RESUMEN
Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients.
Asunto(s)
Resistencia a Antineoplásicos/genética , Genoma Humano , Acumulación de Mutaciones , Tasa de Mutación , Línea Celular Tumoral , Humanos , Modelos Genéticos , Mutación PuntualRESUMEN
With the identification of therapeutic targets for lung adenocarcinoma, it has become mandatory to distinguish it from other entities. Some cases remain classified as non-small cell lung carcinoma, not otherwise specified (NSCLC-NOS) with immunohistochemistry. Electron microscopy (EM) can be useful, allowing the identification of glandular differentiation. The aim of this study was to determine the complementary value of immunohistochemistry and EM.Forty-eight NSCLC-NOS cases were selected (PSMAR-Biobank, Barcelona, Spain). Immunohistochemistry (TTF-1, p40) was performed. Tissue was retrieved from paraffin blocks. Results were compared to the final diagnosis, derived from combination of light microscopy, immunohistochemistry, EM, molecular studies and resection specimen.Immunohistochemistry concurred with final diagnosis in 36 cases (75%, Kappa = 0.517). EM agreed with final diagnosis in 35 (72.9%, Kappa = 0.471). Immunohistochemistry had a sensitivity = 73%, specificity = 100%, positive predictive value (PPV) = 100% and negative predictive value (NPV) = 52.4% for adenocarcinoma. All adenocarcinoma cases not solved by immunohistochemistry (n = 10) were classified by EM, and vice versa. Data from EM were identical to those of immunohistochemistry: sensitivity = 73%, specificity = 100%, PPV = 100% and NPV = 52.4%. Combining both techniques, 47 cases were coincident with final diagnosis (97.9%, Kappa = 0.943).EM can provide valuable information in subtyping NSCLC-NOS, being particularly useful when immunohistochemistry is inconclusive. EM could be considered as a complementary tool for decision-making in NSCLC-NOS.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Inmunohistoquímica/métodos , Neoplasias Pulmonares/diagnóstico , Microscopía Electrónica de Transmisión/métodos , Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/clasificación , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/patología , Terapia Molecular DirigidaRESUMEN
INTRODUCTION: Guidelines for surveillance after polypectomy are lacking in strong evidence. Our aim was to identify some precursors of colorectal cancer lesions at 3 years after polypectomy to improve stratification and surveillance programs. METHODS: We included patients with high-risk lesions (HRLs), defined as advanced adenoma (AA), large serrated polyps (SPs), and multiplicity (≥3 of any adenomas/SPs). Data on age, sex, cardiovascular risk factors, pharmacological treatment, and the histological characteristics in each individual, and mutations in genes involved in the most advanced index polyp, were collected. Parameters independently associated with a metachronous HRL diagnosis were evaluated through univariate and multivariate analyses. The results are reported as odds ratios and 95% confidence intervals along with P values. RESULTS: A total of 537 cases (median age: 60.7 years; 66% male) were included. Dyslipidemia and smoking correlated with metachronous HRLs. Multivariate logistic regression analysis showed that the presence of multiplicity with ≥3 polyps on the index colonoscopy was significantly associated with metachronous HRL, AA, proximal AA, and ≥3 polyps at 3 years. In addition, independent predictors of metachronous proximal AA were increasing age, female sex, and the loss of expression of the MLH1 protein. DISCUSSION: Multiplicity was a strong predictor of HRLs at 3 years, although the inclusion of other clinical variables (age, sex, smoking status, and dyslipidemia) improves surveillance recommendations. Without these risk factors, the surveillance could be extended to 5 years; we propose examining the somatic expression of MHL1 in all patients.
Asunto(s)
Adenoma/diagnóstico , Pólipos del Colon/complicaciones , Pólipos del Colon/cirugía , Colonoscopía , Neoplasias Colorrectales/diagnóstico , Neoplasias Primarias Secundarias/diagnóstico , Factores de Edad , Anciano , Pólipos del Colon/genética , Pólipos del Colon/patología , Dislipidemias/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Factores Sexuales , Fumar/efectos adversosRESUMEN
PURPOSE: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dephosphorylates mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38], mediates breast cancer chemoresistance, and is repressible by doxorubicin in breast cancer cells. We aimed to characterize doxorubicin effects on MKP-1 and phospho-MAPKs in human breast cancers and to further study the clinical relevance of MKP-1 expression in this disease. EXPERIMENTAL DESIGN: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a panel of human breast cancer cells by Western blot and in human breast cancer were assayed ex vivo by immunohistochemistry (n = 50). MKP-1 expression was also assayed in a range of normal to malignant breast lesions (n = 30) and in a series of patients (n = 96) with breast cancer and clinical follow-up. RESULTS: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in approximately 50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin down-regulated MKP-1, and up-regulated p-ERK1/2 and p-JNK, in the majority of cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or phospho-MAPKs. With regard to patient outcome, MKP-1 overexpression was an adverse prognostic factor for relapse both by univariate (P < 0.001) and multivariate analysis (P = 0.002). CONCLUSIONS: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in many human breast cancers.
Asunto(s)
Neoplasias de la Mama/patología , Doxorrubicina/farmacología , Fosfatasa 1 de Especificidad Dual/metabolismo , Antibióticos Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Análisis por Conglomerados , Fosfatasa 1 de Especificidad Dual/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Estimación de Kaplan-Meier , Recurrencia Local de Neoplasia , Fosforilación/efectos de los fármacos , Valor Predictivo de las Pruebas , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVES: KRAS mutations are one of the most prevalent alterations in non-small cell lung cancer. However, patients with this driver alteration present heterogeneous clinical outcomes. In this study, we have explored the potential clinical impact of coexisting alterations in this subset of patients. MATERIALS AND METHODS: Samples from a cohort of 69 lung adenocarcinoma patients homogenously treated with platinum doublet as first-line therapy were evaluated using targeted next generation sequencing (NGS). Mutations and copy number alterations were assessed in 37 advanced KRAS-mutant (KRASm) and in 32 KRAS wild-type (KRASwt). RESULTS: TP53 was the most frequent additional alteration found in both cohorts. Interestingly, TP53 mutations were more frequent in KRASwt than in KRASm patients (80 % vs. 34 %; pâ¯<⯠0.05) as well as STK11 mutations (17 % vs 8 %, p=NS). FGFR3 mutations were only found concomitantly with KRASm (11 %). No genomic co-alteration had an impact on overall survival within the KRASm patients treated with chemotherapy. CONCLUSIONS: KRAS mutated lung adenocarcinoma is a heterogeneous entity and comprehensive characterization of co-alterations using NGS may lead to more accurate patient stratification.
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Adenocarcinoma del Pulmón/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Mutación , Platino (Metal)/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Cohortes , Estudios de Seguimiento , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer.
Asunto(s)
Resistencia a Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Gástricas/metabolismo , Regulación hacia Arriba , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Serina-Treonina Quinasas TOR/metabolismo , TrastuzumabRESUMEN
BACKGROUND: More than 60% of patients with lung cancer are diagnosed at advanced stages. The introduction of targeted therapies requires molecular diagnosis to guide treatment. The aim of this study was to evaluate the feasibility of performing mutational analysis with brushing specimens obtained by radial-miniprobe endobronchial ultrasound (R-EBUS) plus fluoroscopy-guided bronchoscopy in patients with peripheral pulmonary adenocarcinoma. METHODS: Brushing specimens were deposited on cytological slides and were conserved in Roswell Park Memorial Institute (RPMI) culture medium. DNA was isolated to perform a mutational analysis with real-time quantitative polymerase chain reaction and Sanger sequencing for epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS). RESULTS: Thirty patients with adenocarcinoma were prospectively included. In 100% of the patients, the molecular study was viable with brushing specimens. In 16 (53.3%), KRAS or EGFR mutations were detected: 10 KRAS mutations (33.3%) and 6 EGFR mutations (20%). In a comparison with histological samples, a correlation of 86.6% was detected, and only 2 patients with wild-type results from brushing specimens presented with an EGFR mutation in histological samples. CONCLUSIONS: Brushing specimens conserved in RPMI medium and obtained by R-EBUS plus fluoroscopy-guided bronchoscopy are valid for molecular studies. They allow the detection of EGFR/KRAS mutations in patients with peripheral adenocarcinoma. In addition, invasive techniques are avoided, the risk of complications is reduced, and the obtained samples are optimized.
Asunto(s)
Adenocarcinoma del Pulmón/diagnóstico , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico/métodos , Fluoroscopía/métodos , Biopsia Guiada por Imagen/métodos , Neoplasias Pulmonares/diagnóstico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Broncoscopía/métodos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Citodiagnóstico , Análisis Mutacional de ADN , Receptores ErbB/genética , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
BACKGROUND: The assessment of epidermal growth factor receptor (EGFR) mutations is crucial for the management of patients with lung adenocarcinoma. Circulating tumor DNA (ctDNA)-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. PATIENTS AND METHODS: Consecutive patients diagnosed with EGFR-mutant lung adenocarcinoma in tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. EGFR mutations in plasma were quantified using BEAMing (beads, emulsions, amplification, and magnetics) or digital PCR and were correlated with mutations in tumor and with radiologic response and progression. RESULTS: Two hundred twenty-one plasma samples from 33 patients were analyzed. EGFR mutations in plasma were detected in 83% of all patients and 100% of those with extrathoracic metastases. The dynamics of the EGFR mutation load predicted response in 93% and progression in 89% of cases well in advance of radiologic evaluation. Progression-free survival for patients in whom ctDNA was not detected in plasma during treatment was significantly longer than for those in whom ctDNA remained detectable (295 vs. 55 days; hazard ratio, 17.1; P < .001). CONCLUSION: The detection of EGFR mutations in ctDNA showed good correlation with that in tumor biopsy and predicted tumor response and progression in most patients. The liquid biopsy for ctDNA-based assessment of EGFR mutations is a reliable technique for diagnosis and follow-up in patients with EGFR-mutant lung adenocarcinoma in routine clinical practice.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ácidos Nucleicos Libres de Células/genética , ADN de Neoplasias/genética , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/patología , Mutación , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/sangre , ADN de Neoplasias/sangre , Progresión de la Enfermedad , Receptores ErbB/genética , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Approximately 50 % of malignant melanomas harbor activating point mutations in the BRAF gene. Typically, these mutations result in the substitution of the amino acid valine at codon 600 of the gene, and 90-95 % of mutations are either BRAF (V600E) or BRAF (V600K). Specific BRAF inhibitors such as dabrafenib and vemurafenib are the mainstays of treatment in patients with metastatic BRAF-mutant malignant melanomas. The third most common BRAF mutation is V600R, which also leads to increased BRAF signaling. Although evidence exists about the activity of dabrafenib and vemurafenib in patients with the BRAF (V600R) mutation, these patients have been systematically excluded from recent trials with targeted therapies. CASE PRESENTATION: Here, we report the positive results in terms of survival and quality of life obtained with dabrafenib in an 80-year-old Caucasian male patient with a Charlson Comorbidity Index of 8 diagnosed with metastatic malignant melanoma harboring the BRAF (V600R) mutation. Our patient was treated with dabrafenib for 7 months with minimal toxicity. We also report exploratory analyses of circulating tumor DNA during targeted treatment. Interestingly, the mutation was not detected after starting treatment and became detectable before radiological disease progression. CONCLUSIONS: Our report suggests that (1) a relevant benefit can be obtained with a BRAF inhibitor in real-world patients with a malignant melanoma harboring a BRAF (V600R) mutation, and that (2) circulating tumor DNA detection might be of help in assessing tumor burden in everyday clinical practice. The results reported here should encourage the inclusion of patients with BRAF (V600R)-mutated malignant melanomas in future prospective clinical trials with BRAF inhibitors.
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Antineoplásicos/uso terapéutico , Neoplasias del Oído/tratamiento farmacológico , Imidazoles/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Oximas/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Anciano de 80 o más Años , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/secundario , Neoplasias del Oído/genética , Neoplasias del Oído/patología , Humanos , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/secundario , Imagen por Resonancia Magnética , Masculino , Melanoma/diagnóstico por imagen , Melanoma/genética , Melanoma/secundario , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Calidad de Vida , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
PURPOSE: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. EXPERIMENTAL DESIGN: Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. RESULTS: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. CONCLUSIONS: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Células 3T3 , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB/genética , Receptores ErbB/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Panitumumab , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
PURPOSE: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. EXPERIMENTAL DESIGN: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. RESULTS: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. CONCLUSIONS: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.
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Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Genes erbB-1/genética , Mutación , Antineoplásicos/uso terapéutico , Western Blotting , Línea Celular Tumoral , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Análisis Mutacional de ADN , Espacio Extracelular/genética , Citometría de Flujo , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
NF-кB has been linked to doxorubicin resistance in breast cancer patients. NF-кB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-кB-dependent genes and the biological consequences are unclear. We studied NF-кB-dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-кB-dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKß-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-кB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF-кB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of the NF-кB-dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-кB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-кB/p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-кB-response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior.
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Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Doxorrubicina/farmacología , FN-kappa B/genética , Proteína p53 Supresora de Tumor/deficiencia , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Células MCF-7 , Ratones , FN-kappa B/metabolismo , Pronóstico , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
AIM: Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. METHODS: We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. RESULTS: In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. CONCLUSION: Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN , Femenino , Histonas/metabolismo , Humanos , Indoles/administración & dosificación , Indoles/farmacología , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Ftalazinas/administración & dosificación , Ftalazinas/farmacología , Piperazinas/administración & dosificación , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trastuzumab , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting.
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Proteínas 14-3-3/metabolismo , Neoplasias de la Mama/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Proteínas 14-3-3/genética , Transporte Activo de Núcleo Celular , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Análisis por Conglomerados , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Ratones , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Pronóstico , Unión Proteica/efectos de los fármacos , Análisis de Supervivencia , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Antibodies against epidermal growth factor receptor (EGFR)--cetuximab and panitumumab--are widely used to treat colorectal cancer. Unfortunately, patients eventually develop resistance to these agents. We describe an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab. Cells with this mutation, however, retain binding to and are growth inhibited by panitumumab. Two of ten subjects studied here with disease progression after cetuximab treatment acquired this mutation. A subject with cetuximab resistance harboring the S492R mutation responded to treatment with panitumumab.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Cetuximab , Neoplasias Colorrectales/genética , Epítopos/genética , Gefitinib , Humanos , Mutación Missense/genética , Panitumumab , Quinazolinas/uso terapéuticoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto/métodos , Receptores ErbB/antagonistas & inhibidores , Oncología Médica/métodos , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Cetuximab , Resistencia a Antineoplásicos , Receptores ErbB/inmunología , Receptores ErbB/fisiología , Femenino , Humanos , Lapatinib , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/fisiología , Neoplasias/enzimología , Neoplasias/genética , Panitumumab , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Receptor ErbB-2/inmunología , Receptor ErbB-2/fisiología , Proyectos de Investigación , Transducción de Señal/efectos de los fármacos , Investigación Biomédica Traslacional , TrastuzumabRESUMEN
The NF kappa B family is composed by five subunits (p65/RelA, c-Rel, RelB, p105-p50/NF kappa B(1), p100-p52/NF kappa B(2)) and controls the expression of many genes that participate in cell cycle, apoptosis, and other key cellular processes. In a canonical pathway, NF kappa B activation depends on the IKK complex activity, which is formed by three subunits (IKKalpha and IKKbeta and IKKgamma/NEMO). There is an alternative NF kappa B activation pathway that does not require IKKbeta or IKKgamma/NEMO, in which RelB is a major player. We report in a panel of human breast cancer cells that the IKK/NF kappa B system is generally overexpressed in breast cancer cells and there is heterogeneity in expression levels of individual members between different cell lines. Doxorubicin, an anticancer agent used in patients with breast cancer, activated NF kappa B and appeared to be less effective in cells expressing predominantly members of the canonical IKK/NF kappa B. Two NF kappa B inhibitors, bortezomib and NEMO-Binding Domain Inhibitory Peptide, prevented doxorubicin-induced NF kappa B activation and increased doxorubicin antitumor effects in BT-474 cells. Transient down-regulation of members of the canonical pathway (p65, p52, c-Rel and IKKgamma/NEMO) by siRNA in HeLa cells increased doxorubicin cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no evident effects on doxorubicin cytotoxicity. To conclude, NF kappa B inhibition sensitized cells to doxorubicin, implying directly p65, p52, c-Rel and IKKgamma/NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the canonical NF kappa B pathway is sufficient to improve doxorubicin antitumor effects.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Quinasa I-kappa B/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Quinasa I-kappa B/fisiología , FN-kappa B/fisiología , Transducción de Señal/fisiologíaRESUMEN
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