RESUMEN
OBJECTIVES: There is some controversy surrounding theBRAFV600E mutation in patients with lung adenocarcinomas. Although the BRAFV600E mutation is sensitive to BRAF inhibitors, the efficiency of these inhibitors on patients harboring an EGFRL858R/del19/EGFRT790M/BRAFV600E pattern remains unknown. MATERIALS AND METHODS: Here, we presented the case of a patient with initial response followed by progression on osimertinib. Resistance mutations (EGFRT790M, EGFRC797S, BRAFV600E, MET amp and HER2 amp) were assessed in the tissue or plasma DNA using NGS and digital droplet PCR at progression and during osimertinib treatment. RESULTS: Resistance to osimertinib coincided with the emergence of an additional tumor cell subpopulation carrying the knownBRAFV600E resistance mutation. The patient exhibited two tumor subclones (EGFRdel19/T790M and BRAFV600E) that displayed distinct responses to successive tyrosine kinase inhibitors. CONCLUSION: We report the first successful example of using sequential treatment with dabrafetinib/trametinib and osimertinib. Our finding provided that unique tumor biopsies deliver incomplete genetic information, and highlighted the complementary role of circulating tumor DNA to tissue biopsies and CT-scans to efficiently monitor response to osimertinib.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Biopsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Acrilamidas/uso terapéutico , Anciano , Compuestos de Anilina/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Ácidos Nucleicos Libres de Células/análisis , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
The zinc-finger protein A20/TNFAIP3, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, has been shown to protect MCF-7 breast carcinoma cells from TNFalpha-induced apoptosis. As estrogen receptor (ER) status is an important parameter in the development and progression of breast cancer, we analysed the effect of 17beta-estradiol (E2) treatment on the expression of A20. We found that A20 is a new E2-regulated gene, whose expression correlates with ER expression in both cell lines and tumor samples. With the aim of investigating the impact of A20 expression on MCF-7 cells in response to ER ligands, we established stably transfected-MCF-7 cells overexpressing A20 (MCF-7-A20). These cells exhibited a phenotype of resistance to the 4-hydroxy-tamoxifen cytostatic and pro-apoptotic actions and of hyper-response to E2. Dysregulations in bax, bcl2, bak, phospho-bad, cyclin D1, cyclin E2, cyclin D2 and cyclin A2 proteins expression were shown to be related to the resistant phenotype developed by the MCF-7-A20 cells. Interestingly, we found that A20 was also overexpressed in MVLN and VP tamoxifen-resistant cell lines. Furthermore, high A20 expression levels were observed in more aggressive breast tumors (ER-negative, progesterone receptor-negative and high histological grade). These overall findings strongly suggest that A20 is a key protein involved in tamoxifen resistance, and thus represents both a new breast cancer marker and a promising target for developing new strategies to prevent the emergence of acquired mechanisms of drug resistance in breast cancer.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Estradiol/farmacología , Antagonistas de Estrógenos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Tamoxifeno/análogos & derivados , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclinas/metabolismo , Proteínas de Unión al ADN , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E2) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based gene-selection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E2, OH-Tam or both E2 and OH-Tam. Compiled data revealed a hyper-response to E2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor alpha on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E2. Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Estradiol/uso terapéutico , Tamoxifeno/análogos & derivados , Tamoxifeno/uso terapéutico , Antineoplásicos Hormonales/agonistas , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Quimioterapia Combinada , Femenino , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamoxifeno/agonistas , Células Tumorales CultivadasRESUMEN
We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.