RESUMO
Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of â¼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Células-Tronco Embrionárias/enzimologia , Regulação Enzimológica da Expressão Gênica , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Camundongos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis and few therapeutic options. Stathmin1 (STMN1) is a cytosolic protein involved in microtubule dynamics through inhibition of tubulin polymerization and promotion of microtubule depolymerization, which has been implicated in carcinogenesis and aggressive behavior in multiple epithelial malignancies. We aimed to evaluate expression of STMN1 in ACC and to elucidate how this may contribute to its malignant phenotype. METHODS: STMN1 was identified by RNA sequencing as a highly differentially expressed gene in human ACC samples compared with benign adrenal tumors. Expression was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical (IHC) staining of a tissue microarray (TMA) from two independent cohorts. The biologic relevance of STMN1 was investigated in NCI-H295R cells by lentivirus-mediated silencing. RESULTS: Differential gene expression demonstrated an eightfold increase in STMN1 messenger RNA (mRNA) in malignant compared with benign adrenal tissue. IHC showed significantly higher expression of STMN1 protein in ACC compared with normal and benign tissues. STMN1 knockdown in an ACC cell line resulted in decreased cell viability, cell-cycle arrest at G0/G1, and increased apoptosis in serum-starved conditions compared with scramble short hairpin RNA (shRNA) controls. STMN1 knockdown also decreased migration, invasion, and anchorage-independent growth compared with controls. CONCLUSIONS: STMN1 is overexpressed in human ACC samples, and knockdown of this target in vitro resulted in a less aggressive phenotype of ACC, particularly under serum-starved conditions. Further study is needed to investigate the feasibility of interfering with STMN1 as a potential therapeutic target.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Biomarcadores Tumorais/metabolismo , Estatmina/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/cirurgia , Adrenalectomia , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/cirurgia , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Feminino , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Prognóstico , Estatmina/genética , Células Tumorais CultivadasRESUMO
BACKGROUND AND OBJECTIVES: Hürthle cell carcinoma (HCC) is an unusual and relatively rare type of differentiated thyroid cancer. Currently, cytologic analysis of fine-needle aspiration biopsy is limited in distinguishing benign Hürthle cell neoplasms from malignant ones. The aim of this study was to determine whether differences in the expression of specific genes could differentiate HCC from benign Hürthle cell nodules by evaluating differential gene expression in Hürthle cell disease. METHODS: Eighteen benign Hürthle cell nodules and seven HCC samples were analyzed by whole-transcriptome sequencing. Bioinformatics analysis was carried out to identify candidate differentially expressed genes. Expression of these candidate genes was re-examined by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was quantified by immunohistochemistry. RESULTS: Close homolog of L1 (CHL1) was identified as overexpressed in HCC. CHL1 was found to have greater than 15-fold higher expression in fragments per kilobase million in HCC compared with benign Hurthle cell tumors. This was confirmed by qRT-PCR. Moreover, the immunoreactivity score of the CHL1 protein was significantly higher in HCC compared with benign Hürthle cell nodules. CONCLUSIONS: CHL1 expression may represent a novel and useful prognostic biomarker to distinguish HCC from benign Hürthle cell disease.
Assuntos
Adenoma Oxífilo/metabolismo , Moléculas de Adesão Celular/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/metabolismo , Adenoma Oxífilo/diagnóstico , Adenoma Oxífilo/patologia , Idoso , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologiaRESUMO
Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Células Germinativas/metabolismo , Células-Tronco/metabolismo , Animais , Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/deficiência , Gametogênese/genética , Componentes do Gene , Instabilidade Genômica/genética , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Masculino , Camundongos , Testes para Micronúcleos , Proteínas de Manutenção de Minicromossomo , Chaperonas Moleculares , Mutação/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ionizing radiation has garnered considerable attention as a combination partner for immunotherapy due to its potential immunostimulatory effects. In contrast to the more commonly used external beam radiation, we explored the feasibility of combining chimeric antigen receptor (CAR) T cell therapy with targeted radionuclide therapy (TRT), which is achieved by delivering ß-emitting 177Lu-DOTATATE to tumor via tumor-infiltrating CAR T cells that express somatostatin receptor 2 (SSTR2). We hypothesized that the delivery of radiation to tumors could synergize with CAR T therapy, resulting in enhanced antitumor immunity and tumor response. To determine the optimal dosage and timing of 177Lu-DOTATATE treatment, we measured CAR T cell infiltration and expansion in tumors longitudinally through positron emission tomography (PET) using a SSTR2-specific positron-emitting radiotracer,18F-NOTA-Octreotide. In animals receiving CAR T cells and a low-dose (2.5 Gy) of TRT following the administration of 177Lu-DOTATATE, we observed a rapid regression of large subcutaneous tumors, which coincided with a dramatic increase in serum proinflammatory cytokines. Tumor burden was also reduced when a higher radiation dose (6 Gy) was delivered to the tumor. However, this higher dose led to cell death in both the tumor and CAR T cells. Our study suggests that there may exist an optimum range of TRT dosage that can enhance T cell activity and sensitize tumor cells to T cell killing, which may result in more durable tumor control compared to a higher radiation dose.
Assuntos
Neoplasias , Animais , Neoplasias/tratamento farmacológico , Octreotida/uso terapêutico , Linfócitos T , Imunoterapia , Radioisótopos/uso terapêuticoRESUMO
CONTEXT: Pancreatic neuroendocrine tumors (PNETs) exhibit a wide range of behavior from localized disease to aggressive metastasis. A comprehensive transcriptomic profile capable of differentiating between these phenotypes remains elusive. OBJECTIVE: Use machine learning to develop predictive models of PNET metastatic potential dependent upon transcriptomic signature. METHODS: RNA-sequencing data were analyzed from 95 surgically-resected primary PNETs in an international cohort. Two cohorts were generated with equally balanced metastatic PNET composition. Machine learning was used to create predictive models distinguishing between localized and metastatic tumors. Models were validated on an independent cohort of 29 formalin-fixed, paraffin-embedded samples using NanoString nCounter®, a clinically-available mRNA quantification platform. RESULTS: Gene expression analysis identified concordant differentially expressed genes between the two cohorts. Gene set enrichment analysis identified additional genes that contributed to enriched biologic pathways in metastatic PNETs. Expression values for these genes were combined with an additional 7 genes known to contribute to PNET oncogenesis and prognosis, including ARX and PDX1. Eight specific genes (AURKA, CDCA8, CPB2, MYT1L, NDC80, PAPPA2, SFMBT1, ZPLD1) were identified as sufficient to classify the metastatic status with high sensitivity (87.5% - 93.8%) and specificity (78.1% - 96.9%). These models remained predictive of the metastatic phenotype using NanoString nCounter® on the independent validation cohort, achieving a median AUROC of 0.886. CONCLUSIONS: We identified and validated an eight-gene panel predictive of the metastatic phenotype in PNETs, which can be detected using the clinically-available NanoString nCounter® system. This panel should be studied prospectively to determine its utility in guiding operative versus non-operative management.
RESUMO
P-TEFb complex, a heterodimer of the kinase CDK9 and Cyclin T, is a critical factor that stimulates the process of transcription elongation. Here, we explored a fast and large-scale screening method to induce a temperature-dependent conditional disruption of the CDK9/Cyclin T interaction and developed an assay to validate their mutant phenotypes in a biological context. First, we used the yeast two-hybrid system to screen Drosophila melanogaster Cyclin T mutants at a large scale for temperature or cold sensitive (TS or CS) CDK9 interaction phenotypes. The isolated P-TEFb TS mutants were then expressed in Drosophila cells and were investigated for their effects on Drosophila hsp70 transcriptional activity. Our results showed that these P-TEFb TS mutants had a reduced level of hsp70 transcription at restrictive temperatures. A model structure of the Cyclin T and CDK9 complex suggested that the key TS mutations were found within the α2- and α3-helices at the interface of the complex, which may disrupt the binding of Cyclin T to CDK9 directly or indirectly by affecting the conformation of Cyclin T. The yeast two-hybrid-based screening strategy described here for isolating TS or CS interaction phenotypes can be directly applicable to other complexes in higher organisms. The use of TS or CS mutants will enable a 'real-time and reversible perturbation' restricted to specific protein-protein interactions, providing a mechanistic insight into the biological process mediated by a target complex.
Assuntos
Proteínas de Drosophila/genética , Mutação , Fator B de Elongação Transcricional Positiva/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Ciclina T/química , Ciclina T/genética , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Modelos Moleculares , Dados de Sequência Molecular , Fator B de Elongação Transcricional Positiva/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Análise de Sequência de DNA , Temperatura , Transcrição GênicaRESUMO
Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells.
RESUMO
The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Interleucina-12/genética , Molécula de Adesão da Célula Epitelial , Imunoterapia Adotiva , Neoplasias/genética , Neoplasias/terapia , Antígenos de Neoplasias/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
A subset of thyroid cancers, recurrent differentiated thyroid cancers and anaplastic thyroid cancer (ATC), are difficult to treat by thyroidectomy and systemic therapy. A common mutation in thyroid cancer, BRAFV600E, has targetable treatment options; however, the results have been disappointing in thyroid cancers compared with BRAFV600E melanoma, as thyroid cancers quickly become resistant to BRAFV600E inhibitor (BRAFi). Here, we studied the molecular pathway that is induced in BRAFV600E thyroid cancer cells and patient-derived tumor samples in response to BRAFi, vemurafenib, using RNA-sequencing and molecular analysis. Both inducible response to BRAFi and acquired BRAFi resistance in BRAFV600E thyroid cancer cells showed significant activation of the JAK/STAT pathway. Functional analyses revealed that the combination of BRAFi and inhibitors of JAK/STAT pathway controlled BRAFV600E thyroid cancer cell growth. The Cancer Genome Atlas data analysis demonstrated that potent activation of the JAK/STAT signaling was associated with shorter recurrence rate in patients with differentiated thyroid cancer. Analysis of tumor RNA expression in patients with poorly differentiated thyroid cancer and ATC also support that enhanced activity of JAK/STAT signaling pathway is correlated with worse prognosis. Our study demonstrates that JAK/STAT pathway is activated as BRAFV600E thyroid cancer cells develop resistance to BRAFi and that this pathway is a potential target for anticancer activity and to overcome drug resistance that commonly develops to treatment with BRAFi in thyroid cancer. IMPLICATIONS: Dual inhibition of BRAF and JAK/STAT signaling pathway is a potential therapeutic treatment for anticancer activity and to overcome drug resistance to BRAFi in thyroid cancer.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Janus Quinases/genética , Janus Quinases/metabolismo , Janus Quinases/uso terapêutico , Sulfonamidas/farmacologia , Transdução de Sinais , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição STAT/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Mutação , RNA , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular TumoralRESUMO
Decades of research in reproductive biology have led to the generally accepted belief that in female mammals, all surviving germ cells enter meiosis at the end of fetal development and as a result, the postnatal ovary harbours a limited supply of oocytes that cannot be replenished or regenerated if lost to injury or disease. However, recent reports have challenged this view, suggesting instead that oocyte production is maintained through continual seeding of the ovary by circulating, bone-marrow-derived germ cells. To test directly the physiological relevance of circulating cells for female fertility, we established transplantation and parabiotic mouse models to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. Our studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes.
Assuntos
Linhagem da Célula , Oócitos/citologia , Oócitos/fisiologia , Ovulação/fisiologia , Óvulo/citologia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Leucócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/patologia , Ovário/fisiologia , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Sex-based differences in survival have emerged among patients with pancreatic neuroendocrine tumors (PNETs). Mechanisms driving these differences remain poorly understood. We aimed to further characterize sex-based clinicopathologic and survival differences among patients with PNETs and correlate divergent mutational signatures in these patients. METHODS: The National Cancer Database (NCDB) was queried for PNET patients diagnosed 2004-2017 who underwent surgery. Clinicopathologic features were analyzed by sex. The overall survival (OS) of men and women by disease stage was compared using the Kaplan-Meier method. Differences in PNET mutational signatures were analyzed by querying the American Association for Cancer Research Genomics Evidence Neoplasia Information (AACR-GENIE) Cohort v11.0-public. Frequencies of mutational signatures were compared by Fischer's exact (FE) test, adjusting for multiple testing via the Benjamini-Hochberg correction. RESULTS: About 15,202 patients met inclusion criteria from the NCDB; 51.9% were men and 48.1% were women. Men more frequently had tumors > 2 cm than women and more commonly had poorly or undifferentiated tumors. Despite this, lymph node positivity and distant metastases were similar. Differences in OS were only seen among those with early stage rather than stage 3 or 4 disease. MEN1 and DAXX mutations were more frequent among men with PNETs, whereas TP53 mutations were more frequent among women when assessed by FE test. However, neither of these mutational differences maintained statistical significance when adjusted for multiple testing. CONCLUSION: Compared to women, men have larger tumors but similar rates of distant metastases at time of surgery. OS differences appear to be driven by patients with early-stage disease without clearly identifiable differences in mutational signatures between the sexes.
Assuntos
Tumores Neuroectodérmicos Primitivos , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Masculino , Humanos , Feminino , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/cirurgia , Tumores Neuroendócrinos/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/patologia , Estudos de Coortes , Mutação , Estudos RetrospectivosRESUMO
Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-Seq, cancer and T cell profiling, and pharmacologic perturbations. RNA-Seq analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared with localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, and this augmentation was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.
Assuntos
Tumores Neuroendócrinos , Neoplasias Pancreáticas , Animais , Camundongos , Linfócitos T , Quimiocinas , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente TumoralRESUMO
The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Smu tandem repeats show shifts indicating that a 4-5-kb DNA domain (bounded upstream by the Imu promoter) is accessible for switching independent of Smu sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4-5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Smu tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.
Assuntos
Diversidade de Anticorpos/genética , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina/imunologia , Região de Troca de Imunoglobulinas/genética , Modelos Genéticos , Proteínas Proto-Oncogênicas/metabolismo , Animais , Primers do DNA , Proteínas de Ligação a DNA/genética , Switching de Imunoglobulina/genética , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Sequências de Repetição em Tandem/genéticaRESUMO
Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of "stemness" have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP(+) BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.
Assuntos
Células-Tronco Embrionárias/citologia , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco/citologia , Telomerase/metabolismo , Animais , Biomarcadores/metabolismo , Medula Óssea/metabolismo , Separação Celular , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Testículo/metabolismoRESUMO
Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.
Assuntos
Molécula de Adesão da Célula Epitelial/metabolismo , Imunoterapia Adotiva/métodos , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Deriva e Deslocamento Antigênicos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Molécula de Adesão da Célula Epitelial/genética , Humanos , Imunoterapia Adotiva/efeitos adversos , Molécula 1 de Adesão Intercelular/genética , Masculino , Camundongos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting â¼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Regulação Neoplásica da Expressão Gênica , Expressão Gênica , Imunoterapia/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/terapia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/terapia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Prognóstico , Carcinoma Anaplásico da Tireoide/imunologia , Neoplasias da Glândula Tireoide/imunologiaRESUMO
BACKGROUND: The majority of papillary thyroid cancers are driven by acquired mutations typically in the BRAF or RAS genes that aberrantly activate the mitogen-activated protein kinase pathway. This process leads to malignant transformation, dedifferentiation, and a decrease in the expression of the sodium-iodide symporter (NIS; SLC5A5), which results in resistance to radioactive iodine therapy. We sought to determine whether inhibition of aberrant mitogen-activated protein kinase-signaling can restore NIS expression. METHODS: We prospectively developed cultures of papillary thyroid cancers derived from operative specimens and applied drug treatments for 24 hours. Samples were genotyped to identify BRAF and RAS mutations. We performed quantitative PCR to measure NIS expression after treatment. RESULTS: We evaluated 24 patient papillary thyroid cancer specimens; BRAFV600E mutations were identified in 18 out of 24 (75.0%); 1 patient tumor had an HRAS mutation, and the remaining 5 were BRAF and RAS wildtype. Dual treatment with dabrafenib and trametinib increased NIS expression (mean fold change 4.01 ± 2.04, P < .001), and single treatment with dabrafenib had no effect (mean fold change 0.98 ± 0.42, P = .84). Tumor samples that had above-median NIS expression increases came from younger patients (39 vs 63 years, P < .05). CONCLUSION: Dual treatment with BRAF and MEK inhibitors upregulated NIS expression, suggesting that this treatment regimen may increase tumor iodine uptake. The effect was greatest in tumor cells from younger patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Radioisótopos do Iodo/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Simportadores/metabolismo , Câncer Papilífero da Tireoide/terapia , Neoplasias da Glândula Tireoide/terapia , Adulto , Fatores Etários , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Radioisótopos do Iodo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Oximas/farmacologia , Oximas/uso terapêutico , Cultura Primária de Células , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridonas , Pirimidinonas , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Cancer therapy utilizing adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated remarkable clinical outcomes in hematologic malignancies. However, CAR T cell application to solid tumors has had limited success, partly due to the lack of tumor-specific antigens and an immune-suppressive tumor microenvironment. From the tumor tissues of gastric cancer patients, we found that intercellular adhesion molecule 1 (ICAM-1) expression is significantly associated with advanced stage and shorter survival. In this study, we report a proof-of-concept study using ICAM-1-targeting CAR T cells against gastric cancer. The efficacy of ICAM-1 CAR T cells showed a significant correlation with the level of ICAM-1 expression in target cells in vitro. In animal models of human gastric cancer, ICAM-1-targeting CAR T cells potently eliminated tumors that developed in the lungs, while their efficacy was more limited against the tumors in the peritoneum. To augment CAR T cell activity against intraperitoneal tumors, combinations with paclitaxel or CAR activation-dependent interleukin (IL)-12 release were explored and found to significantly increase anti-tumor activity and survival benefit. Collectively, ICAM-1-targeting CAR T cells alone or in combination with chemotherapy represent a promising strategy to treat patients with ICAM-1+ advanced gastric cancer.