RESUMO
The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient-derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.
RESUMO
PURPOSE: To identify prognostic circulating biomarkers to cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i), we performed a mutational analysis on circulating tumor DNA (ctDNA) samples from patients included in the TREnd trial, which randomly assigned patients to receive the CDK4/6i palbociclib alone or with the endocrine treatment (ET) to which they had progressed. METHODS: Forty-six patients were enrolled in this substudy. Plasma was collected before treatment (T0), after the first cycle of therapy (T1), and at the time of progression (T2). ctDNA hybridization and capture were performed using the Illumina TruSight Tumor 170 Kit. Acquired mutations were confirmed by digital polymerase chain reaction. Progression-free survival analysis was estimated using the Kaplan-Meier method and compared with the log-rank test. RESULTS: The most frequently mutated genes at T0 were ESR1 (23%), PIK3CA (17%), AR, FGFR2, and TP53 (10%). Mutations in ESR1 at T0 conferred higher risk of progression in the entire population (P = .02) and in patients treated with palbociclib + ET (P = .04). ESR1 mutation effect remained significant after correction for clinical variables (P = .03). PIK3CA mutations at T0 were not prognostic, but higher risk of progression was observed when a broader analysis of PI3K pathway was performed (P = .04). At T2, we observed the emergence of nine new mutations in seven genes. CONCLUSION: Mutations in ESR1 and in PI3K pathway genes at T0 were associated with worse prognosis in palbociclib-treated patients. We describe the emergence of newly acquired mutations in palbociclib-treated patients, which might potentially affect subsequent treatment.
Assuntos
Neoplasias da Mama , DNA Tumoral Circulante , Piperazinas , Piridinas , Receptor ErbB-2 , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , DNA Tumoral Circulante/genética , Fosfatidilinositol 3-Quinases , Receptores de Estrogênio/genéticaRESUMO
PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.
Assuntos
Apoptose , Neoplasias da Mama , Ciclo Celular , Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-myc , Receptores de Estrogênio , Transdução de Sinais , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Resistencia a Medicamentos Antineoplásicos/genética , Apoptose/efeitos dos fármacos , Animais , Camundongos , Receptores de Estrogênio/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Sistemas CRISPR-CasRESUMO
Cyclin-dependent kinases 4/6 (CDK4/6) inhibitors such as palbociclib are approved for the treatment of metastatic estrogen receptor-positive (ER+) breast cancer in combination with endocrine therapies and significantly improve outcomes in patients with this disease. However, given the large number of possible pairwise drug combinations and administration schedules, it remains unclear which clinical strategy would lead to best survival. Here, we developed a computational, cell cycle-explicit model to characterize the pharmacodynamic response to palbociclib-fulvestrant combination therapy. This pharmacodynamic model was parameterized, in a Bayesian statistical inference approach, using in vitro data from cells with wild-type estrogen receptor (WT-ER) and cells expressing the activating missense ER mutation, Y537S, which confers resistance to fulvestrant. We then incorporated pharmacokinetic models derived from clinical data into our computational modeling platform. To systematically compare dose administration schedules, we performed in silico clinical trials based on integrating our pharmacodynamic and pharmacokinetic models as well as considering clinical toxicity constraints. We found that continuous dosing of palbociclib is more effective for lowering overall tumor burden than the standard, pulsed-dose palbociclib treatment. Importantly, our mathematical modeling and statistical analysis platform provides a rational method for comparing treatment strategies in search of optimal combination dosing strategies of other cell-cycle inhibitors in ER+ breast cancer. SIGNIFICANCE: We created a computational modeling platform to predict the effects of fulvestrant/palbocilib treatment on WT-ER and Y537S-mutant breast cancer cells, and found that continuous treatment schedules are more effective than the standard, pulsed-dose palbociclib treatment schedule.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Fulvestranto , Receptores de Estrogênio/análise , Teorema de BayesRESUMO
Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, the key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) enriched in ER+ endocrine-resistant (EndoR) cells. We identify Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, as a strong predictor for poor outcomes of ER+ BC. It is elevated in ER+ metastases vs. primary tumors, irrespective of ESR1 mutations. Genomic ER binding near Secretome14 genes is also increased in mutant ER-expressing or mitogen-treated ER+ BC cells and in ER+ metastatic vs. primary tumors, suggesting a convergent pathway including high growth factor receptor signaling in activating pro-metastatic secretome genes. Our findings uncover H-FOXA1-induced ER reprogramming that drives EndoR and metastasis partly via an H-FOXA1/ER-dependent secretome.
RESUMO
Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated ß2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of ß2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer.
Assuntos
Neoplasias da Mama , NF-kappa B , Humanos , Feminino , NF-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias da Mama/patologia , Apresentação de Antígeno , Proteínas Reguladoras de Apoptose , Apoptose , Linhagem Celular Tumoral , Proteínas Mitocondriais/metabolismo , Microambiente TumoralRESUMO
DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients' circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Humanos , Feminino , Ácidos Nucleicos Livres/genética , Medicina de Precisão , Metilação de DNA , Neoplasias da Mama/genética , Biomarcadores Tumorais/genética , DNA de Neoplasias/genéticaRESUMO
PURPOSE: Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated. PATIENTS AND METHODS: We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2- advanced breast cancer who progressed on prior ET (N = 36; NCT02448771). RESULTS: The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0-7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5-13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment. CONCLUSIONS: The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2- breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sequenciamento do Exoma , Biópsia Líquida , Receptor ErbB-2/análise , Receptores de Estrogênio/genética , Resultado do TratamentoRESUMO
Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor-positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1-estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1-ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. SIGNIFICANCE: A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Lobular , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Cromatina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Prognóstico , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
PURPOSE: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor-positive (ER+)/HER2- breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. EXPERIMENTAL DESIGN: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation-resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. RESULTS: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an "IFN-related palbociclib-resistance Signature" (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. CONCLUSIONS: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Neoplasias da Mama/química , Feminino , Humanos , Receptores de Estrogênio/análise , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.
Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Gênica , Estrogênios/metabolismo , Hormônios Esteroides Gonadais/genética , Histona Acetiltransferases/genética , Humanos , Conformação Molecular , Proteínas Nucleares/antagonistas & inibidores , Ligação Proteica/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Análise de Célula ÚnicaRESUMO
Forkhead box A1 (FOXA1) is a pioneer factor that facilitates chromatin binding and function of lineage-specific and oncogenic transcription factors. Hyperactive FOXA1 signaling due to gene amplification or overexpression has been reported in estrogen receptor-positive (ER+) endocrine-resistant metastatic breast cancer. However, the molecular mechanisms by which FOXA1 up-regulation promotes these processes and the key downstream targets of the FOXA1 oncogenic network remain elusive. Here, we demonstrate that FOXA1 overexpression in ER+ breast cancer cells drives genome-wide enhancer reprogramming to activate prometastatic transcriptional programs. Up-regulated FOXA1 employs superenhancers (SEs) to synchronize transcriptional reprogramming in endocrine-resistant breast cancer cells, reflecting an early embryonic development process. We identify the hypoxia-inducible transcription factor hypoxia-inducible factor-2α (HIF-2α) as the top high FOXA1-induced SE target, mediating the impact of high FOXA1 in activating prometastatic gene sets and pathways associated with poor clinical outcome. Using clinical ER+/HER2- metastatic breast cancer datasets, we show that the aberrant FOXA1/HIF-2α transcriptional axis is largely nonconcurrent with the ESR1 mutations, suggesting different mechanisms of endocrine resistance and treatment strategies. We further demonstrate the selective efficacy of an HIF-2α antagonist, currently in clinical trials for advanced kidney cancer and recurrent glioblastoma, in reducing the clonogenicity, migration, and invasion of endocrine-resistant breast cancer cells expressing high FOXA1. Our study has uncovered high FOXA1-induced enhancer reprogramming and HIF-2α-dependent transcriptional programs as vulnerable targets for treating endocrine-resistant and metastatic breast cancer.
RESUMO
Circulating tumor cell clusters (CTCcl) have a higher metastatic potential compared to single CTCs and predict long-term outcomes in breast cancer (BC) patients. Because of the rarity of CTCcls, molecular characterization of primary tumors that give rise to CTCcl hold significant promise for better diagnosis and target discovery to combat metastatic BC. In our study, we utilized the reverse-phase protein array (RPPA) and transcriptomic (RNA-Seq) data of 10 triple-negative BC patient-derived xenograft (TNBC PDX) transplantable models with CTCs and evaluated expression of upregulated candidate protein Bcl2 (B-cell lymphoma 2) by immunohistochemistry (IHC). The sample-set consisted of six CTCcl-negative (CTCcl-) and four CTCcl-positive (CTCcl+) models. We analyzed the RPPA and transcriptomic profiles of CTCcl- and CTCcl+ TNBC PDX models. In addition, we derived a CTCcl-specific gene signature for testing if it predicted outcomes using a publicly available dataset from 360 patients with basal-like BC. The RPPA analysis of CTCcl+ vs. CTCcl- TNBC PDX tumors revealed elevated expression of Bcl2 (false discovery rate (FDR) < 0.0001, fold change (FC) = 3.5) and reduced acetyl coenzyme A carboxylase-1 (ACC1) (FDR = 0.0005, FC = 0.3) in CTCcl+ compared to CTCcl- tumors. Genome-wide transcriptomic analysis of CTCcl+ vs. CTCcl- tumors revealed 549 differentially expressed genes associated with the presence of CTCcls. Apoptosis was one of the significantly downregulated pathways (normalized enrichment score (NES) = -1.69; FDR < 0.05) in TNBC PDX tumors associated with CTCcl positivity. Two out of four CTCcl+ TNBC PDX primary tumors had high Bcl2 expression by IHC (H-score > 34); whereas, only one of six CTCcl- TNBC PDX primary tumors met this criterion. Evaluation of epithelial-mesenchymal transition (EMT)-specific signature did not show significant differences between CTCcl+ and CTCcl- tumors. However, a gene signature associated with the presence of CTCcls in TNBC PDX models was associated with worse relapse-free survival in the publicly available dataset from 360 patients with basal-like BC. In summary, we identified the multigene signature of primary PDX tumors associated with the presence of CTCcls. Evaluation of additional TNBC PDX models and patients can further illuminate cellular and molecular pathways facilitating CTCcl formation.
RESUMO
Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab-resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab-resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab-resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ-mTORC1-Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. IMPLICATIONS: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Ácido Mevalônico/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Transdução de Sinais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Lapatinib/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Trastuzumab/farmacologiaRESUMO
BACKGROUND: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system. METHODS: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. RESULTS: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. CONCLUSIONS: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Células Neoplásicas Circulantes/metabolismo , Análise de Célula Única/métodos , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais/sangue , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Separação Celular , Classe I de Fosfatidilinositol 3-Quinases/sangue , Feminino , Humanos , Queratinas/sangue , Antígenos Comuns de Leucócito/sangue , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Células Neoplásicas Circulantes/efeitos dos fármacos , Análise de Sequência de DNARESUMO
BACKGROUND: The oestrogen receptor (ER) is an important therapeutic target in ER-positive (ER+) breast cancer. The selective ER degrader (SERD), fulvestrant, is effective in patients with metastatic breast cancer, but its intramuscular route of administration and low bioavailability are major clinical limitations. METHODS: Here, we studied the pharmacology of a new oral SERD, AZD9496, in a panel of in vitro and in vivo endocrine-sensitive and -resistant breast cancer models. RESULTS: In endocrine-sensitive models, AZD9496 inhibited cell growth and blocked ER activity in the presence or absence of oestrogen. In vivo, in the presence of oestrogen, short-term AZD9496 treatment, like fulvestrant, resulted in tumour growth inhibition and reduced expression of ER-dependent genes. AZD9496 inhibited cell growth in oestrogen deprivation-resistant and tamoxifen-resistant cell lines and xenograft models that retain ER expression. AZD9496 effectively reduced ER levels and ER-induced transcription. Expression analysis of short-term treated tumours showed that AZD9496 potently inhibited classic oestrogen-induced gene transcription, while simultaneously increasing expression of genes negatively regulated by ER, including genes potentially involved in escape pathways of endocrine resistance. CONCLUSIONS: These data suggest that AZD9496 is a potent anti-oestrogen that antagonises and degrades ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Cinamatos/administração & dosagem , Indóis/administração & dosagem , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptores de Estrogênio/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estradiol/genética , Estradiol/metabolismo , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Fulvestranto/administração & dosagem , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Neoplasias Hormônio-Dependentes/genética , Receptores de Estrogênio/genética , Tamoxifeno/administração & dosagemRESUMO
The surface glycoprotein THY is a marker of myoepithelial precursor cells, which are basal cells with epithelial-mesenchymal intermediate phenotype originating from the ectoderm. Myoepithelial precursor cells are lost during progression from in situ to invasive carcinoma. To define the functional role of Thy1-positive cells within the myoepithelial population, we tracked Thy1 expression in human breast cancer samples, isolated THY1-positive myoepithelial progenitor cells (CD44+/CD24low/CD90+), and established long-term cultures (parental cells). Parental cells were used to generate a xenograft model to examine Thy1 expression during tumor formation. Post-transplantation cell cultures lost THY1 expression through methylation at the THY1 locus and this is associated with an increase in EGFR and NOTCH1 transcript levels. Thy1-low cells are sensitive to the EGFR/HER2 dual inhibitor lapatinib. High THY1 expression is associated with poorer relapse-free survival in patients with breast cancer. THY1 methylation may track the shift of bipotent progenitors into differentiated cells. Thy1 is a good candidate biomarker in basal-like breast cancer. IMPLICATIONS: Our findings provide evidence that THY1 expression is lost in xenografts due to promoter methylation. Thy1-low cells with increased EGFR and Notch1 expression are responsive to target therapy. Because DNA methylation is often altered in early cancer development, candidate methylation markers may be exploited as biomarkers for basal-like breast cancer.
Assuntos
Células Epiteliais/metabolismo , Receptores de Hialuronatos/metabolismo , Receptor Notch1/metabolismo , Antígenos Thy-1/genética , Animais , Antígeno CD24/metabolismo , Metilação de DNA , Epigênese Genética , Células Epiteliais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas , Receptor Notch1/genética , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Antígenos Thy-1/metabolismoRESUMO
Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.
Assuntos
Alelos , Cromatina/metabolismo , Receptor alfa de Estrogênio/genética , Mutação/genética , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Camundongos TransgênicosRESUMO
Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2-irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivoConclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123-34. ©2017 AACR.