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1.
Nature ; 512(7513): 155-60, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-25079324

RESUMO

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.


Assuntos
Neoplasias da Mama/genética , Evolução Clonal , Genoma/genética , Linhagem Celular Tumoral , Impressões Digitais de DNA , Feminino , Variação Genética , Humanos , Modelos Teóricos , Mutação/genética , Análise de Sequência de DNA , Análise de Célula Única , Neoplasias de Mama Triplo Negativas/genética
2.
Leukemia ; 37(10): 2017-2026, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37604981

RESUMO

This is the primary report of the randomized, placebo-controlled phase 3 BRIGHT AML 1019 clinical trial of glasdegib in combination with intensive chemotherapy (cytarabine and daunorubicin) or non-intensive chemotherapy (azacitidine) in patients with untreated acute myeloid leukemia. Overall survival (primary endpoint) was similar between the glasdegib and placebo arms in the intensive (n = 404; hazard ratio [HR] 1.05; 95% confidence interval [CI]: 0.782-1.408; two-sided p = 0.749) and non-intensive (n = 325; HR 0.99; 95% CI: 0.768-1.289; two-sided p = 0.969) studies. The proportion of patients who experienced treatment-emergent adverse events was similar for glasdegib versus placebo (intensive: 99.0% vs. 98.5%; non-intensive: 99.4% vs. 98.8%). The most common treatment-emergent adverse events were nausea, febrile neutropenia, and anemia in the intensive study and anemia, constipation, and nausea in the non-intensive study. The addition of glasdegib to either cytarabine and daunorubicin or azacitidine did not significantly improve overall survival and the primary efficacy endpoint for the BRIGHT AML 1019 phase 3 trial was not met. Clinical trial registration: ClinicalTrials.gov: NCT03416179.


Assuntos
Anemia , Leucemia Mieloide Aguda , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Daunorrubicina , Citarabina , Azacitidina/uso terapêutico , Anemia/tratamento farmacológico , Náusea/tratamento farmacológico
3.
Science ; 381(6657): 515-524, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37535729

RESUMO

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.


Assuntos
Polaridade Celular , Quimiocina CXCL9 , Neoplasias de Cabeça e Pescoço , Macrófagos , Osteopontina , Carcinoma de Células Escamosas de Cabeça e Pescoço , Microambiente Tumoral , Humanos , Quimiocina CXCL9/análise , Quimiocina CXCL9/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/patologia , Macrófagos/imunologia , Osteopontina/análise , Osteopontina/metabolismo , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Polaridade Celular/imunologia
4.
Cancer Res ; 82(21): 3917-3931, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36040373

RESUMO

Lung adenocarcinoma (LUAD) is one of the most common cancer types and has various treatment options. Better biomarkers to predict therapeutic response are needed to guide choice of treatment modality and to improve precision medicine. Here, we used a consensus hierarchical clustering approach on 509 LUAD cases from The Cancer Genome Atlas to identify five robust LUAD expression subtypes. Genomic and proteomic data from patient samples and cell lines was then integrated to help define biomarkers of response to targeted therapies and immunotherapies. This approach defined subtypes with unique proteogenomic and dependency profiles. Subtype 4 (S4)-associated cell lines exhibited specific vulnerability to loss of CDK6 and CDK6-cyclin D3 complex gene (CCND3). Subtype 3 (S3) was characterized by dependency on CDK4, immune-related expression patterns, and altered MET signaling. Experimental validation showed that S3-associated cell lines responded to MET inhibitors, leading to increased expression of programmed death-ligand 1 (PD-L1). In an independent real-world patient dataset, patients with S3 tumors were enriched with responders to immune checkpoint blockade. Genomic features in S3 and S4 were further identified as biomarkers for enabling clinical diagnosis of these subtypes. Overall, our consensus hierarchical clustering approach identified robust tumor expression subtypes, and our subsequent integrative analysis of genomics, proteomics, and CRISPR screening data revealed subtype-specific biology and vulnerabilities. These LUAD expression subtypes and their biomarkers could help identify patients likely to respond to CDK4/6, MET, or PD-L1 inhibitors, potentially improving patient outcome. SIGNIFICANCE: Integrative analysis of multiomic and drug dependency data uncovers robust lung adenocarcinoma expression subtypes with unique therapeutic vulnerabilities and subtype-specific biomarkers of response.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Proteômica , Biomarcadores Tumorais/genética , Mutação , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/patologia , Prognóstico , Perfilação da Expressão Gênica
5.
Cancer Discov ; 11(3): 678-695, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33203734

RESUMO

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 521.


Assuntos
Ferroptose/genética , Lipogênese/genética , Melanoma/genética , Melanoma/metabolismo , Células Neoplásicas Circulantes/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Transferrina/metabolismo , Biomarcadores Tumorais , Células Cultivadas , Suscetibilidade a Doenças , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Melanoma/patologia , Mutação , Células Neoplásicas Circulantes/patologia , Transdução de Sinais , Análise de Célula Única , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
6.
Nat Med ; 27(8): 1432-1441, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239137

RESUMO

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.


Assuntos
Antígeno CTLA-4/imunologia , Microbioma Gastrointestinal , Receptor de Morte Celular Programada 1/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-1beta/imunologia , Melanoma , Camundongos , Camundongos Endogâmicos C57BL
8.
Clin Cancer Res ; 26(18): 4852-4862, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32571788

RESUMO

PURPOSE: Plasma genotyping may identify mutations in potentially "actionable" cancer genes, such as BRCA1/2, but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC). EXPERIMENTAL DESIGN: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation. RESULTS: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients (P = 0.008), those with triple-negative disease (P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the "BRCA" mutational signature (COSMIC Signature 3) were present in BRCA1/2-mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC. CONCLUSIONS: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , DNA Tumoral Circulante/genética , Idoso , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Estudos Retrospectivos , Sequenciamento do Exoma
9.
Genome Biol ; 21(1): 271, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33148332

RESUMO

BACKGROUND: Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. RESULTS: We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. CONCLUSIONS: Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.


Assuntos
Terapia de Imunossupressão , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástase Neoplásica/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Transcriptoma , Sequenciamento do Exoma
10.
Nat Commun ; 11(1): 1839, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296058

RESUMO

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response.


Assuntos
Genômica/métodos , Melanoma/metabolismo , Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA/genética , Humanos , Melanoma/genética , Mutação/genética , Ativação de Neutrófilo/genética , Ativação de Neutrófilo/fisiologia , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia
11.
Cancer Discov ; 8(5): 556-567, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29496665

RESUMO

Combined MEK and CDK4/6 inhibition (MEKi + CDK4i) has shown promising clinical outcomes in patients with NRAS-mutant melanoma. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi + CDK4i therapy but subsequently developed resistance. Whole-exome sequencing and functional validation identified an acquired PIK3CAE545K mutation as conferring drug resistance. We demonstrate that PIK3CAE545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CAE545K being nonuniformly distributed. This resistant population rapidly expanded after the initiation of MEKi + CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy and distant metastasis. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CAE545K-induced resistance. These results demonstrate that difficult-to-detect preexisting resistance mutations may exist more often than previously appreciated and also posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways.Significance: We report the first characterization of clinical acquired resistance to MEKi + CDK4i, identifying a rare preexisting PIK3CAE545K subpopulation that expands upon therapy and exhibits drug resistance. We suggest that single-region pretreatment biopsy is insufficient to detect rare, spatially segregated drug-resistant subclones. Inhibition of S6K1 is able to resensitize PIK3CAE545K-expressing NRAS-mutant melanoma cells to MEKi + CDK4i. Cancer Discov; 8(5); 556-67. ©2018 AACR.See related commentary by Sullivan, p. 532See related article by Teh et al., p. 568This article is highlighted in the In This Issue feature, p. 517.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/genética , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/genética , Mutação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Melanoma/diagnóstico , Melanoma/tratamento farmacológico , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Fosforilação , Tomografia por Emissão de Pósitrons , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos
12.
Sci Transl Med ; 9(379)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28251903

RESUMO

Immune checkpoint blockade produces clinical benefit in many patients. However, better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen-4 (CTLA-4) followed by programmed death receptor-1 (PD-1) and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T cell receptor sequencing and whole-exome sequencing within the same cohort and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of CNAs identified a higher burden of copy number loss in nonresponders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was nonredundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy.


Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Biópsia , Antígeno CTLA-4/metabolismo , Células Clonais , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Dosagem de Genes , Genoma , Humanos , Mutação/genética , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-28819565

RESUMO

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (<8% on average) across the cohort. Variables related to treatment response were identified via these approaches and through novel radiomic assessment. These data yield insight into differential therapeutic responses to targeted therapy and immune checkpoint blockade in melanoma, and have key translational implications in the age of precision medicine.

14.
Cancer Discov ; 6(8): 827-37, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27301722

RESUMO

UNLABELLED: Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. SIGNIFICANCE: These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827-37. ©2016 AACR.See related commentary by Teng et al., p. 818This article is highlighted in the In This Issue feature, p. 803.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Biomarcadores , Resistencia a Medicamentos Antineoplásicos , Imunomodulação/efeitos dos fármacos , Neoplasias/imunologia , Neoplasias/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Biópsia , Antígeno CTLA-4/antagonistas & inibidores , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
15.
PLoS One ; 7(9): e45023, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028742

RESUMO

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI(50); 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Naftoquinonas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Técnicas de Silenciamento de Genes , Haploinsuficiência/genética , Heterozigoto , Humanos , Células MCF-7 , Modelos Biológicos , Mutação/genética , Naftoquinonas/química , Naftoquinonas/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos
16.
Langmuir ; 25(5): 3102-7, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19437776

RESUMO

This article describes a method for creating submicrometer surface patterns on cubes and microspheres. In this method, PDMS cubes and microspheres are exposed to oxygen plasma, which creates a very thin, hard, surface-modified layer on a compliant substrate. These are then compressed, causing the layer to crack in patterns dictated by the distribution of tensile stresses in the surface layer. Cracks with submicrometer widths were generated on 1 cm3 cubes and 800-microm-diameter microspheres, and the resulting crack patterns were observed. Finite-element simulations of the tensile stress distributions reveal that the fracture patterns arise from different mechanisms in the cubes and spheres. In particular, pattern formation is associated with frictional contact in the cubes but not in the microspheres, where geometrical effects associated with changes in the cross-sectional area along the axis lead to the generation of tensile stress. These observations and analyses provide a foundation on which to predict and guide crack pattern formation on a wide variety of small 3D objects. In anticipation of future applications in materials science and biology, we demonstrate the selective deposition of compounds into the cracks to make them functionally differentiable from the rest of the surface.


Assuntos
Dimetilpolisiloxanos/química , Dióxido de Silício/química , Acetonitrilas/química , Aminas/química , Cromatografia em Camada Fina/métodos , Coloides/química , Força Compressiva , Difusão , Íons , Espectroscopia de Ressonância Magnética/métodos , Microesferas , Modelos Químicos , Propilaminas , Silanos/química , Propriedades de Superfície
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