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Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance.
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The frequent activation of the PI3K/AKT/mTOR pathway and its crucial role in estrogen receptor-positive (ER+) breast cancer tumorigenesis and drug resistance has made it a highly attractive therapeutic target in this breast cancer subtype. Consequently, the number of new inhibitors in clinical development targeting this pathway has drastically increased. Among these, the PIK3CA isoform-specific inhibitor alpelisib and the pan-AKT inhibitor capivasertib were recently approved in combination with the estrogen receptor degrader fulvestrant for the treatment of ER+ advanced breast cancer after progression on an aromatase inhibitor. Nevertheless, the clinical development of multiple inhibitors of the PI3K/AKT/mTOR pathway, in parallel with the incorporation of CDK4/6 inhibitors into the standard of care treatment in ER+ advanced breast cancer, has led to a multitude of available therapeutic agents and many possible combined strategies which complicate personalizing treatment. Here, we review the role of the PI3K/AKT/mTOR pathway in ER+ advanced breast cancer, highlighting the genomic contexts in which the various inhibitors of this pathway may have superior activity. We also discuss selected trials with agents targeting the PI3K/AKT/mTOR and related pathways as well as the rationale supporting the clinical development of triple combination therapy targeting ER, CDK4/6 and PI3K/AKT/mTOR in ER+ advanced breast cancer.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is a heterogeneous disease that accounts for 10-15% of all breast cancer cases. Within TNBC, the treatment of basal B is the most challenging due to its highly invasive potential, and thus treatments to suppress metastasis formation in this subgroup are urgently needed. However, the mechanisms underlying the metastatic ability of TNBC remain unclear. In the present study, we investigated the role of Aurora A and Bcl-xL in regulating basal B cell invasion. We found gene amplification and elevated protein expression in the basal B cells, which also showed increased invasiveness in vitro, compared to basal A cells. Chemical inhibition of Aurora A with alisertib and siRNA-mediated knockdown of BCL2L1 decreased the number of invading cells compared to non-treated cells in basal B cell lines. The analysis of the correlation between AURKA and BCL2L1 expression in TNBC and patient survival revealed significantly decreased relapse-free survival (n = 534, p = 0.012) and distant metastasis-free survival (n = 424, p = 0.017) in patients with primary tumors exhibiting a high combined expression of AURKA and BCL2L1. Together, our findings suggest that high levels of Aurora A and Bcl-xL promote metastasis, and inhibition of these proteins may suppress metastasis and improve patient survival in basal B TNBC.
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Aurora Quinasa A/metabolismo , Metástasis de la Neoplasia , Neoplasias de la Mama Triple Negativas , Proteína bcl-X/metabolismo , Aurora Quinasa A/genética , Línea Celular Tumoral , Humanos , Recurrencia Local de Neoplasia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteína bcl-X/genéticaRESUMEN
Resistance to aromatase inhibitor (AI) treatment and combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) are crucial clinical challenges in treating estrogen receptor-positive (ER+) breast cancer. Understanding the resistance mechanisms and identifying reliable predictive biomarkers and novel treatment combinations to overcome resistance are urgently needed. Herein, we show that upregulation of CDK6, p-CDK2, and/or cyclin E1 is associated with adaptation and resistance to AI-monotherapy and combined CDK4/6i and ET in ER+ advanced breast cancer. Importantly, co-targeting CDK2 and CDK4/6 with ET synergistically impairs cellular growth, induces cell cycle arrest and apoptosis, and delays progression in AI-resistant and combined CDK4/6i and fulvestrant-resistant cell models and in an AI-resistant autocrine breast tumor in a postmenopausal xenograft model. Analysis of CDK6, p-CDK2, and/or cyclin E1 expression as a combined biomarker in metastatic lesions of ER+ advanced breast cancer patients treated with AI-monotherapy or combined CDK4/6i and ET revealed a correlation between high biomarker expression and shorter progression-free survival (PFS), and the biomarker combination was an independent prognostic factor in both patients cohorts. Our study supports the clinical development of therapeutic strategies co-targeting ER, CDK4/6 and CDK2 following progression on AI-monotherapy or combined CDK4/6i and ET to improve survival of patients exhibiting high tumor levels of CDK6, p-CDK2, and/or cyclin E1.
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The introduction of CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) has revolutionized the treatment landscape for patients with estrogen receptor-positive (ER+) advanced breast cancer (ABC) and has become the new standard treatment. However, resistance to this combined therapy inevitably develops and represents a major clinical challenge in the management of ER+ ABC. Currently, elucidation of the resistance mechanisms, identification of predictive biomarkers, and development of novel effective combined targeted treatments to overcome the resistance are active areas of research. Given the heterogeneity of the resistance mechanisms towards combined CDK4/6i and ET, identification of a single universal predictive biomarker of resistance is unlikely. Novel approaches are being explored, including examination of multiple genetic alterations in circulating cell-free tumor DNA in liquid biopsies from ABC patients with disease progression on combined CDK4/6i and ET treatment. Here, we review the molecular basis of the main known resistance mechanisms towards combined CDK4/6i and ET and associated potential biomarkers. As inhibiting key molecules in the pathways driving resistance may play an important role in the selection of therapeutic strategies for patients who experience disease progression on combined CDK4/6i and ET, we also review preclinical and early phase clinical data on novel combination therapies for these patients.
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CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome.
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Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Fulvestrant/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Quimioterapia Combinada , Femenino , Humanos , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
BACKGROUND: Resistance to endocrine treatment in metastatic breast cancer is a major clinical challenge. Clinical tools to predict both drug resistance and possible treatment combination approaches to overcome it are lacking. This unmet need is mainly due to the heterogeneity underlying both the mechanisms involved in resistance development and breast cancer itself. METHODS: To study the complexity of the mechanisms involved in the resistance to the selective estrogen receptor degrader (SERD) fulvestrant, we performed comprehensive biomarker analyses using several in vitro models that recapitulate the heterogeneity of developed resistance. We further corroborated our findings in tissue samples from patients treated with fulvestrant. RESULTS: We found that different in vitro models of fulvestrant resistance show variable stability in their phenotypes, which corresponded with distinct genomic alterations. Notably, the studied models presented adaptation at different cell cycle nodes to facilitate progression through the cell cycle and responded differently to CDK inhibitors. Cyclin E2 overexpression was identified as a biomarker of a persistent fulvestrant-resistant phenotype. Comparison of pre- and post-treatment paired tumor biopsies from patients treated with fulvestrant revealed an upregulation of cyclin E2 upon development of resistance. Moreover, overexpression of this cyclin was found to be a prognostic factor determining resistance to fulvestrant and shorter progression-free survival. CONCLUSIONS: These data highlight the complexity of estrogen receptor positive breast cancer and suggest that the development of diverse resistance mechanisms dictate levels of ER independence and potentially cross-resistance to CDK inhibitors.
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Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Antagonistas del Receptor de Estrógeno/farmacología , Fulvestrant/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Estrógenos/metabolismo , Antineoplásicos Hormonales/farmacología , Biomarcadores de Tumor , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Mutación , Polimorfismo de Nucleótido Simple , Transducción de SeñalRESUMEN
Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER+ breast cancer. We discovered that ER+ breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in the cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser315,33), CHK1(Ser317), and cdc25b(Ser323), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Interestingly, the MCM3 levels did not affect the efficacy of growth inhibitory by CDK4/6 inhibitors. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic marker adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER+ breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER+ breast cancer patients.
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BACKGROUND: Endocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance. METHODS: In the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting. RESULTS: SNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004). CONCLUSIONS: Our results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.
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Antineoplásicos Hormonales/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Factores de Transcripción de la Familia Snail/genética , Adulto , Anciano , Antineoplásicos Hormonales/efectos adversos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Femenino , Fulvestrant/administración & dosificación , Fulvestrant/efectos adversos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células MCF-7 , Persona de Mediana Edad , Metástasis de la NeoplasiaRESUMEN
Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer, however, tamoxifen resistance is frequently observed. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. Although the target genes affected by the altered miRNA in the three TamRs differed, good agreement in terms of affected molecular pathways was observed. Moreover, we found evidence of miRNA-mediated regulation of ESR1, PGR1, FOXM1 and 14-3-3 family genes. Integrating the inferred miRNA-target relationships, we investigated the functional importance of 2 central genes, SNAI2 and FYN, which showed increased expression in TamR cells, while their corresponding regulatory miRNA were downregulated. Using specific chemical inhibitors and siRNA-mediated gene knockdown, we showed that both SNAI2 and FYN significantly affect the growth of TamR cell lines. Finally, we show that a combination of 2 miRNAs (miR-190b and miR-516a-5p) exhibiting altered expression in TamR cell lines were predictive of treatment outcome in a cohort of ER+ breast cancer patients receiving adjuvant tamoxifen mono-therapy. Our results provide new insight into the molecular mechanisms of tamoxifen resistance and may form the basis for future medical intervention for the large number of women with tamoxifen-resistant ER+ breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/metabolismo , Tamoxifeno/farmacología , Proteínas 14-3-3/metabolismo , Antineoplásicos Hormonales/farmacología , Mama/metabolismo , Línea Celular Tumoral , Estudios de Cohortes , Receptor alfa de Estrógeno/metabolismo , Femenino , Proteína Forkhead Box M1/metabolismo , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/farmacología , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
PURPOSE: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER+ advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined. EXPERIMENTAL DESIGN: We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. RESULTS: We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively). CONCLUSIONS: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Clin Cancer Res; 22(22); 5514-26. ©2016 AACR.