RESUMEN
Mogamulizumab is a humanized anti-CC chemokine receptor 4 (CCR4) antibody approved for the treatment of mycosis fungoides and Sézary syndrome. Despite almost universal expression of CCR4 in these diseases, most patients eventually develop resistance to mogamulizumab. We tested whether resistance to mogamulizumab is associated with loss of CCR4 expression. We identified 17 patients with mycosis fungoides or Sézary syndrome who either were intrinsically resistant or acquired resistance to mogamulizumab. Low expression of CCR4 by immunohistochemistry or flow cytometry was found in 65% of patients. Novel emergent CCR4 mutations targeting the N-terminal and transmembrane domains were found in 3 patients after disease progression. Emerging CCR4 copy number loss was detected in 2 patients with CCR4 mutations. Acquisition of CCR4 genomic alterations corresponded with loss of CCR4 antigen expression. We also report on outcomes of 3 cutaneous T-cell lymphoma (CTCL) patients with gain-of-function CCR4 mutations treated with mogamulizumab. Our study indicates that resistance to mogamulizumab in CTCL frequently involves loss of CCR4 expression and emergence of CCR4 genomic alterations. This finding has implications for management and monitoring of CTCL patients on mogamulizumab and development of future CCR4-directed therapies.
Asunto(s)
Resistencia a Antineoplásicos , Linfoma Cutáneo de Células T , Receptores CCR4 , Neoplasias Cutáneas , Anticuerpos Monoclonales Humanizados , Humanos , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/genética , Receptores CCR4/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genéticaRESUMEN
Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III ß-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other ß-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking ß-tubulin isotype compensation visible at the level of total ß-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.
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Neoplasias Ováricas , Tubulina (Proteína) , Humanos , Femenino , Carboplatino/farmacología , Carboplatino/uso terapéutico , Regulación hacia Arriba , Tubulina (Proteína)/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Activación TranscripcionalRESUMEN
BACKGROUND: ABCB1 expression is uncommon in ovarian cancers in the clinical setting so we investigated non-MDR mechanisms of resistance to taxanes. METHODS: We established eight taxane-resistant variants from the human ovarian carcinoma cell lines A2780/1A9, ES-2, MES-OV and OVCAR-3 by selection with paclitaxel or docetaxel, with counter-selection by the transport inhibitor valspodar. RESULTS: Non-MDR taxane resistance was associated with reduced intracellular taxane content compared to parental controls, and cross-resistance to other microtubule stabilising drugs. Collateral sensitivity to depolymerising agents (vinca alkaloids and colchicine) was observed with increased intracellular vinblastine. These variants exhibited marked decreases in basal tubulin polymer and in tubulin polymerisation in response to taxane exposure. TUBB3 content was increased in 6 of the 8 variants. We profiled gene expression of the parental lines and resistant variants, and identified a transcriptomic signature with two highly significant networks built around FN1 and CDKN1A that are associated with cell adhesion, cell-to-cell signalling, and cell cycle regulation. miR-200 family members miR-200b and miR-200c were downregulated in resistant cells, associated with epithelial to mesenchymal transition (EMT), with increased VIM, FN1, MMP2 and/or MMP9. CONCLUSIONS: These alterations may serve as biomarkers for predicting taxane effectiveness in ovarian cancer and should be considered as therapeutic targets.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal , Neoplasias Ováricas/tratamiento farmacológico , Tubulina (Proteína)/metabolismo , Antineoplásicos Fitogénicos/uso terapéutico , Cadherinas/genética , Carcinoma/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Docetaxel , Femenino , Fibronectinas/genética , Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , MicroARNs/genética , Neoplasias Ováricas/genética , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Polimerizacion/efectos de los fármacos , Taxoides/farmacología , Taxoides/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Vimentina/genética , Vinblastina/farmacologíaRESUMEN
Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.
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Inmunoterapia , Linfoma Cutáneo de Células T , Humanos , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/terapia , Linfoma Cutáneo de Células T/patología , Inmunoterapia/métodos , Interferón gamma/metabolismo , Interferón gamma/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/tratamiento farmacológico , Masculino , Femenino , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , MultiómicaRESUMEN
The PD-1 inhibitor pembrolizumab is effective in treating Sézary syndrome, a leukemic variant of cutaneous T-cell lymphoma. Our purpose was to investigate the effects of pembrolizumab on healthy and malignant T cells in Sézary syndrome and to discover characteristics that predict pembrolizumab response. Samples were analyzed before and after 3 weeks of pembrolizumab treatment using single-cell RNA-sequencing of 118,961 peripheral blood T cells isolated from six Sézary syndrome patients. T-cell receptor clonotyping, bulk RNA-seq signatures, and whole-exome data were integrated to classify malignant T-cells and their underlying subclonal heterogeneity. We found that responses to pembrolizumab were associated with lower KIR3DL2 expression within Sézary T cells. Pembrolizumab modulated Sézary cell gene expression of T-cell activation associated genes. The CD8 effector populations included clonally expanded populations with a strong cytotoxic profile. Expansions of CD8 terminal effector and CD8 effector memory T-cell populations were observed in responding patients after treatment. We observed intrapatient Sézary cell heterogeneity including subclonal segregation of a coding mutation and copy number variation. Our study reveals differential effects of pembrolizumab in both malignant and healthy T cells. These data support further study of KIR3DL2 expression and CD8 immune populations as predictive biomarkers of pembrolizumab response in Sézary syndrome.
Asunto(s)
Síndrome de Sézary , Neoplasias Cutáneas , Anticuerpos Monoclonales Humanizados , Variaciones en el Número de Copia de ADN , Humanos , ARN , Receptores KIR3DL2/genética , Síndrome de Sézary/tratamiento farmacológico , Síndrome de Sézary/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genéticaRESUMEN
AIM: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs. METHODS: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute. RESULTS: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance. CONCLUSION: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.
RESUMEN
CONTEXT: Glioblastomas--uniformly fatal brain tumors--often have both monosomy of chromosome 10 and gains of the epidermal growth factor receptor (EGFR) gene locus on chromosome 7, an association for which the mechanism is poorly understood. OBJECTIVES: To assess whether coselection of EGFR gains on 7p12 and monosomy 10 in glioblastomas promotes tumorigenic epidermal growth factor (EGF) signaling through loss of the annexin A7 (ANXA7) gene on 10q21.1-q21.2 and whether ANXA7 acts as a tumor suppressor gene by regulating EGFR in glioblastomas. DESIGN, SETTING, AND PATIENTS: Multidimensional analysis of gene, coding sequence, promoter methylation, messenger RNA (mRNA) transcript, protein data for ANXA7 (and EGFR), and clinical patient data profiles of 543 high-grade gliomas from US medical centers and The Cancer Genome Atlas pilot project (made public 2006-2008; and unpublished, tumors collected 2001-2008). Functional analyses using LN229 and U87 glioblastoma cells. MAIN OUTCOME MEASURES: Associations among ANXA7 gene dosage, coding sequence, promoter methylation, mRNA transcript, and protein expression. Effect of ANXA7 haploinsufficiency on EGFR signaling and patient survival. Joint effects of loss of ANXA7 and gain of EGFR expression on tumorigenesis. RESULTS: Heterozygous ANXA7 gene deletion is associated with significant loss of ANXA7 mRNA transcript expression (P = 1 x 10(-15); linear regression) and a reduction (mean [SEM]) of 91.5% (2.3%) of ANXA7 protein expression compared with ANXA7 wild-type glioblastomas (P = .004; unpaired t test). ANXA7 loss of function stabilizes the EGFR protein (72%-744% increase in EGFR protein abundance) and augments EGFR transforming signaling in glioblastoma cells. ANXA7 haploinsufficiency doubles tumorigenic potential of glioblastoma cells, and combined ANXA7 knockdown and EGFR overexpression promotes tumorigenicity synergistically. The heterozygous loss of ANXA7 in approximately 75% of glioblastomas in the The Cancer Genome Atlas plus infrequency of ANXA7 mutation (approximately 6% of tumors) indicates its role as a haploinsufficiency gene. ANXA7 mRNA transcript expression, dichotomized at the median, associates with patient survival in 191 glioblastomas (log-rank P = .008; hazard ratio [HR], 0.667; 95% confidence interval [CI], 0.493-0.902; 46.9 vs 74.8 deaths/100 person-years for high vs low ANXA7 mRNA expression) and with a separate group of 180 high-grade gliomas (log-rank P = .00003; HR, 0.476; 95% CI, 0.333-0.680; 21.8 vs 50.0 deaths/100 person-years for high vs low ANXA7 mRNA expression). Deletion of the ANXA7 gene associates with poor patient survival in 189 glioblastomas (log-rank P = .042; HR, 0.686; 95% CI, 0.476-0.989; 54.0 vs 80.1 deaths/100 person-years for wild-type ANXA7 vs ANXA7 deletion). CONCLUSION: Haploinsufficiency of the tumor suppressor ANXA7 due to monosomy of chromosome 10 provides a clinically relevant mechanism to augment EGFR signaling in glioblastomas beyond that resulting from amplification of the EGFR gene.
Asunto(s)
Anexina A7/genética , Neoplasias Encefálicas/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 10/genética , Receptores ErbB/genética , Genes Supresores de Tumor , Glioblastoma/genética , Anexina A7/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Cromosomas Humanos Par 7 , Factor de Crecimiento Epidérmico/metabolismo , Epigénesis Genética , Receptores ErbB/metabolismo , Femenino , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Monosomía , Mutación , Fosfohidrolasa PTEN/genética , ARN Mensajero/análisis , Transducción de Señal , Análisis de SupervivenciaRESUMEN
In a previously published study, higher levels of spleen tyrosine kinase (Syk) were observed in recurrent post-chemotherapy ovarian cancers compared to primary tumors. Syk inhibition was found to stabilize microtubules and potentiate paclitaxel activity in cellular models of taxane-resistant ovarian cancers. We further studied the effects of Syk inhibition on paclitaxel activity in Syk(+) ovarian cancer cell models and in variants selected for taxane resistance. Syk inhibition was accomplished using RNAi and by exposure to the small molecule competitive inhibitor R406, the active metabolite of fostamatinib. Exposure to R406 or to a SYK-specific pool of siRNAs did not alter taxane activity in the OVCAR-3 cell line, which has the most Syk content in our panel of nine human ovarian cancer cell lines. However, treatment with R406 sensitised the multidrug resistant (MDR) variants MES-SA/Dx5 and SK-OV-3/TR to paclitaxel in a dose-dependent manner resulting from the inhibition of the ABCB1/P-glycoprotein (P-gp) drug transporter. These observations are Syk-independent since both MDR cell models are Syk negative. R406 modulated resistance to other known P-gp substrates, and we observed orthovanadate-sensitive ATPase stimulation resulting from treatment with R406. These data indicate that the chemo-sensitizing effect of R406 in taxane-resistant cells previously reported was not associated with Syk but resulted from the modulation of P-gp-mediated MDR.
Asunto(s)
Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Oxazinas/farmacología , Piridinas/farmacología , Quinasa Syk/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Paclitaxel/farmacología , ARN Interferente Pequeño/genética , Quinasa Syk/genética , Taxoides/farmacologíaRESUMEN
PURPOSE: The primary aim of this study was to determine cabazitaxel's affinity for the ABCB1/P-glycoprotein (P-gp) transporter compared to first-generation taxanes. METHODS: We determined the kinetics of drug accumulation and retention using [14C]-labeled taxanes in multidrug-resistant (MDR) cells. In addition, membrane-enriched fractions isolated from doxorubicin-selected MES-SA/Dx5 cells were used to determine sodium orthovanadate-sensitive ATPase stimulation after exposure to taxanes. Custom [3H]-azido-taxane analogues were synthesized for the photoaffinity labeling of P-gp. RESULTS: The maximum intracellular drug concentration was achieved faster with [14C]-cabazitaxel (5 min) than [14C]-docetaxel (15-30 min). MDR cells accumulated twice as much cabazitaxel than docetaxel, and these levels could be restored to parental levels in the presence of the P-gp inhibitor PSC-833 (valspodar). Efflux in drug-free medium confirmed that MDR cells retained twice as much cabazitaxel than docetaxel. There was a strong association (r2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. One cell model expressing low levels of P-gp was not cross-resistant to cabazitaxel while demonstrating modest resistance to docetaxel. Furthermore, there was a 1.9 × reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel compared to docetaxel. We calculated a dissociation constant (Kd) value of 1.7 µM for [3H]-azido-docetaxel and ~ 7.5 µM for [3H]-azido-cabazitaxel resulting in a 4.4 × difference in P-gp labeling, and cold docetaxel was a more effective competitor than cabazitaxel. CONCLUSION: Our studies confirm that cabazitaxel is more active in ABCB1(+) cell models due to its reduced affinity for P-gp compared to docetaxel.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Docetaxel/farmacología , Taxoides/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Ciclosporinas/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factores de TiempoRESUMEN
We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.
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Adenocarcinoma de Células Claras/genética , Adenocarcinoma/genética , Neoplasias de la Mama/genética , Carcinoma Papilar/genética , Proteínas Nucleares , Neoplasias Ováricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Células Claras/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Papilar/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Efrina-B1/genética , Efrina-B1/metabolismo , Femenino , Factor de Transcripción GATA3 , Proteínas Ligadas a GPI , Humanos , Inmunohistoquímica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mesotelina , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/metabolismo , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Factores de Transcripción del Factor Regulador X , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
The origin of drug-resistant cells in human cancers has been a fundamental problem of cancer pharmacology. Two major contrasting hypotheses (genetics versus epigenetics) have been proposed to elucidate the mechanisms of acquired drug resistance. In this study, we answer these fundamental questions through investigation of the genetic and epigenetic pathways that control the origin of ABCB1 (MDR1) gene activation with acquired multidrug resistance in drug-sensitive human sarcoma (MES-SA cells). The genetic and epigenetic bases of this selected activation involve the initiation of transcription at a site 112 kb upstream of the ABCB1 proximal promoter (P1) in the drug-resistant cells. This activation was associated with a chromatin-remodeling process characterized by an increase in acetylated histone H3 within a 968-bp region 5' of the ABCB1 upstream promoter. These alterations provide both genetic and epigenetic susceptibility for ABCB1 expression in drug-resistant cells. Complete activation of the ABCB1 gene through the coding region was proposed by interactions of selected trans-alterations or epigenetic changes on the ABCB1 proximal promoter, which occurred during initial drug exposure. Thus, our data provide evidence for a major genomic alteration that changes the chromatin structure of the ABCB1 upstream promoter via acetylation of histone H3 initiating ABCB1 activation, further elucidating the genetic and epigenetic bases that determine chemotherapeutic response in drug-resistant derivatives of MES-SA cells.
Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Genes MDR/genética , Sarcoma/tratamiento farmacológico , Sarcoma/genética , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/genética , Acetilación , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Línea Celular Tumoral , Cromatina/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma/metabolismo , Activación Transcripcional , Neoplasias Uterinas/metabolismoRESUMEN
The goal of this study was to determine the prevalence of sequence variants in the class I beta-tubulin (clone m40) gene and their occurrence in human tumors and cancer cell lines. DNA was isolated from 93 control individuals representing a wide variety of ethnicities, 49 paclitaxel-naive specimens (16 ovarian cancers, 17 non-small cell lung cancers, and 16 ovarian cancer cell lines), and 30 paclitaxel-resistant specimens (9 ovarian cancers, 9 ovarian cancer cell lines, and 12 ovarian cancer xenografts in nude mice). Denaturing high-performance liquid chromatography and direct sequence analysis detected two silent polymorphisms in exon 4, Leu217Leu (CTG/CTA) and Gly400Gly (GGC/GGT), with minor allele frequencies of 17 and 0.5%, respectively. Five nucleotide substitutions and one single-base deletion were detected in introns 1, 2, and 3 and in the 3' untranslated region. Analysis of 49 paclitaxel-naive and 30 paclitaxel-resistant specimens revealed no additional polymorphisms in the coding region. In addition, no amino acid replacements were found in chimpanzee, gorilla, and orangutan in comparison to human. Our data demonstrate a very high degree of sequence conservation in class I beta-tubulin, suggesting that all residues are important in tubulin structure and function. Individual variation in response to treatment with paclitaxel is not likely to be caused by genetic variations in the beta-tubulin drug target. Moreover, acquired mutations in class I beta-tubulin are unlikely to be a clinically relevant cause of drug resistance.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Neoplasias Pulmonares/genética , Mutación/genética , Neoplasias Ováricas/genética , Paclitaxel/farmacología , Tubulina (Proteína)/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/etnología , Estudios de Casos y Controles , Secuencia Conservada , Análisis Mutacional de ADN , Cartilla de ADN , Femenino , Variación Genética , Haplotipos , Heterocigoto , Humanos , Neoplasias Pulmonares/etnología , Ratones , Neoplasias Ováricas/etnología , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Tubulina (Proteína)/clasificación , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We studied the role of miRNA-200 family members in cellular sensitivity to paclitaxel and carboplatin, using two ovarian cancer cell lines, OVCAR-3 and MES-OV, and their paclitaxel resistant variants OVCAR-3/TP and MES-OV/TP. Both resistant variants display a strong epithelial-mesenchymal transition (EMT) phenotype, with marked decreases in expression of miR-200c and miR-141 in OVCAR-3/TP, and down-regulation of all five members of the miR-200 family in MES-OV/TP. Lentiviral transfection of inhibitors of miR-200c or miR-141 in parental OVCAR-3 triggered EMT and rendered the cells resistant to paclitaxel and carboplatin. Conversely, the infection of OVCAR-3/TP cells with retroviral particles carrying the miR-200ab429 and 200c141 clusters triggered a partial mesenchymal to epithelial transition (MET). This partial MET was not sufficient to re-sensitize OVCAR-3/TP cells to paclitaxel. However, the miR-200c/miR-141 cluster transfectants became 6-8x resistant to carboplatin, an unexpected result, whereas miR-200a/miR-200b/miR-429 had no effect. Transfecting the OVCAR-3/TP GFP cells with specific miRNA mimics confirmed these data. MiR-200c and miR-141 mimics conferred resistance to carboplatin in MES-OV/TP cells, similar to OVCAR-3/TP, but sensitized MES-OV to paclitaxel. Several genes involved in balancing oxidative stress were altered in OVCAR-3/TP 200c141 cells compared to controls. The miR-200 family plays major, cell-context dependent roles in regulating EMT and sensitivity to carboplatin and paclitaxel in OVCAR-3 and MES-OV cells.
Asunto(s)
Carboplatino/uso terapéutico , Cistadenocarcinoma Seroso/genética , Resistencia a Antineoplásicos/genética , MicroARNs/fisiología , Neoplasias Ováricas/genética , Paclitaxel/uso terapéutico , Línea Celular Tumoral , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/genética , Familia de Multigenes , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patologíaRESUMEN
We studied mechanisms of resistance to the novel taxane cabazitaxel in established cellular models of taxane resistance. We also developed cabazitaxel-resistant variants from MCF-7 breast cancer cells by stepwise selection in drug alone (MCF-7/CTAX) or drug plus the transport inhibitor PSC-833 (MCF-7/CTAX-P). Among multidrug-resistant (MDR) variants, cabazitaxel was relatively less cross-resistant than paclitaxel and docetaxel (15- vs. 200-fold in MES-SA/Dx5 and 9- vs. 60-fold in MCF-7/TxT50, respectively). MCF-7/TxTP50 cells that were negative for MDR but had 9-fold resistance to paclitaxel were also 9-fold resistant to cabazitaxel. Selection with cabazitaxel alone (MCF-7/CTAX) yielded 33-fold resistance to cabazitaxel, 52-fold resistance to paclitaxel, activation of ABCB1, and 3-fold residual resistance to cabazitaxel with MDR inhibition. The MCF-7/CTAX-P variant did not express ABCB1, nor did it efflux rhodamine-123, BODIPY-labeled paclitaxel, and [(3)H]-docetaxel. These cells are hypersensitive to depolymerizing agents (vinca alkaloids and colchicine), have reduced baseline levels of stabilized microtubules, and impaired tubulin polymerization in response to taxanes (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Class III ß-tubulin (TUBB3) RNA and protein were elevated in both MCF-7/CTAX and MCF-7/CTAX-P. Decreased BRCA1 and altered epithelial-mesenchymal transition (EMT) markers are also associated with cabazitaxel resistance in these MCF-7 variants, and may serve as predictive biomarkers for its activity in the clinical setting. In summary, cabazitaxel resistance mechanisms include MDR (although at a lower level than paclitaxel and docetaxel), and alterations in microtubule dynamicity, as manifested by higher expression of TUBB3, decreased BRCA1, and by the induction of EMT.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Taxoides/farmacología , Tubulina (Proteína)/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclosporinas/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Tubulina (Proteína)/metabolismoRESUMEN
Idarubicin (IDA) is an anthracycline anticancer drug utilized in the treatment of acute leukemias. There are conflicting data published with regard to the cross-resistance of IDA in multidrug-resistant (MDR) cells expressing P-glycoprotein (P-gp). We evaluated the cytotoxicity and cellular accumulation of IDA in a panel of anthracycline-selected MDR cell lines. Leukemia K562/R7 cells and sarcoma MES-SA/Dx5 cells expressing high levels of the MDR1 (ABCB1) gene were resistant to IDA (42-fold and 150-fold, respectively). In both of these cell lines, resistance to IDA was equivalent to that for doxorubicin, the drug used to select for the MDR variants. The P-gp inhibitor PSC 833 (valspodar) at 2 microM completely restored sensitivity to IDA. IDA accumulation was decreased 12-fold in MES-SA/Dx5 cells vs parental cell line, and drug uptake was restored to control levels by PSC 833. Reduced intracellular IDA was correlated with P-gp content by flow cytometry. Experiments in NIH3T3 murine cells transfected with the human MDR1 gene substantiated the findings of cross-resistance to IDA and reversal of resistance by PSC 833. Our data indicate that IDA is a high-affinity substrate for P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Antibióticos Antineoplásicos/farmacología , Ciclosporinas/farmacología , Idarrubicina/farmacología , Células K562/efectos de los fármacos , Antibióticos Antineoplásicos/uso terapéutico , Ciclosporinas/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Idarrubicina/uso terapéuticoRESUMEN
A vinblastine resistant cell line, KCVB2, was established by co-selecting the parental erythroleukemic cell line K562 with step-wise increased concentrations of vinblastine (Velban) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KCVB2 cells are 8-fold resistant to the selecting agent, vinblastine, but also exhibit significant resistance to other vinca alkaloids, including 14-fold resistance to vinorelbine, as well as 6-fold cross-resistance to paclitaxel. Doubling time and morphology were similar to the parental K562 cells. Rt-PCR analysis revealed no alterations in the expression of the mdr1 and MRP genes. Intracellular vinblastine accumulation was unchanged. Disruption of the mitotic spindles and multiple mitotic asters occurred in both cell lines but required higher concentrations of vinblastine in KCVB2 cells than in K562 cells. Significant differences were observed in the tubulin content of KCVB2 cells: reduction of total tubulin content, increased polymerized fraction of total tubulin, and overexpression of class III beta-tubulin which is expressed at very low levels in the parental K562 cells. K562 cells transfected with murine class III beta-tubulin did not display the resistance pattern observed in KCVB2 cells. Revertants of KCVB2 manifested reversion to parental drug sensitivity, an increase in total tubulin level, and a decrease in polymerized tubulin. In conclusion, the KCVB2 cell line displays a novel mechanism of resistance to both depolymerizing and stabilizing microtubule-targeted cytotoxins which does not involve altered cellular drug accumulation, but corresponds to alterations in the total tubulin content and polymerization status, and may involve an effect on microtubule dynamics.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Resistencia a Antineoplásicos/fisiología , Leucemia/tratamiento farmacológico , Microtúbulos/efectos de los fármacos , Tubulina (Proteína)/efectos de los fármacos , Vinblastina/farmacología , Antineoplásicos Fitogénicos/farmacocinética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclosporinas/efectos de los fármacos , Ciclosporinas/metabolismo , Genes MDR , Humanos , Leucemia/metabolismo , Microtúbulos/genética , Mitosis/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de Proteína , Huso Acromático/efectos de los fármacos , Transfección/métodos , Tubulina (Proteína)/análisis , Tubulina (Proteína)/genética , Vinblastina/farmacocinéticaRESUMEN
Ovarian cancer is associated with a leukocyte infiltrate and high levels of chemokines such as CCL2. We tested the hypothesis that CCL2 inhibition can enhance chemotherapy with carboplatin and paclitaxel. Elevated CCL2 expression was found in three non-MDR paclitaxel resistant ovarian cancer lines ES-2/TP, MES-OV/TP and OVCAR-3/TP, compared to parental cells. Mice xenografted with these cells were treated with the anti-human CCL2 antibody CNTO 888 and the anti-mouse MCP-1 antibody C1142, with and without paclitaxel or carboplatin. Our results show an additive effect of CCL2 blockade on the efficacy of paclitaxel and carboplatin. This therapeutic effect was largely due to inhibition of mouse stromal CCL2. We show that inhibition of CCL2 can enhance paclitaxel and carboplatin therapy of ovarian cancer.
Asunto(s)
Anticuerpos/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carboplatino/uso terapéutico , Quimiocina CCL2/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Anticuerpos/administración & dosificación , Anticuerpos/inmunología , Anticuerpos/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carboplatino/administración & dosificación , Carboplatino/farmacología , Línea Celular Tumoral , Quimiocina CCL2/inmunología , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Ovario/efectos de los fármacos , Ovario/inmunología , Ovario/patología , Paclitaxel/administración & dosificación , Paclitaxel/farmacologíaRESUMEN
The microtubule-associated protein Tau has been reported to be a predictive factor for clinical response to taxanes in metastatic breast cancer. We generated a panel of eight taxane-resistant variants from four human breast cancer cell lines (MCF-7, T-47D, MDA-MB-231, and BT-549). Four variants had higher levels of Tau compared with their T-47D and MDA-MB-231 parental cells. Using isoform-specific primers, we found that Tau 0N, 1N, 2N, 3R, and 4R isoforms are overexpressed in the resistant variants, as is Tau exon 6 but not exons 4A or 8. To determine whether Tau overexpression produces resistance to taxanes, we derived three independent T-47D clones stably overexpressing Tau 3R and 4R isoforms. Tau overexpression did not result in taxane resistance compared with parental cells transfected with vector alone. We then knocked down Tau expression in three cell lines that expressed Tau constitutively (MCF-7 and ZR-75-1 breast cancer cells, and OVCAR-3 ovarian cancer cells). Lentivirus-mediated silencing of Tau expression in MCF-7 and OVCAR-3 cells did not result in increased taxane sensitivity compared with luciferase short hairpin RNA-infected cells and uninfected parental cells. Transient silencing using Tau-specific small interfering RNAs also did not alter taxane sensitivity relative to nontargeting controls in both MCF-7 and ZR-75-1 cells. These results show that neither overexpression nor depletion of Tau modulates cellular sensitivity to taxanes. Although Tau overexpression has been reported to be a predictive marker of taxane resistance, it is not likely to be a direct mechanism of taxane resistance in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Proteínas tau/antagonistas & inhibidores , Proteínas tau/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/fisiología , Humanos , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/farmacología , Taxoides/farmacología , Transfección , Células Tumorales Cultivadas , Proteínas tau/metabolismoRESUMEN
Zosuquidar is a potent and specific inhibitor of P-glycoprotein (P-gp). In preliminary experiments, blockade of P-gp for at least 12 h was required to reverse daunorubicin resistance. Because of the short half-life of zosuquidar, we performed a phase I trial of this drug as a 72-h infusion (CIV) in 16 patients during leukemic induction with daunorubicin and cytarabine. Study goals were to establish safety and determine the dose required for P-gp inhibition in NK cells and AML blasts. > 90% P-gp inhibition was achieved within 2h at a plasma threshold of 132 ng/ml zosuquidar. The recommended phase II dose of zosuquidar is 700 mg/day.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Citarabina/administración & dosificación , Citarabina/efectos adversos , Daunorrubicina/administración & dosificación , Daunorrubicina/efectos adversos , Dibenzocicloheptenos/administración & dosificación , Dibenzocicloheptenos/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Quinolinas/administración & dosificación , Quinolinas/efectos adversos , Factores de TiempoRESUMEN
Taxanes are important drugs in the treatment of ovarian and other cancers, but their efficacy is limited by intrinsic and acquired drug resistance. Expression of the multidrug transporter P-glycoprotein, encoded by the MDR1 (ABCB1) gene, is one of the causes of clinical drug resistance to taxanes. To study the mechanisms of MDR1 activation related to taxanes, we established 11 multidrug-resistant variants from six ovarian cancer cell lines by continuous exposure to either paclitaxel or docetaxel. We profiled gene expression and gene copy number alterations in these cell lines using cDNA microarrays and identified a cluster of genes coactivated with MDR1 in 7q21.11-13. Regional activation was evident in nine resistant variants displaying a coexpression pattern of up to 22 genes over an 8-Mb area, including SRI, MGC4175, CLDN12, CROT, and CDK6. In six of these variants, regional activation was driven by gene copy number alterations, with low-level gains or high-level amplifications spanning the involved region. However, three variants displayed regional increases in gene expression even without concomitant gene copy number changes. These results suggest that regional gene activation may be a fundamental mechanism for acquired drug resistance, with or without changes in gene dosage. In addition to numerical and structural chromosomal changes driven by genome instability in cancer cells, other mechanisms might be involved in MDR1 regional activation, such as chromatin remodeling and DNA or histone modifications of the 7q21 region.