚnicaRESUMO
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
Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins.
Assuntos
Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Mucoproteínas/imunologia , Mucoproteínas/metabolismo , Proteínas Oncogênicas/imunologia , Proteínas Oncogênicas/metabolismoRESUMO
PURPOSE: To investigate the presence of ESR1 mutations in primary estrogen-receptor-positive (ER+) breast cancer treated with extended (>4 weeks) neoadjuvant (presurgical) aromatase inhibitor (NAI) therapy and to identify patients who may gain less benefit from aromatase inhibition (AI) alone based upon on-treatment changes in gene expression. EXPERIMENTAL DESIGN: We evaluated ER, progesterone receptor, and Ki67 by immunostaining, ESR1 mutations by droplet-digital PCR and expression of over 800 key breast cancer genes in paired pre- and post-NAI tumor samples from 87 ER+ breast cancer patients. RESULTS: Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment of ESR1 mutations was found in five tumors and predominantly in patients receiving therapy for >6 months. ESR1-mutant tumors showed increased expression of ESR1 transcript and limited suppression of ERGs and proliferation-associated genes in response to NAI. ESR1 wild-type tumors with high residual proliferation (Ki67r ≥10%; 15/87 tumors) showed lower ESR1/ER expression pre- and post-therapy and lower ERGs. Tumors with ESR1 mutations or Ki67r ≥10% showed less inhibition of estrogen response, cell cycle, and E2F-target genes. CONCLUSIONS: Ligand-independent ER signaling, as a result of ESR1 mutation or reduced ER dependence, identified after extended NAI therapy, can guide early selection of patients who would benefit from combination therapy.
Assuntos
Inibidores da Aromatase/administração & dosagem , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Mutação , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Resultado do TratamentoRESUMO
Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, â¼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Feminino , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Prognóstico , Transcriptoma/genética , Transplante HeterólogoRESUMO
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
The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.