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High grade serous ovarian carcinoma (HGSC) without identifiable serous tubal intraepithelial carcinoma (STIC) within the fallopian tube (FT) occurs in approximately 50% of patients. The objective of this study was to use a multisite tumor sampling approach to study HGSC with and without STIC. RNAseq analysis of HGSC samples collected from multiple sites e.g. ovary, FT and peritoneum, revealed moderate levels of intrapatient heterogeneity in gene expression that could influence molecular profiles. Mixed-model ANOVA analysis of gene expression in tumor samples from patients with multiple tumor sites (n = 13) and patients with a single site tumor sample (n = 11) to compare HGSC-STIC to HGSC-NOSTIC identified neurotensin (NTS) as significantly higher (> two-fold change, False Discovery Rate (FDR) < 0.10) in HGSC-STIC. This data was validated using publicly available RNA-Seq datasets. Concordance between higher NTS gene expression and NTS peptide levels in HGSC-STIC samples was demonstrated by immunohistochemistry. To determine the role of NTS in HGSC, five ovarian cancer (OvCa) cell lines were screened for expression of NTS and its receptors, NTSR1 and NTSR3. Increased expression of NTS and NSTR1 was observed in several of the OvCa cells, whereas the NTSR3 receptor was lower in all OvCa cells, compared to immortalized FT epithelial cells. Treatment with NTSR1 inhibitor (SR48692) decreased cell proliferation, but increased cell migration in OvCa cells. The effects of SR48692 were receptor mediated, since transient RNAi knockdown of NTSR1 mimicked the migratory effects and knockdown of NTSR3 mimicked the anti-proliferative effects. Further, knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial or mesenchymal, respectively. Taken together, our results reveal a difference in a biologically active pathway between HGSC with and without STIC. Furthermore, we identify neurotensin signaling as an important pathway involved in cell proliferation and epithelial-mesenchymal transition in HGSC-STIC which warrants further study as a potential therapeutic target. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Carcinoma Epitelial de Ovario/patología , Neoplasias de las Trompas Uterinas/patología , Neurotensina/metabolismo , Neoplasias Ováricas/patología , Carcinoma in Situ/patología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Células Epiteliales/patología , Neoplasias de las Trompas Uterinas/genética , Trompas Uterinas/patología , Femenino , Humanos , Inmunohistoquímica/métodos , Neoplasias Ováricas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.
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Antígenos de Neoplasias/química , Puntos de Control del Ciclo Celular/fisiología , Inestabilidad Cromosómica/fisiología , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/química , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiología , Línea Celular , Daño del ADN , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/fisiología , ADN Complementario/genética , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Resistencia a Antineoplásicos , Fibroblastos , Células HL-60 , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
BACKGROUND: Homologous recombination deficiency (HRD) is shown to predict response to DNA-damaging therapies in patients with high-grade serous ovarian cancer (HGSOC); however, changes in HRD during progression remains unknown. METHODS: HRD scores were evaluated in paired primary and/or recurrent HGSOC samples (N = 107) from 54 patients with adjuvant platinum-based chemotherapy. BRCA1/2 mutation, BRCA1 methylation, loss of heterozygosity (LOH), and HRD scores were characterised using tumour DNA-based next-generation sequencing assays. RESULTS: Among 50 evaluable pairs (N = 100 samples), high intra-patient correlation in HRD score was observed (r2 = 0.93). BRCA1/2 mutations, BRCA1/2 LOH, and HRD were maintained between primary and recurrent samples, except for one pair in which a BRCA1 reversion mutation was identified in the recurrent sample. Despite the reversion, both samples were classified as having high HRD scores ( ≥ 42). All samples with BRCA1/2 mutations exhibited high HRD scores; however, high HRD scores were more prevalent than BRCA1/2 mutations (55% vs. 30%, respectively). CONCLUSION: Markers of HRD were maintained between the primary and recurrent samples, regardless of other genomic changes that occurred during recurrence. HRD score/markers in primary tumours may be valuable and adequate for selection of platinum-based therapy and/or poly-ADP-ribose-polymerase (PARP) inhibitors in recurrent HGSOC.
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Cistadenocarcinoma Seroso/genética , Recombinación Homóloga , Recurrencia Local de Neoplasia/genética , Neoplasias Ováricas/genética , Platino (Metal)/uso terapéutico , Análisis de Secuencia de ADN/métodos , Adulto , Anciano , Proteína BRCA1/genética , Proteína BRCA2/genética , Quimioterapia Adyuvante , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Metilación de ADN , Progresión de la Enfermedad , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Pérdida de Heterocigocidad , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Adulto JovenRESUMEN
OBJECTIVES: Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear. METHODS: HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro. RESULTS: Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05). CONCLUSIONS: HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.
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Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/terapia , Proteínas de Homeodominio/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Secuencia de Bases , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Quimioterapia Adyuvante , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/cirugía , Procedimientos Quirúrgicos de Citorreducción , Supervivencia sin Enfermedad , Femenino , Proteínas de Homeodominio/biosíntesis , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Compuestos Organoplatinos/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/cirugía , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción , TranscriptomaRESUMEN
Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.
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Colágeno Tipo II/genética , Cistadenocarcinoma Seroso/metabolismo , Proteínas de Transporte de Membrana/genética , Recurrencia Local de Neoplasia/metabolismo , Neoplasias Ováricas/metabolismo , Seudogenes , Adulto , Anciano , Cistadenocarcinoma Seroso/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/patología , Análisis de Secuencia de ARNRESUMEN
Homeobox (HOX) genes are a family of transcription factors that are essential regulators of development. HOX genes play important roles in normal reproductive physiology, as well as in the development and progression of serous carcinomas, the predominant and most aggressive subtype of epithelial ovarian cancer (EOC). This review discusses aberrant HOX gene expression in serous EOC and its impact on tumor development and progression. Further identification of HOX target genes may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of patients with serous EOC.
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Genes Homeobox , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Animales , Carcinoma Epitelial de Ovario , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patologíaRESUMEN
PURPOSE: Biomarkers that predict response or toxicity to antiangiogenic therapy are sought to favorably inform the risk/benefit ratio. This study evaluated the association of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) genetic polymorphisms with the development of hypertension (HTN) and clinical outcome in metastatic clear cell renal cell carcinoma (MCCRCC) patients treated with sunitinib. PATIENT AND METHODS: Sixty-three MCCRCC patients receiving sunitinib (50 mg 4/2) with available blood pressure (BP) data and germline DNA were retrospectively identified. A panel of candidate VEGF and VEGFR2 single nucleotide polymorphisms (SNPs) were evaluated for associations with the development of hypertension and clinical outcome. RESULTS: VEGF SNP -634 genotype was associated with the prevalence and duration of sunitinib-induced hypertension (as defined by systolic pressure ≥150 mmHg and/or diastolic pressure ≥90 mmHg) in both univariable analysis (P = .03 and .01, respectively) and multivariable analysis, which adjusted for baseline BP and use of antihypertension medication (P = .05 and .02, respectively). Patients with the GG genotype were estimated to have a greater likelihood of being hypertensive during treatment compared with patients with the CC genotype (odds ratio of 13.62, 95% confidence interval [CI] 3.71-50.04). No single VEGF or VEGFR SNPs were found to correlate with clinical outcome. However, the combination of VEGF SNP 936 and VEGFR2 SNP 889 were associated with overall survival after adjustment for prognostic risk group (P = .03). CONCLUSIONS: In MCCRCC patients treated with sunitinib, VEGF SNP -634 is associated with hypertension and a combination of VEGF SNP 936 and VEGFR2 SNP 889 genotypes is associated with overall survival.
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Carcinoma de Células Renales/tratamiento farmacológico , Hipertensión/inducido químicamente , Hipertensión/genética , Indoles/efectos adversos , Neoplasias Renales/tratamiento farmacológico , Pirroles/efectos adversos , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Renales/complicaciones , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/secundario , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Sunitinib , Resultado del TratamientoRESUMEN
Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.
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Artefactos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Isoenzimas/metabolismo , Tirosina/metabolismo , Anticuerpos Fosfo-Específicos/metabolismo , Biocatálisis , Western Blotting , Dicroismo Circular , Bromuro de Cianógeno/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Células HL-60 , Halogenación , Humanos , Isoenzimas/genética , Modelos Moleculares , Mutación , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tripsina/metabolismo , Tirosina/genéticaRESUMEN
We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
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Antígenos de Neoplasias/metabolismo , Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/metabolismo , División del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Serina/metabolismo , Antígenos de Neoplasias/química , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Cinasa 1 épsilon/genética , Quinasa de la Caseína I/genética , Quinasa Idelta de la Caseína/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/química , Regulación hacia Abajo , Etopósido/farmacología , Células HL-60 , Humanos , Péptidos/química , Péptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
Angiogenesis is central to the growth of normal tissues and tumors. Inhibiting this pathway has been a strategy for drug development for tumors not responsive to most agents used in chemotherapy. Notably, signaling mediated by vascular endothelial growth factor (VEGF) is a key target because aberrant signaling via this pathway is frequently associated with neoangiogenesis in tumors. The drug-discovery effort to blunt VEGF signaling has led to the approval of bevacizumab and several receptor tyrosine kinase inhibitors (TKIs) that have shown efficacy in the clinical management of breast, colorectal, lung, and kidney cancer. Understanding the genetic variability in VEGF and VEGF receptor has led to identifying genotypic variations (single nucleotide polymorphisms [SNPs]) associated with treatment outcome and toxicity. Notably, identification of SNPs in VEGF associated with angiogenesis inhibitor treatment-induced hypertension and outcome provides exciting opportunities for personalized medicine to improve outcome and reduced toxicity with these novel TKIs.
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Hipertensión/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Polimorfismo Genético/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factores de Crecimiento Endotelial Vascular/genética , Animales , HumanosRESUMEN
Immune cell infiltrates within the tumor microenvironment can influence treatment response and outcome in several cancers. In this study, we developed RNA-based immune signatures from pan-cancer analysis that could serve as potential markers across tumor types and tested them for association with outcome in high-grade serous ovarian cancer (HGSOC) and other female cancers. Pan-cancer RNA-Seq cluster analysis of immune-related gene expression profiles in The Cancer Genome Atlas (TCGA) from 29 different solid tumors (4446 specimens) identified distinct but concordant gene signatures. Among these immune signatures, Cytotoxic Lymphocyte Immune Signature (CLIS), T-cell trafficking (TCT), and the TCT to M2 tumor-associated macrophage (M2TAM) ratio (TCT:M2TAM) were significantly (p < 0.05) associated with overall survival (OS), using multivariable Cox proportional hazards regression models, in a discovery cohort and two independent validation cohorts of HGSOC patients. Notably, the TCT:M2TAM ratio was highly significant (p ≤ 0.000001) in two HGSOC cohorts. Immune signatures were also significant (p < 0.05) in the presence of tumor cytoreduction, BRCA1/2 mutation, and COL2A1 expression. Importantly, the CLIS and TCT signatures were also validated for prognostic significance (p < 0.05) in TCGA cohorts for endometrial and high tumor mutational burden (Hi-TMB) breast cancer. These immune signatures also have the potential for being predictive in other cancers and for patients following different treatment strategies.
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Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.
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Anthracyclines used in the treatment of acute myelogenous leukemia (AML) inhibit the activity of the mammalian topoisomerase II (topo II) isoforms, topo II α and topo IIß. In 230 patients with non-M3 AML who received frontline ara-C/daunorubicin we determined expression of topo IIα and topo IIß by RT-PCR and its relationship to immunophenotype (IP) and outcomes. Treatment outcomes were analyzed by logistic or Cox regression. In 211 patients, available for analysis, topo IIα expression was significantly lower than topo IIß (P < 0.0001). In contrast to topo IIα, topo IIß was significantly associated with blast percentage in marrow or blood (P = 0.0001), CD7 (P = 0.01), CD14 (P < 0.0001) and CD54 (P < 0.0001). Event free survival was worse for CD56-negative compared to CD56-high (HR = 1.9, 95% CI [1.0-3.5], p = 0.04), and overall survival was worse for CD-15 low as compared to CD15-high (HR = 2.2, 95% CI [1.1-4.2], p = 0.02). Ingenuity pathway analysis indicated topo IIß and immunophenotype markers in a network associated with cell-to-cell signaling, hematological system development/function and inflammatory response. Topo IIß expression reflects disease biology of highly proliferative disease and distinct IP but does not appear to be an independent variable influencing outcome in adult AML patients treated with anthracycline-based therapy.
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ADN-Topoisomerasas de Tipo II/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/inmunología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antineoplásicos/uso terapéutico , Estudios de Cohortes , Citarabina/uso terapéutico , ADN-Topoisomerasas de Tipo II/genética , Daunorrubicina/uso terapéutico , Femenino , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Inhibidores de Topoisomerasa II/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients.
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PURPOSE: The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated. MATERIALS AND METHODS: Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --> PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A). RESULTS: In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --> PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (> 3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --> PS-341 exhibited enhanced caspase 9 activity (> 2-fold), caspase 3 (> 2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells. CONCLUSION: In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácidos Borónicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteasoma , Pirazinas/farmacología , Inhibidores de Topoisomerasa I , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ácidos Borónicos/administración & dosificación , Bortezomib , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Camptotecina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Daño del ADN , Sinergismo Farmacológico , Humanos , Proteínas I-kappa B/genética , Proteínas Inhibidoras de la Apoptosis , Irinotecán , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Inhibidor NF-kappaB alfa , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Pirazinas/administración & dosificación , Survivin , TransfecciónRESUMEN
Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.
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Proteínas 14-3-3/fisiología , Apoptosis , Ácidos Borónicos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Daño del ADN , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Neoplasias/fisiología , Inhibidores de Proteasoma , Pirazinas/farmacología , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Bortezomib , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cartilla de ADN , Regulación hacia Abajo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , SurvivinRESUMEN
Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.
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Antineoplásicos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Animales , Antineoplásicos/uso terapéutico , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Cisteína Endopeptidasas , Daño del ADN , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Complejo de la Endopetidasa ProteasomalRESUMEN
BACKGROUND: We previously demonstrated that KN-62, an inhibitor of calcium calmodulin-dependent enzymes, sensitizes human leukemia HL-60 cells resistant to topoisomerase II-targeting drugs. The objective of this study was to determine pathways of apoptosis downstream of DNA damage induced by KN-62 co-treatment with VP-16. MATERIALS AND METHODS: HL-60/Y/DOX0.05 cells were treated with VP-16, KN-62, or VP-16 + KN-62. Following treatment, cells were assayed for c-IAP1, c-IAP2 and XIAP protein expression, as well as caspase activation, cytochrome c release and PARP cleavage. RESULTS: Baseline c-IAP1 protein levels were 2-fold higher in HL60 cells selected for resistance to doxorubicin compared to the parent sensitive line. VP-16 and KN-62 co-treatment was associated with caspase activation via the mitochondrial pathway and significant reductions (p = 0.002) in c-IAP1 protein expression but not with c-IAP2 or XIAP. CONCLUSION: These data suggest that KN-62 co-treatment sensitizes doxorubicin-resistant cells to VP-16-induced apoptosis by enhancing caspase activity and reducing c-IAP1 expression.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Etopósido/farmacología , Biosíntesis de Proteínas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Antibióticos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Caspasas/metabolismo , Daño del ADN , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Etopósido/administración & dosificación , Regulación Leucémica de la Expresión Génica , Células HL-60 , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas/antagonistas & inhibidores , Proteínas/genética , Ubiquitina-Proteína Ligasas , Regulación hacia ArribaRESUMEN
Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIß isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIß is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.