RESUMEN
Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.
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Approximately one-third of endocrine-treated women with estrogen receptor alpha-positive (ER+) breast cancers are at risk of recurrence due to intrinsic or acquired resistance. Thus, it is vital to understand the mechanisms underlying endocrine therapy resistance in ER+ breast cancer to improve patient treatment. Mitochondrial fatty acid ß-oxidation (FAO) has been shown to be a major metabolic pathway in triple-negative breast cancer (TNBC) that can activate Src signaling. Here, we found metabolic reprogramming that increases FAO in ER+ breast cancer as a mechanism of resistance to endocrine therapy. A metabolically relevant, integrated gene signature was derived from transcriptomic, metabolomic, and lipidomic analyses in TNBC cells following inhibition of the FAO rate-limiting enzyme carnitine palmitoyl transferase 1 (CPT1), and this TNBC-derived signature was significantly associated with endocrine resistance in patients with ER+ breast cancer. Molecular, genetic, and metabolomic experiments identified activation of AMPK-FAO-oxidative phosphorylation (OXPHOS) signaling in endocrine-resistant ER+ breast cancer. CPT1 knockdown or treatment with FAO inhibitors in vitro and in vivo significantly enhanced the response of ER+ breast cancer cells to endocrine therapy. Consistent with the previous findings in TNBC, endocrine therapy-induced FAO activated the Src pathway in ER+ breast cancer. Src inhibitors suppressed the growth of endocrine-resistant tumors, and the efficacy could be further enhanced by metabolic priming with CPT1 inhibition. Collectively, this study developed and applied a TNBC-derived signature to reveal that metabolic reprogramming to FAO activates the Src pathway to drive endocrine resistance in ER+ breast cancer. SIGNIFICANCE: Increased fatty acid oxidation induced by endocrine therapy activates Src signaling to promote endocrine resistance in breast cancer, which can be overcome using clinically approved therapies targeting FAO and Src.
Asunto(s)
Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Fosforilación , Transducción de Señal , Ácidos Grasos/metabolismo , Resistencia a Antineoplásicos/genéticaRESUMEN
Breast cancer accounts for 25% of the cancers in women worldwide. The most common subtype of breast cancer diagnosed is hormone receptor positive, which expresses the oestrogen receptor (ER). Targeting of the ER with endocrine therapy (ET) is the current standard of care for ER-positive (ER+) breast cancer, reducing the mortality by up to 40%. Resistance to ET, however, remains a major issue for ER + breast cancer, leading to recurrence and metastasis. One major driver of ET resistance is mutations in the ER gene (ESR1) leading to constitutive transcriptional activity and reduced ET sensitivity. These mutations are particularly detrimental in metastatic breast cancer (MBC) as they are present in as high as 36% of the patients. This review summarises the pre-clinical characterisation of ESR1 mutations and their association with clinical outcomes in MBC and primary disease. The clinically approved and investigational therapeutic options for ESR1 mutant breast cancer and the current clinical trials evaluating ESR1 mutations and ET resistance are also discussed. Finally, this review addresses pre-clinical models and multi-'omics' approaches for developing the next generation of therapeutics for ESR1 mutant and ET-resistant breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptor alfa de Estrógeno/genética , Terapia Molecular Dirigida/métodos , Mutación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Metástasis de la NeoplasiaRESUMEN
Metastasis is the spread of cancer cells to new areas of the body by way of the lymph system or bloodstream. Mechanism-based therapeutics have transformed its treatment. This issue of British Journal of Cancer will highlight recent advances in our understanding of metastasis, and how to block its spread.
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Redes Reguladoras de Genes , Metástasis de la Neoplasia/patología , Neoplasias/patología , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Terapia Molecular Dirigida , Metástasis de la Neoplasia/genética , Neoplasias/genética , Neoplasias/metabolismo , Medicina de PrecisiónRESUMEN
Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.
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Neoplasias de la Mama/tratamiento farmacológico , Receptor alfa de Estrógeno/genética , Receptores Androgénicos/genética , Tamoxifeno/farmacología , Animales , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Estradiol/metabolismo , Estrógenos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mutación/genética , Metástasis de la Neoplasia , Receptores Androgénicos/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Oestrogen Receptor 1 (ESR1) mutations are frequently acquired in oestrogen receptor (ER)-positive metastatic breast cancer (MBC) patients who were treated with aromatase inhibitors (AI) in the metastatic setting. Acquired ESR1 mutations are associated with poor prognosis and there is a lack of effective therapies that selectively target these cancers. METHODS: We performed a proteomic kinome analysis in ESR1 Y537S mutant cells to identify hyperactivated kinases in ESR1 mutant cells. We validated Recepteur d'Origine Nantais (RON) and PI3K hyperactivity through phospho-immunoblot analysis, organoid growth assays, and in an in vivo patient-derived xenograft (PDX) metastatic model. RESULTS: We demonstrated that RON was hyperactivated in ESR1 mutant models, and in acquired palbociclib-resistant (PalbR) models. RON and insulin-like growth factor 1 receptor (IGF-1R) interacted as shown through pharmacological and genetic inhibition and were regulated by the mutant ER as demonstrated by reduced phospho-protein expression with endocrine therapies (ET). We show that ET in combination with a RON inhibitor (RONi) decreased ex vivo organoid growth of ESR1 mutant models, and as a monotherapy in PalbR models, demonstrating its therapeutic efficacy. Significantly, ET in combination with the RONi reduced metastasis of an ESR1 Y537S mutant PDX model. CONCLUSIONS: Our results demonstrate that RON/PI3K pathway inhibition may be an effective treatment strategy in ESR1 mutant and PalbR MBC patients. Clinically our data predict that ET resistance mechanisms can also contribute to CDK4/6 inhibitor resistance.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Ratones , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética , Estrógenos/metabolismo , Hormonas Esteroides Gonadales/genética , Histona Acetiltransferasas/genética , Humanos , Conformación Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Análisis de la Célula IndividualRESUMEN
The acquisition of ligand-independent ESR1 mutations during aromatase inhibitor therapy in metastatic estrogen receptor (ER)-positive breast cancer is a common mechanism of hormonal therapy resistance. Preclinical and clinical studies have demonstrated that ESR1 mutations can preexist in primary tumors and can be enriched during metastasis. Furthermore, ESR1 mutations express a unique transcriptional profile that favors tumor progression, suggesting that selected ESR1 mutations may influence metastasis. Several groups have used sensitive detection methods using patient liquid biopsies to track ESR1 or truncal somatic mutations to predict treatment outcome and tumor progression, and some of these techniques may eventually be used to guide sequential treatment options in patients. Further development and standardization of mutation tracking in circulating tumor DNA is ongoing. Clinically, patients with ESR1 mutations derive clinical benefit when treated with fulvestrant and CDK4/6-targeted therapies, but the development of more potent selective ER degraders and/or new targeted biotherapies are needed to overcome the endocrine-resistant phenotype of ESR1 mutant-bearing tumors. In this review, we discuss the mechanisms of resistance and dissemination of ESR1 mutations as well as the detection methods for ESR1 mutation tracking, newly discovered potential therapeutic targets, and the clinical implications and treatment options for treating patients with ESR1 mutant-bearing tumors.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Perfilación de la Expresión Génica/métodos , Mutación , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Fulvestrant/uso terapéutico , Humanos , Biopsia Líquida , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10-40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Humanos , Células MCF-7 , Unión Proteica/genética , Proteómica , Transcripción Genética/genética , Activación Transcripcional/genética , Translocación Genética/genéticaRESUMEN
After nearly 20 years of research, it is now established that mutations within the estrogen receptor (ER) gene, ESR1, frequently occur in metastatic breast cancer and influence response to hormone therapy. Though early studies presented differing results, sensitive sequencing techniques now show that ESR1 mutations occur at a frequency between 20 and 40% depending on the assay method. Recent studies have focused on several "hot spot mutations," a cluster of mutations found in the hormone-binding domain of the ESR1 gene. Throughout the course of treatment, tumor evolution can occur, and ESR1 mutations emerge and become enriched in the metastatic setting. Sensitive techniques to continually monitor mutant burden in vivo are needed to effectively treat patients with mutant ESR1. The full impact of these mutations on tumor response to different therapies remains to be determined. However, recent studies indicate that mutant-bearing tumors may be less responsive to specific hormonal therapies, and suggest that aromatase inhibitor (AI) therapy may select for the emergence of ESR1 mutations. Additionally, different mutations may respond discretely to targeted therapies. The need for more preclinical mechanistic studies on ESR1 mutations and the development of better agents to target these mutations are urgently needed. In the future, sequential monitoring of ESR1 mutational status will likely direct personalized therapeutic regimens appropriate to each tumor's unique mutational landscape.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Terapia Molecular Dirigida/métodos , Medicina de Precisión/métodos , Femenino , Humanos , MutaciónRESUMEN
PURPOSE: Aberrant activation of the PI3K pathway has been implicated in resistance to HER2-targeted therapy, but results of clinical trials are confounded by the co-administration of chemotherapy. We investigated the effect of perturbations of this pathway in breast cancers from patients treated with neoadjuvant anti-HER2-targeted therapy without chemotherapy. PATIENTS AND METHODS: Baseline tumor samples from patients with HER2-positive breast cancer enrolled in TBCRC006 (NCT00548184), a 12-week neoadjuvant clinical trial with lapatinib plus trastuzumab [plus endocrine therapy for estrogen receptor (ER)-positive tumors], were assessed for PTEN status by immunohistochemistry and PIK3CA mutations by sequencing. Results were correlated with pathologic complete response (pCR). RESULTS: Of 64 evaluable patients, PTEN immunohistochemistry and PIK3CA mutation analysis were performed for 59 and 46 patients, respectively. PTEN status (dichotomized by H-score median) was correlated with pCR (32% in high PTEN vs. 9% in low PTEN, p = 0.04). PIK3CA mutations were identified in 14/46 tumors at baseline (30%) and did not correlate with ER or PTEN status. One patient whose tumor harbored a PIK3CA mutation achieved pCR (p = 0.14). When considered together (43 cases), 1/25 cases (4%) with a PIK3CA mutation and/or low PTEN expression levels had a pCR compared to 7/18 cases (39%) with wild-type PI3KCA and high PTEN expression levels (p = 0.006). CONCLUSION: PI3K pathway activation is associated with resistance to lapatinib and trastuzumab in breast cancers, without chemotherapy. Further studies are warranted to investigate how to use these biomarkers to identify upfront patients who may respond to anti-HER2 alone, without chemotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfohidrolasa PTEN/genética , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Persona de Mediana Edad , Mutación , Terapia Neoadyuvante/efectos adversos , Quinazolinas/administración & dosificación , Quinazolinas/efectos adversos , Receptor ErbB-2/genética , Trastuzumab/administración & dosificación , Trastuzumab/efectos adversosRESUMEN
BACKGROUND: The progesterone receptor (PR) is expressed by â¼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples. METHODS: Archived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade. RESULTS: A total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p<0.0001). Cumulative progression rate and DFS were similar irrespective of APR status. Both APRA and APRB were independent of HER2, ERα and Ki67 expression. CONCLUSIONS: APR had a binary mode of expression in the breast cancer specimens tested, allowing separation into two tumour subsets. APR is an independent target at the cellular and tumour level and may therefore be a suitable predictive marker for antiprogestins, such as onapristone. Using the described technique, a companion diagnostic is under development to identify APR in solid tumours.
RESUMEN
The purpose of this study was to address the role of ESR1 hormone-binding mutations in breast cancer. Soft agar anchorage-independent growth assay, Western blot, ERE reporter transactivation assay, proximity ligation assay (PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR (ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers; however, we do not yet know how to best treat these patients. We have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and Western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R, and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Mutación , Receptores de Somatomedina/genética , Tamoxifeno/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Modelos Genéticos , Receptor IGF Tipo 1 , Receptores de Estrógenos/metabolismo , Receptores de Somatomedina/metabolismo , Transducción de Señal , Tamoxifeno/uso terapéuticoRESUMEN
Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1-S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942-53. ©2016 AACR.
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Proteínas de Neoplasias/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Tirosina Fosfatasas/genética , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Proteínas de Neoplasias/metabolismo , Pronóstico , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
Wang and colleagues demonstrate that digital droplet PCR (ddPCR) identified ESR1 mutations in 7% of primary breast cancers. ESR1 mutations were also readily detected in metastatic tissues and circulating tumor DNA in the blood. These results suggest that ddPCR may be amendable for monitoring tumor burden, and to predict relapse. See related article by Wang et al., p. 1130.
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Neoplasias de la Mama , Recurrencia Local de Neoplasia , ADN de Neoplasias/sangre , Humanos , MutaciónRESUMEN
Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) α-positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI) α, a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDIα and seek to determine AR's contribution to resistance. We engineered ERα-positive cell lines with stable knockdown (KD) of Rho GDIα (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Tam-resistant tumors and cell lines with low Rho GDIα levels exhibited upregulated AR expression. Microarray of Rho GDIα KD cells indicated that activation of EGFR and ERα was associated with Tam treatment. When AR levels were elevated, interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ERα. Tam exhibited agonist activity in AR overexpressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ERα-positive cells with low expression of Rho GDIα.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptores ErbB/genética , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores Androgénicos/metabolismo , Tamoxifeno/farmacología , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Unión Proteica , Receptores Androgénicos/genética , Tamoxifeno/uso terapéutico , Activación Transcripcional , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
The purpose of this study was to discover novel nuclear receptor targets in triple-negative breast cancer. Expression microarray, Western blot, qRT-PCR analyses, MTT growth assay, soft agar anchorage-independent growth assay, TRE reporter transactivation assay, and statistical analysis were performed in this study. We performed microarray analysis using 227 triple-negative breast tumors, and clustered the tumors into five groups according to their nuclear receptor expression. Thyroid hormone receptor beta (TRß) was one of the most differentially expressed nuclear receptors in group 5 compared to other groups. TRß low expressing patients were associated with poor outcome. We evaluated the role of TRß in triple-negative breast cancer cell lines representing group 5 tumors. Knockdown of TRß increased soft agar colony and reduced sensitivity to docetaxel and doxorubicin treatment. Docetaxel or doxorubicin long-term cultured cell lines also expressed decreased TRß protein. Microarray analysis revealed cAMP/PKA signaling was the only KEGG pathways upregulated in TRß knockdown cells. Inhibitors of cAMP or PKA, in combination with doxorubicin further enhanced cell apoptosis and restored sensitivity to chemotherapy. TRß-specific agonists enhanced TRß expression, and further sensitized cells to both docetaxel and doxorubicin. Sensitization was mediated by increased apoptosis with elevated cleaved PARP and caspase 3. TRß represents a novel nuclear receptor target in triple-negative breast cancer; low TRß levels were associated with enhanced resistance to both docetaxel and doxorubicin treatment. TRß-specific agonists enhance chemosensitivity to these two agents. Mechanistically enhanced cAMP/PKA signaling was associated with TRß's effects on response to chemotherapy.