RESUMO
Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.
Assuntos
Neoplasias da Mama , Pequeno RNA não Traduzido , Humanos , Feminino , Neoplasias da Mama/genética , Pequeno RNA não Traduzido/genética , Genes Reguladores , Metástase Linfática/genética , RNA Nucleolar Pequeno/genéticaRESUMO
The deregulation of microRNAs (miRNAs) is associated with the various steps of the metastatic process. In addition, circulating miRNAs are remarkably stable in peripheral blood, making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to determine whether tumor-tissue-derived miRNAs are traceable to plasma in ER-positive early breast cancer patients. We performed RNA-sequencing on 30 patients for whom plasma, sentinel lymph nodes (SLNs) and tumor tissue were available. We carried out differential expression, gene ontology and enrichment analyses. Our results show that circulating miRNAs are inversely expressed compared with tumor tissue or SLNs obtained from the same patients. Our differential expression analysis shows the overall downregulation of circulating miRNAs. However, the expression of miR-643a-3p and miR-223 was up-regulated in patients with positive SLNs. Furthermore, gene ontology analysis showed the significant enrichment of biological processes associated with the regulation of epithelial cell proliferation and transcriptional regulation commonly involved in the promotion of metastases. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant miRNAs and to validate our results in a larger cohort of patients.
Assuntos
Neoplasias da Mama , MicroRNA Circulante , MicroRNAs , Linfonodo Sentinela , Humanos , Feminino , Receptores de Estrogênio/genética , Neoplasias da Mama/genética , MicroRNAs/genética , MicroRNA Circulante/genéticaRESUMO
BACKGROUND: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. METHODS: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. RESULTS: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. CONCLUSIONS: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ésteres do Colesterol/metabolismo , Acetil-CoA C-Acetiltransferase/biossíntese , Idoso , Neoplasias da Mama/patologia , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , Receptores de LDL/biossíntese , Receptores Depuradores Classe B/biossínteseRESUMO
Disruption of the microtubule cytoskeleton impairs tumor angiogenesis by inhibiting the hypoxia-inducible factor (HIF-1α) pathway. However, the signaling cascade linking microtubule disruption to HIF-1α inactivation has not been elucidated. Here, we show that microtubule-targeting drug (MTD) treatment impaired HIF-1α protein nuclear translocation, which significantly down-regulated HIF transcriptional activity. We provide strong evidence that HIF-1α protein associates with polymerized microtubules and traffics to the nucleus, with the aid of the dynein motor protein. Together, these data suggest that microtubules are critically involved in the nuclear trafficking and transcriptional activity of HIF-1α. We also show that the connection between the microtubule cytoskeleton and HIF-1α regulation is lost in renal cell carcinoma (RCC), where HIF-1α is overexpressed because of mutations in the von Hippel Lindau (VHL) tumor suppressor protein. Specifically, we show that MTD treatment of RCC cells did not impair HIF-1α nuclear accumulation or transcriptional activity, and had no effect on the polysome association profile of HIF-1α. Interestingly, we found that HIF-1α protein did not bind microtubules in RCC. Moreover, restoration of VHL function failed to restore the ability of MTDs to inhibit HIF-1α, suggesting that VHL does not contribute to this phenotype. Together, these results suggest that HIF-1α regulation is microtubule-independent, and likely contributes to the chemoresistant nature of RCCs. Further understanding of the microtubule-dependent HIF-1α regulation, and lack thereof in RCC, is essential given the importance of HIF-1α in tumor biology, and the widespread use of MTDs in clinical oncology.
Assuntos
Transporte Ativo do Núcleo Celular , Antineoplásicos/farmacologia , Carcinoma de Células Renais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microtúbulos/fisiologia , Taxoides/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Dineínas/metabolismo , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta , Transcrição Gênica , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.
Assuntos
Proteínas de Ligação a DNA/efeitos dos fármacos , Estradiol/farmacologia , Microtúbulos/efeitos dos fármacos , Neovascularização Patológica , Proteínas Nucleares/efeitos dos fármacos , Fatores de Transcrição , 2-Metoxiestradiol , Animais , Northern Blotting , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Estradiol/análogos & derivados , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/efeitos dos fármacos , Linfocinas/genética , Linfocinas/metabolismo , Camundongos , Microscopia Confocal , Modelos Animais , Proteínas Nucleares/metabolismo , RNA Mensageiro , Transcrição Gênica , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Lonafarnib (LNF) is a protein farnesyl transferase (FTase) inhibitor that has shown synergistic activity with taxanes in preclinical models and early stage clinical trials. Preclinical findings suggested tubulin acetylation and FTase expression levels may be important determinants of drug sensitivity that would help identify patient populations more likely to benefit from this regimen. This pilot study evaluated the biological effects of LNF and docetaxel (DTX) combination therapy in refractory solid tumors by comparing pretreatment and post-treatment tumor biopsies. METHODS: Patients with histologically confirmed locally advanced or metastatic solid malignancies refractory to standard therapies or with no effective therapies available were eligible. Patients were randomized to 1 of 4 dosing cohorts: 1) 30 mg/m², 100 mg; 2) 36 mg/m², 100 mg; 3) 30 mg/m², 150 mg; or 4) 36 mg/m², 150 mg of DTX intravenously weekly, LNF orally twice daily, respectively. RESULTS: Of the 38 patients enrolled, 36 were treated, and 29 were evaluable for toxicity and response assessment. The combination of LNF and DTX was tolerated in all cohorts with the exception of a 28% incidence of grade 3/4 diarrhea, which was manageable with aggressive antidiarrheal regimens. Seven patients derived clinically meaningful benefit from this combination treatment; these patients had significantly lower basal FTase-beta mRNA expression levels than the mean study population level (P < .05). Correlation of clinical benefit with tubulin acetylation content as well as basal acetyl-tubulin content were evaluated. However, no significant correlation was found. CONCLUSIONS: Despite the small number of patients, these findings support our preclinical mechanistic studies and warrant further clinical investigations using FTase-beta mRNA expression as a potential predictive biomarker to select for an enriched patient population to study the effects of taxane and FTase inhibitor combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Farnesiltranstransferase/sangue , Neoplasias/tratamento farmacológico , Piperidinas/administração & dosagem , Piridinas/administração & dosagem , Taxoides/administração & dosagem , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Docetaxel , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
MicroRNAs have emerged as important regulators of the metastatic process. In addition, circulating miRNAs appear to be surprisingly stable in peripheral blood making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to investigate the expression profile of circulating miRNAs and their association with the metastatic lymph node status in early breast cancer patients. Sentinel lymph node status was detected by one-step nucleic acid (OSNA) analysis. We performed RNA-sequencing in 16 plasma samples and validated the results by qPCR. Gene Ontology term enrichment and KEGG pathway analyses were carried out using DAVID tools. We found16 differentially expressed miRNAs (q < 0.01) in patients with positive SLNs. Fourteen miRNAs were down-regulated (miR-339-5p, miR-133a-3p, miR-326, miR-331-3p, miR-369-3p, miR-328-3p, miR-26a-3p, miR-139-3p, miR-493-3p, miR-664a-5p, miR-146a-5p, miR-323b-3p, miR-1307-3p and miR-423-3p) and 2 were up-regulated (miR-101-3pand miR-144-3p). Hierarchical clustering using differentially expressed miRNAs clearly distinguished patients according to their lymph node status. Gene ontology analysis showed a significant enrichment of biological processes associated with the regulation of the epithelial mesenchymal transition, cell proliferation and transcriptional regulation. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further validation in a larger cohort of patients will be necessary to confirm our results.
RESUMO
Evidence that microRNAs (miRNAs) regulate the various steps of metastasis is increasing. Several studies have looked at the miRNA expression profile in primary breast tumors but few have compared primary tumor and sentinel lymph node (SLN) metastasis. We correlated the expression of miRNAs with the SLN status and the outcome of axillary lymph node dissection (ALND) in 60 patients with early breast cancer. We profiled the expression of miRNAs in paired breast tumor samples and SLNs using the NextSeq500 Illumina platform and key findings were validated by qPCR. MultiMiR Bioconductor and Reactome pathways analysis were performed to identify target genes and signaling pathways affected by altered expressed miRNAs. Our results show that nine miRNAs were differentially expressed in tumor tissues (q ≤ 0.05). In tumor samples, a 13.5-fold up-regulation of miR-7641-2 (q < 0.001) and a 2.9-fold down-regulation of miR-1291 (q < 0.001) were associated with tumors with positive SLNs. However, only down-regulation of miR-1291 (q = 0.048) remained significant in paired SLNs samples. Interestingly, a 10.5 up-regulation of miR-1291 in SLNs samples was associated with additional axillary lymph node involvement (q < 0.001). The enrichment analyses showed that canonical and non-canonical WNT pathways and negative regulation of various receptor tyrosine kinases signaling pathways were targets of miR-1291 and supports the role of miR-1291 as a tumor suppressor gene (TSG). Further studies are warranted to investigate the use of miR-1291 as a surrogate biomarker of SLN node metastasis in patients with early-stage breast cancer.
RESUMO
Grade 4 malignant glioma (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The hallmark of GBM tumors is the presence of pseudopalisading necrosis and microvascular proliferation. These tumor cells are hypoxic and express hypoxia-inducible factor-1 (HIF-1), a prosurvival transcription factor that promotes formation of neovasculature through activation of target genes, such as vascular endothelial growth factor. Here, we evaluated whether 2-methoxyestradiol, a microtubule and HIF-1 inhibitor, would have therapeutic potential for this disease in a 9L rat orthotopic gliosarcoma model using a combination of noninvasive imaging methods: magnetic resonance imaging to measure the tumor volume and bioluminescence imaging for HIF-1 activity. After imaging, histologic data were subsequently evaluated to elucidate the drug action mechanism in vivo. Treatment with 2-methoxyestradiol (60-600 mg/kg/d) resulted in a dose-dependent inhibition of tumor growth. This effect was also associated with improved tumor oxygenation as assessed by pimonidazole staining, decreased HIF-1alpha protein levels, and microtubule destabilization as assessed by deacetylation. Our results indicate that 2-methoxyestradiol may be a promising chemotherapeutic agent for the treatment of malignant gliomas, with significant growth inhibition. Further studies are needed to assess the effect of low or intermediate doses of 2-methoxyestradiol in combination with chemotherapeutic agents in clinical studies focused on malignant gliomas. In addition to showing tumor growth inhibition, we identified three potential surrogate biomarkers to determine the efficacy of 2-methoxyestradiol therapy: decreased HIF-1alpha levels, alpha-tubulin acetylation, and degree of hypoxia as determined by pimonidazole staining.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Estradiol/análogos & derivados , Glioma/tratamento farmacológico , Moduladores de Tubulina/uso terapêutico , 2-Metoxiestradiol , Animais , Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estradiol/uso terapêutico , Glioma/patologia , Imageamento por Ressonância Magnética , Ratos , Ratos Endogâmicos F344RESUMO
We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Microtúbulos/efeitos dos fármacos , 2-Metoxiestradiol , Alcanos/farmacologia , Carbamatos/farmacologia , Linhagem Celular Tumoral , Colchicina/farmacologia , Docetaxel , Regulação para Baixo/efeitos dos fármacos , Epotilonas/farmacologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactonas/farmacologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Pironas/farmacologia , Taxoides/farmacologia , Vincristina/farmacologiaRESUMO
2-Methoxyestradiol is an estradiol metabolite with significant antiproliferative and antiangiogenic activity independent of estrogen receptor status. To identify a molecular basis for acquired 2-methoxyestradiol resistance, we generated a stable 2-methoxyestradiol-resistant (2ME2R) MDA-MB-435 human cancer cell line by stepwise exposure to increasing 2-methoxyestradiol concentrations. 2ME2R cells maintained in the presence of the drug and W435 cells maintained in the absence of the drug showed 32.34- to 40.07-fold resistance to 2-methoxyestradiol. Cross-resistance was observed to Vinca alkaloids, including vincristine, vinorelbine, and vinblastine (4.29- to 6.40-fold), but minimal resistance was seen to colchicine-binding agents including colchicine, colcemid, and AVE8062A (1.72- to 2.86-fold). No resistance was observed to paclitaxel and epothilone B, polymerizing agents (0.89- to 1.14-fold). Genomic sequencing identified two different heterozygous point mutations in the class I (M40) isotype of beta-tubulin at amino acids 197 (Dbeta197N) and 350 (Kbeta350N) in 2ME2R cells. Tandem mass spectrometry confirmed the presence of both wild-type and the mutant beta-tubulin in 2ME2R cells at the protein level. Consistently, treatment of parental P435 cells with 2-methoxyestradiol resulted in a dose-dependent depolymerization of microtubules, whereas 2ME2R cells remained unaffected. In contrast, paclitaxel affected both cell lines. In the absence of 2-methoxyestradiol, 2ME2R cells were characterized by an elevated level of detyrosination. Upon 2-methoxyestradiol treatment, levels of acetylated and detyrosinated tubulins decreased in P435 cells, while remaining constant in 2ME2R cells. These results, together with our structure-based modeling, show a tight correlation between the antitubulin and antiproliferative effects of 2-methoxyestradiol, consistent with acquired tubulin mutations contributing to 2-methoxyestradiol resistance.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Estradiol/análogos & derivados , Mutação Puntual , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/genética , 2-Metoxiestradiol , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos , Estradiol/farmacologia , Humanos , Espectrometria de Massas , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
Hypoxia, a reduction in the normal level of tissue oxygen tension, occurs in most solid tumors in regions where tumor growth outstrips new blood vessel formation. Hypoxic cancer cells are resistant to both chemotherapy and radiation and are a major reason for the failure of cancer therapy. The cellular response to hypoxia is mediated through the hypoxia-inducible transcription factor-1 (HIF-1). HIF-1 is critically important for tumor progression and angiogenesis. In fact, HIF-1alpha is overexpressed in 70% of human cancers and their metastases. Thus, agents that inhibit angiogenesis and tumor growth via inhibition of HIF-1 represent an attractive yet unexplored new modality for cancer treatment. We will overview inhibitors of HIF-1alpha and will discuss their potential use for cancer therapy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Neoplasias/terapia , Neovascularização Patológica/prevenção & controle , Fatores de Transcrição/metabolismo , Hipóxia Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Gene methylation and K-ras mutations were examined in tumor and paired serum DNA of 50 resected non-small-cell lung cancer patients. RASSF1A, death associated protein kinase and target of methylation-induced silencing were methylated in 17/50 (34%), 23/50 (45%) and 18/50 (35%) tumors, respectively, and in 17/50 (34%), 20/50 (40%) and 17/50 (34%) sera, respectively. Methylation in tumor and serum were closely correlated (P=0.001), but no correlation was found with survival. Twelve K-ras mutations (cysteine) were found in serum and nine mutations were found in tumor (five cysteine, one alanine, one aspartic, one arginine, and one valine). K-ras mutations in serum correlated significantly with survival (P=0.01).
Assuntos
Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Genes ras/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Mutação , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/sangue , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Códon , DNA/metabolismo , Análise Mutacional de DNA , Proteínas Quinases Associadas com Morte Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Fatores de TempoRESUMO
Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P = .026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P = .054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P = .007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis.
Assuntos
Antraciclinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Centrômero/efeitos dos fármacos , Cromossomos Humanos Par 17 , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibióticos Antineoplásicos/farmacologia , Antígenos de Neoplasias/genética , Neoplasias da Mama/mortalidade , Duplicação Cromossômica , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Terapia Neoadjuvante , Proteínas de Ligação a Poli-ADP-Ribose , Prognóstico , Receptor ErbB-2/genética , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Molecular differentiation between invasive lobular carcinomas (ILCs) and invasive ductal carcinomas (IDCs) of the breast has not been well defined. We investigated gene expression differences between ILCs and IDCs and their correlation with variations in invasiveness and tumor growth. Total RNA was isolated from 30 frozen tumor samples: 10 from ILCs and 20 from IDCs. Gene expression was investigated using the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Data and validation were performed by reverse transcriptase polymerase chain reaction and immunohistochemistry. Gene expression differences between ILCs and IDCs were found in 140 genes. Overall, ILCs showed up-regulation of genes related with cell migration, lipid and fatty acid metabolism, and some transcription factors and showed down-regulation of cell adhesion, actin cytoskeleton, cell proliferation, and energetic metabolism of the tumor cells. Our reverse transcriptase polymerase chain reaction results showed that PLEKHA and TMSB10 expression discriminated ILCs from luminal A IDCs, whereas PLEKHA7, TMSB10, PRDX4, and SERPINB5 discriminated ILCs from luminal B IDCs. At the protein level, Plekha7 was overexpressed in ILCs but not in normal tissue or low-grade IDCs. Moreover, Plekha7 overexpression had an inverse relation with E-cadherin expression. The gene expression profile in ILCs and IDCs differs in several signaling pathways. Our findings suggest that overexpression of PLEKHA7 is common in ILCs and could be a molecular marker to differentiate ILCs from IDCs.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Lobular/diagnóstico , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Regulação para Cima , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Técnicas de Diagnóstico Molecular , Invasividade Neoplásica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: The mechanism of progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) remains largely unknown. We compared gene expression in tumors with simultaneous DCIS and IDC to decipher how diverse proteins participate in the local invasive process.Twenty frozen tumor specimens with concurrent, but separated, DCIS and IDC were microdissected and evaluated. Total RNA was extracted and microarray analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Microarray data were validated by quantitative real time reverse transcription-PCR (qRT-PCR) and immunohistochemistry. Controls included seven pure in situ carcinomas, eight fragments from normal breast tissue, and a series of mouse breast carcinomas (MMTV-PyMT).Fifty-six genes were differentially expressed between DCIS and IDC samples. The genes upregulated in IDC samples, and probably associated with invasion, were related to the epithelial-mesenchymal transition (ASPN, THBS2, FN1, SPARC, and COL11A1), cellular adhesion (GJB2), cell motility and progression (PLAUR, PLAU, BGN, ADAMTS16, and ENPP2), extracellular matrix degradation (MMP11, MMP13, and MMP14), and growth/proliferation (ST6GAL2). qRT-PCR confirmed the expression patterns of ASPN, GJB2, ENPP2, ST6GAL2, and TMBS10. Expression of the ASPN and GJB2 gene products was detected by immunohistochemistry in invasive carcinoma foci. The association of GJB2 protein expression with invasion was confirmed by qRT-PCR in mouse tumors (P < 0.05). CONCLUSIONS: The upregulation of ASPN and GJB2 may play important roles in local invasion of breast ductal carcinomas.
RESUMO
Low-density lipoprotein receptor-related protein 1, a member of the low-density lipoprotein cholesterol receptor family, has been implicated in the progression of certain tumors; but it remains unclear whether it plays a role in infiltrating ductal breast carcinomas. We studied a series of 81 ductal breast tumors to determine the correlation of low-density lipoprotein receptor-related protein 1 overexpression with clinicopathologic and immunohistochemical characteristics associated with prognosis. Low-density lipoprotein receptor-related protein 1 overexpression was identified in 14% (11/81) of tumors and was correlated with a high nuclear grade (P = .043), high mitotic index (P = .006), and Ki-67 greater than 20% (P = .047). Furthermore, low-density lipoprotein receptor-related protein 1 expression was associated with aggressive carcinomas (triple-negative tumors [21%, 7/33] and Her-2/neu tumors [17%, 4/24]) but not with hormone-dependent carcinomas (0%, 0/24) (P = .040). There was no correlation between low-density lipoprotein receptor-related protein 1 expression and survival, but a trend was found between low-density lipoprotein receptor-related protein 1 overexpression and tumor recurrence. Low-density lipoprotein receptor-related protein 1 overexpression was related to proliferation and invasiveness in Her-2/neu and triple-negative breast carcinoma. Moreover, patients with low-density lipoprotein receptor-related protein 1-positive tumors had higher cholesterol levels (62.5%, 5/8) than those with low-density lipoprotein receptor-related protein 1-negative tumors (40%, 19/47). Nevertheless, the correlation between low-density lipoprotein receptor-related protein 1 and hypercholesterolemia was not statistically significant; but cholesterol levels were higher in patients with triple-negative breast carcinoma (60%, 15/25) and Her-2/neu carcinomas (40%, 6/15) than in luminal-A carcinomas (20%, 3/15) (P = .046). These findings suggest a relationship between hypercholesterolemia and aggressiveness of ductal breast carcinomas.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/complicações , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/sangue , Carcinoma Ductal de Mama/complicações , Carcinoma Ductal de Mama/metabolismo , Colesterol/sangue , Feminino , Humanos , Hipercolesterolemia/etiologia , Pessoa de Meia-Idade , Invasividade Neoplásica , PrognósticoRESUMO
It has been suggested that down-regulation of E-cadherin in invasive breast ductal carcinomas is mediated by the aberrant expression of several of its transcriptional repressors, but their inhibitory role and clinical importance are not yet well established. We investigated gene and protein expression patterns of the E-cadherin repressors SNAIL, ZEB1, and TWIST in relation to clinicopathologic parameters, in a series of 88 patients with invasive breast ductal carcinomas. Up-regulation of SNAIL messenger RNA (P = .008) and down-regulation of TWIST (P = .022) were associated with triple-negative tumors, whereas ZEB1 gene expression was more frequent in hormone-positive tumors (P = .004). Loss of E-cadherin was found in 19% of the tumors, but it did not correlate with aberrant expression of any of the repressors investigated herein. Nonetheless, we found that ZEB-1 protein overexpression inversely correlated with high tumor grade (P = .018), nuclear grade (P = .002), and presence of lymph nodes (P = .001), and these data were consistent with the gene expression data for ZEB1. Clinically, down-regulation of ZEB1 messenger RNA was associated with poor overall survival (P = .011) and disease-free survival (P = .053), whereas patients with TWIST negative tumors had a worse overall survival (P = .008) and disease-free survival (P = .006). Our data indicate that deregulation of TWIST is somehow important in the aggressiveness of triple-negative carcinomas and poor patient outcome, whereas down-regulation of ZEB1 seems to play a role in tumor spread, metastases, and poor survival.
Assuntos
Neoplasias da Mama/metabolismo , Caderinas/biossíntese , Carcinoma Ductal de Mama/metabolismo , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/patologia , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição da Família Snail , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on ß-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the ßI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a ß-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the ß-tubulin isotype composition of the cells was examined. Increased expression of ßII- and ßIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Lactonas/farmacologia , Macrolídeos/farmacologia , Mutação/genética , Neoplasias Ovarianas/genética , Tubulina (Proteína)/genética , Antineoplásicos/metabolismo , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Lactonas/metabolismo , Macrolídeos/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired ß-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy.