RESUMO
Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Antígenos/química , Antígenos/imunologia , Biomarcadores Tumorais , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunomodulação , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/mortalidade , Peptídeos/química , Peptídeos/imunologia , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacos , Resultado do Tratamento , Microambiente Tumoral/imunologiaRESUMO
Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Gástricas/genética , Linfócitos T Reguladores/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Animais , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL10/biossíntese , Quimiocina CXCL11/biossíntese , Ácidos Graxos não Esterificados/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/imunologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.
Assuntos
Hemangiossarcoma , Histiocitoma Fibroso Benigno , Sequenciamento por Nanoporos , Proteínas de Fusão Oncogênica , Adulto , Feminino , Humanos , Hemangiossarcoma/genética , Hemangiossarcoma/diagnóstico , Hemangiossarcoma/patologia , Histiocitoma Fibroso Benigno/genética , Histiocitoma Fibroso Benigno/diagnóstico , Histiocitoma Fibroso Benigno/patologia , Sequenciamento por Nanoporos/métodos , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/diagnóstico , Tetraspanina 30/genética , Tetraspanina 30/metabolismoRESUMO
An aneurysmal bone cyst (ABC) is a benign bone neoplasm that typically occurs during the first and second decades of life. ABC usually presents as a rapidly growing intramedullary expansile mass with multiple blood-filled cysts in the metaphysis of the long tubular bones. Here, we report a case of a periosteal solid ABC that was initially diagnosed as a high-grade surface osteosarcoma. A 10-year-old male was referred to our hospital for swelling and tenderness of the left upper arm. Radiography revealed periosteal mass without fluid-fluid levels. On performing open biopsy, the tumor showed hypercellular proliferation of uniform spindle to epithelioid cells with brisk mitotic activity (up to 12/2 mm2) and lace-like osteoid formation, which was diagnosed as a high-grade surface osteosarcoma. After one course of chemotherapy using adriamycin and cisplatin, peripheral sclerosis was conspicuous, which led to pathological review and revision of diagnosis as "possibly osteoblastoma." The patient was disease-free for 4 years after marginal resection and curettage. Retrospective nanopore DNA sequencing unexpectedly detected a PAFAH1B1::USP6 rearrangement. The fusion gene was further validated using reverse transcription-polymerase chain reaction and the diagnosis was revised to ABC. Chromothripsis involving chromosome 17 has also been identified. Methylation analysis classified the present tumor as an ABC or non-ossifying fibroma using t-distributed stochastic neighbor embedding and unsupervised hierarchical clustering. This case report highlights the utility of nanopore DNA sequencing for soft tissue and bone tumor diagnosis.
Assuntos
Cistos Ósseos Aneurismáticos , Cromotripsia , Sequenciamento por Nanoporos , Osteossarcoma , Ubiquitina Tiolesterase , Humanos , Masculino , Cistos Ósseos Aneurismáticos/genética , Cistos Ósseos Aneurismáticos/patologia , Cistos Ósseos Aneurismáticos/diagnóstico , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/diagnóstico , Ubiquitina Tiolesterase/genética , Criança , Sequenciamento por Nanoporos/métodos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/diagnóstico , Rearranjo GênicoRESUMO
By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.
Assuntos
Carcinoma de Células Escamosas , Modelos Animais de Doenças , Neoplasias Cutâneas , Quinases Ativadas por p21 , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Camundongos , Linhagem Celular Tumoral , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Feminino , Diferenciação Celular/efeitos dos fármacos , Acetato de Tetradecanoilforbol , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.
Assuntos
Isocitrato Desidrogenase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Doença Aguda , Adolescente , Adulto , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Criança , Humanos , Isocitrato Desidrogenase/metabolismo , Pessoa de Meia-Idade , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , Transcriptoma , Adulto JovemRESUMO
Myxoid liposarcoma (MLPS) is a rare sarcoma, typically arising in deep soft tissues during the fourth to fifth decades of life. Histologically, MLPS is composed of uniform oval cells within a background of myxoid stroma and chicken-wire capillaries. Genetically, MLPS is characterized by the FUS/EWSR1::DDIT3 fusion gene, which generally results from balanced interchromosomal translocation and is detectable via DDIT3 break-apart fluorescence in situ hybridization (FISH). Here, we report an unusual intra-articular MLPS case, negative for DDIT3 break-apart FISH but positive for EWSR1::DDIT3. An 18-year-old female was referred to our hospital complaining of an intra-articular mass in the right knee joint. Histologically, the tumor was mainly composed of mature adipocytes, brown fat-like cells, and lipoblasts. Nanopore sequencing detected DNA rearrangements between EWSR1 and DDIT3 and clustered complex rearrangements involving multiple chromosomes, suggesting chromoplexy. Methylation classification using random forest, t-distributed stochastic neighbor embedding, and unsupervised hierarchical clustering correctly classified the tumor as MLPS. The copy number was almost flat. The TERT promoter C-124T was also detected. This report highlights, for the first time, the potential value of a fast and low-cost nanopore sequencer for diagnosing sarcomas.
RESUMO
Hyalinizing clear cell carcinoma (HCCC) is a rare indolent malignant tumor of minor salivary gland origin with EWSR1::ATF1 rearrangement. Pathologically, the tumor cells possess a clear cytoplasm in a background of hyalinized stroma. Generally, the tumor cells are positive for p63 and p40 and negative for s100 and α-smooth muscle actin, suggesting that they differentiate into squamous epithelium and not into myoepithelium. In this study, we performed a detailed histopathological and genomic analysis of 6 cases of HCCC, including 2 atypical subtypes-a case of "high-grade transformation" and 1 "possessing a novel partner gene for EWSR1." We performed a sequential analysis of the primary and recurrent tumor by whole-exome sequencing, RNA sequencing, Sanger sequencing, and fluorescence in situ hybridization to investigate the effect of genomic changes on histopathology and clinical prognosis. A fusion gene involving the EWSR1 gene was detected in all cases. Five cases, including the "high-grade transformation," harbored a known EWSR1::ATF1 fusion gene; however, 1 case harbored a novel EWSR1::LARP4 fusion gene. This novel EWSR1::LARP4-fused HCCC has a SOX10-positive staining, which is different from the EWSR1::ATF1-fused HCCC. According to whole-exome sequencing and fluorescence in situ hybridization analysis, the "whole-genome doubling" and focal deletion involving CDKN2A, CDKN2B, and PTEN were detected in HCCC with "high-grade transformation." Conclusively, we identified a novel partner gene for EWSR1, LARP4, in indolent HCCC. Importantly, "high-grade transformation" and poor prognosis were caused by whole-genome doubling and subsequent genomic aberrations.
Assuntos
Adenocarcinoma de Células Claras , Carcinoma , Neoplasias das Glândulas Salivares , Humanos , Hibridização in Situ Fluorescente , Proteína EWS de Ligação a RNA/genética , Glândulas Salivares/patologia , Sequência de Bases , Genes cdc , Proteínas de Fusão Oncogênica/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Fatores de Transcrição SOXE/genéticaRESUMO
The cellular origins of cervical cancer and the histological differentiation of human papillomavirus (HPV)-infected cells remain unexplained. To gain new insights into the carcinogenesis and histological differentiation of HPV-associated cervical cancer, we focused on cervical cancer with mixed histological types. We conducted genomic and transcriptomic analyses of cervical cancers with mixed histological types. The commonality of the cellular origins of these cancers was inferred using phylogenetic analysis and by assessing the HPV integration sites. Carcinogenesis was estimated by analyzing human gene expression profiles in different histological types. Among 42 cervical cancers with known HPV types, mixed histological types were detected in four cases, and three of them were HPV18-positive. Phylogenetic analysis of these three cases revealed that the different histological types had a common cell of origin. Moreover, the HPV-derived transcriptome and HPV integration sites were common among different histological types, suggesting that HPV integration could occur before differentiation into each histological type. Human gene expression profiles indicated that HPV18-positive cancer retained immunologically cold components with stem cell properties. Mixed cervical cancer has a common cellular origin among different histological types, and progenitor cells with stem-like properties may be associated with the development of HPV18-positive cervical cancer.
Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/patologia , Papillomavirus Humano 18/genética , Filogenia , Papillomaviridae/genética , DNA Viral/genéticaRESUMO
BACKGROUND: Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME). METHODS: We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets. RESULTS: We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/ß-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types. CONCLUSIONS: We propose a novel concept of "paradoxical neoantigenic mutations" that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.
Assuntos
Neoplasias Colorretais , Neoplasias , Humanos , Receptor de Morte Celular Programada 1 , Microambiente Tumoral , Neoplasias/genética , Mutação , Instabilidade de Microssatélites , Neoplasias Colorretais/patologiaRESUMO
BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Instabilidade de Microssatélites , Neoplasias Colorretais/patologia , Neoplasias do Colo/genética , Mutação , Apresentação de Antígeno , Repetições de Microssatélites/genéticaRESUMO
BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Genes MHC Classe I/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microglobulina beta-2/genética , Alelos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , Epigênese Genética , Expressão Gênica , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Humanos , Imunogenética , Linfócitos do Interstício Tumoral , Instabilidade de Microssatélites , Complexo de Endopeptidases do Proteassoma/genética , Fatores de Transcrição de Fator Regulador X/genética , Taxa de Sobrevida , Microglobulina beta-2/metabolismoRESUMO
Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
Assuntos
Autofagia , Sistemas CRISPR-Cas , Amidofosforribosiltransferase/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genéticaRESUMO
Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Líquida/métodos , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico/genética , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Separação Celular/métodos , Crizotinibe/uso terapêutico , DNA/isolamento & purificação , Resistencia a Medicamentos Antineoplásicos/genética , Éxons/genética , Feminino , Amplificação de Genes , Deleção de Genes , Genes erbB-1 , Humanos , Separação Imunomagnética/métodos , Queratinas , Antígenos Comuns de Leucócito , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes , Proteínas de Fusão Oncogênica/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.
Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Ácidos Nucleicos Livres , Fusão Gênica , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biópsia , Carbazóis/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácidos Nucleicos Livres/isolamento & purificação , Crizotinibe/uso terapêutico , Proteínas do Citoesqueleto/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Biópsia Líquida/métodos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/isolamento & purificação , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , RNA Mensageiro/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.
Assuntos
Proteína BRCA1/genética , Transcriptoma/genética , Neoplasias de Mama Triplo Negativas/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Metilação de DNA/genética , Exoma/genética , Feminino , Amplificação de Genes , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Recombinação Homóloga/genética , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI-H] CRC with CGI methylator phenotype, 28 MSI-H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies.
Assuntos
Neoplasias Colorretais/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Instabilidade de Microssatélites , Fenótipo , Transcriptoma/genéticaRESUMO
Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Fusão Gênica , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Sequenciamento do ExomaRESUMO
Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.
Assuntos
Tecido Adiposo/patologia , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético , Histona Desmetilases com o Domínio Jumonji/fisiologia , Doenças Metabólicas/patologia , Obesidade/patologia , Adipogenia , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Feminino , Metabolismo dos Lipídeos , Masculino , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismoRESUMO
The major driver mutations of lung cancer, EGFR mutations and EML4-ALK fusion, are mainly detected in terminal respiratory unit (TRU)-type lung adenocarcinomas, which typically show lepidic and/or papillary patterns, but are rarely associated with a solid or invasive mucinous morphology. In order to elucidate the key genetic events in non-TRU-type lung cancer, we carried out whole-exome sequencing on 43 non-TRU-type lung adenocarcinomas based on morphology (17 acinar, nine solid, and two enteric adenocarcinomas, and 15 adenocarcinomas with a mucinous morphology). Our analysis identified mutations in TP53 (16/43, 37.2%), KRAS (13/43, 30.2%), and NKX2-1/TTF-1 (7/43; 16.3%) as the top three significantly mutated genes, while the EGFR mutation was rare (1/43, 2.3%) in this cohort. Eight NKX2-1/TTF-1 mutations (five frameshift, two nonsense, and one missense) were identified, with one case harboring two distinct NKX2-1/TTF-1 mutations (one missense and one frameshift). Functional assays with the NK2 homeobox 1 (NKX2-1)/thyroid transcription factor 1 (TTF-1) mutants revealed that none of them retain the activity as a transcriptional factor. Histologically, invasive mucinous adenocarcinomas accounted for most of the NKX2-1/TTF-1 mutations (five cases), as well as one enteric and one acinar adenocarcinoma. Immunohistochemistry showed that the cohort was largely divided into TTF-1-postive/hepatocyte nuclear factor 4-α (HNF4-α)-negative and TTF-1-negative/HNF4-α-positive groups. NKX2-1/TTF-1 mutations were exclusively found in the latter, in which the gastrointestinal markers, mucin 5AC and cytokeratin 20, were frequently expressed. Bisulfite sequencing revealed that the NKX2-1/TTF-1 gene body was highly methylated in NKX2-1/TTF-1-negative cases, including those without the NKX2-1/TTF-1 mutations. The genetic or epigenetic inactivation of NKX2-1/TTF-1 may play an essential role in the development and aberrant differentiation of non-TRU-type lung adenocarcinomas.