RESUMO
Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy.
Assuntos
Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Análise de Célula Única , Carcinoma de Células Renais/imunologia , Sobrevivência Celular , Células Endoteliais/patologia , Células Epiteliais/patologia , Humanos , Imunoterapia , Rim/patologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Células Mieloides/patologia , Resultado do TratamentoRESUMO
Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies, with an average life expectancy of â¼6 months from the time of diagnosis. The genetic and epigenetic changes that underlie this malignancy are incompletely understood. We found that ASH1-like histone lysine methyltransferase (ASH1L) is overexpressed in ATC relative to the much less aggressive and more common differentiated thyroid cancer. This increased expression was due at least in part to reduced levels of microRNA-200b-3p (miR-200b-3p), which represses ASH1L expression, in ATC. Genetic knockout of ASH1L protein expression in ATC cell lines decreased cell growth both in culture and in mouse xenografts. RNA-Seq analysis of ASH1L knockout versus WT ATC cell lines revealed that ASH1L is involved in the regulation of numerous cancer-related genes and gene sets. The pro-oncogenic long noncoding RNA colon cancer-associated transcript 1 (CCAT1) was one of the most highly (approximately 68-fold) down-regulated transcripts in ASH1L knockout cells. Therefore, we investigated CCAT1 as a potential mediator of the growth-inducing activity of ASH1L. Supporting this hypothesis, CCAT1 knockdown in ATC cells decreased their growth rate, and ChIP-Seq data indicated that CCAT1 is likely a direct target of ASH1L's histone methyltransferase activity. These results indicate that ASH1L contributes to the aggressiveness of ATC and suggest that ASH1L, along with its upstream regulator miR-200b-3p and its downstream mediator CCAT1, represents a potential therapeutic target in ATC.
Assuntos
Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , RNA Helicases DEAD-box/genética , Ribonuclease III/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adolescente , Feminino , Humanos , Masculino , Mutação , Adulto JovemRESUMO
Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.
Assuntos
Neoplasias do Córtex Suprarrenal/classificação , Neoplasias do Córtex Suprarrenal/diagnóstico , Biomarcadores Tumorais/genética , Inclusão em Parafina/métodos , RNA-Seq/métodos , Transcriptoma , Neoplasias do Córtex Suprarrenal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Seguimentos , Formaldeído/química , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5(+) antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5-GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5(+) stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi-colored reporter demonstrated that Notch-activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD-induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper-proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.
Assuntos
Homeostase/fisiologia , Antro Pilórico/citologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Análise de Variância , Animais , Pesos e Medidas Corporais , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Técnicas Histológicas , Hibridização In Situ , Camundongos , Microscopia Confocal , Antro Pilórico/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Serina-Treonina Quinases TOR/metabolismoRESUMO
Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Bases de Dados de Ácidos Nucleicos , Progressão da Doença , Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Transgênicos , Interferência de RNA , Fatores de Risco , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
BACKGROUND & AIMS: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. METHODS: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. RESULTS: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. CONCLUSIONS: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.
Assuntos
Duodeno/metabolismo , Gastrinas/metabolismo , Neuroglia/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neoplasias Duodenais/enzimologia , Neoplasias Duodenais/genética , Neoplasias Duodenais/patologia , Duodeno/efeitos dos fármacos , Duodeno/patologia , Gastrinoma/enzimologia , Gastrinoma/genética , Gastrinoma/patologia , Gastrinas/genética , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hiperplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Proteólise , Proteínas Proto-Oncogênicas/genética , Inibidores da Bomba de Prótons/farmacologia , Receptor de Colecistocinina B/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Fatores de Tempo , UbiquitinaçãoRESUMO
Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.
Assuntos
Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Mutação , Córtex Suprarrenal/metabolismo , Citocromo P-450 CYP11B2/metabolismo , DNA/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Humanos , Hiperaldosteronismo/etiologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Análise de Sequência de DNA , Análise de Sequência de RNA , Transcriptoma , Zona GlomerulosaRESUMO
OBJECTIVE: Correct subtyping of primary aldosteronism (PA) is essential for good surgical outcomes. Adrenal vein sampling (AVS) and/or computed tomography (CT) are used for PA subclassification. Clinical and/or biochemical improvement after surgery, however, is not always achieved in patients with presumed unilateral PA. We aimed to identify the pitfalls in PA subclassification leading to surgical treatment failures. PATIENTS AND DESIGN: We retrospectively studied 208 patients who underwent adrenal vein sampling (AVS) for PA subclassification in a tertiary referral centre, between January 2009 and August 2016. Simultaneous bilateral AVS was performed before and after cosyntropin administration. We implemented immunohistochemistry for aldosterone synthase (CYP11B2) and 17α-hydroxylase/17,20 lyase (CYP17A1) in adrenal glands resected from patients without improvement of PA after surgical treatment and from those with limitations in AVS interpretation. RESULTS: Of 55 patients who underwent adrenalectomy, three (5.5%) had no improvement of PA. All three patients underwent partial adrenalectomy to remove a CT-detected nodule present on the same side with AVS lateralization. Immunohistochemistry revealed a CYP11B2-negative nodule in both cases available. All patients who underwent total adrenalectomy based on AVS lateralization benefitted from surgery, including three patients with unilateral unsuccessful AVS and aldosterone suppression in the catheterized side vs inferior vena cava. CONCLUSIONS: Radiographically identified adrenal nodules are not always a source of PA, even when ipsilateral with AVS lateralization. These data caution against reliance on imaging findings, either alone or in conjunction with AVS, to guide surgery for PA.
Assuntos
Glândulas Suprarrenais/metabolismo , Hiperaldosteronismo/metabolismo , Imuno-Histoquímica/métodos , Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/cirurgia , Adrenalectomia , Adulto , Idoso , Citocromo P-450 CYP11B2/metabolismo , Feminino , Humanos , Hiperaldosteronismo/patologia , Hiperaldosteronismo/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Esteroide 17-alfa-Hidroxilase/metabolismoRESUMO
PURPOSE OF REVIEW: The present review is focused on the recently published study on the genomics of papillary thyroid carcinoma performed by The Cancer Genome Atlas Research Network and its implications for the follicular variant of papillary carcinoma. RECENT FINDINGS: The Cancer Genome Atlas study of papillary thyroid carcinoma comprehensively examined the cancer genome of nearly 500 primary tumors. Using a highly integrated bioinformatic analysis, papillary carcinoma was shown at the genomic level to consist of two highly distinct classes that reflected both tumor histology and underlying genotype. Tumors with true papillary architecture were dominated by BRAF(V600E) mutations and RET kinase fusions and were designated as BRAF(V600E)-like. Tumors with follicular architecture were conversely dominated by RAS mutations and were designated as RAS-like. Given the strong genotype:phenotype correlation known to be present in thyroid cancer, the separation of BRAF(V600E)-like and RAS-like tumors has profound implications for its classification, especially the follicular variant of papillary carcinoma. SUMMARY: The recent genomic characterization of papillary thyroid carcinoma is challenging the established pathological classification of thyroid cancer with significance for the care of patients.
Assuntos
Carcinoma/genética , Carcinoma/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Carcinoma/classificação , Carcinoma Papilar , Genoma Humano , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-ret/genética , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/classificação , Proteínas ras/genéticaRESUMO
Gene amplification is a tumor-specific event during malignant transformation. Recent studies have proposed a lineage-dependency (addiction) model of human cancer whereby amplification of certain lineage transcription factors predisposes a survival mechanism in tumor cells. These tumor cells are derived from tissues where the lineage factors play essential developmental and maintenance roles. Here, we show that recurrent amplification at 18q11.2 occurs in 21% of esophageal adenocarcinomas (EAC). Utilization of an integrative genomic strategy reveals a single gene, the embryonic endoderm transcription factor GATA6, as the selected target of the amplification. Overexpression of GATA6 is found in EACs that contain gene amplification. We find that EAC patients whose tumors carry GATA6 amplification have a poorer survival. We show that ectopic expression of GATA6, together with FGFR2 isoform IIIb, increases anchorage-independent growth in immortalized Barrett's esophageal cells. Conversely, siRNA-mediated silencing of GATA6 significantly reduces both cell proliferation and anchorage-independent growth in EAC cells. We further demonstrate that induction of apoptotic/anoikis pathways is triggered upon silencing of GATA6 in EAC cells but not in esophageal squamous cells. We show that activation of p38α signaling and up-regulation of TNF-related apoptosis-inducing ligand are detected in apoptotic EAC cells upon GATA6 deprivation. We conclude that selective gene amplification of GATA6 during EAC development sustains oncogenic lineage-survival of esophageal adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem da Célula/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Fator de Transcrição GATA6/genética , Apoptose/genética , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 18/genética , Hibridização Genômica Comparativa , Fragmentação do DNA , Fator de Transcrição GATA6/metabolismo , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Genoma Humano/genética , Humanos , RNA Interferente Pequeno/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: When presenting with advanced stage disease, lung cancer patients have <5% 5-y survival. The overexpression of checkpoint kinase 1 (CHK1) is associated with poorer outcomes and may contribute to therapy resistance. Targeting CHK1 with small-molecule inhibitors in p53 mutant tumors might improve the effectiveness of chemotherapy and radiotherapy in non-small cell lung cancer (NSCLC). METHODS: We evaluated CHK1 messenger RNA and protein levels in multiple NSCLC cell lines. We assessed cell line sensitization to gemcitabine, pemetrexed, and radiotherapy by CHK1 inhibition with the small molecule AZD7762 using proliferation and clonogenic cell survival assays. We analyzed CHK1 signaling by Western blotting to confirm that AZD7762 inhibits CHK1. RESULTS: We selected two p53 mutant NSCLC cell lines with either high (H1299) or low (H1993) CHK1 levels for further analysis. We found that AZD7762 sensitized both cell lines to gemcitabine, pemetrexed, and radiotherapy. Chemosensitization levels were greater, however, for the higher CHK1 protein expressing cell line, H1299, when compared with H1993. Furthermore, analysis of the CHK1 signaling pathway showed that H1299 cells have an increased dependence on the CHK1 pathway in response to chemotherapy. There was no increased sensitization to radiation in H1299 versus H1993. CONCLUSIONS: CHK1 inhibition by AZD7762 preferentially sensitizes high CHK1 expressing cells, H1299, to anti-metabolite chemotherapy as compared with low CHK1 expressing H1993 cells. Thus, CHK1 inhibitors may improve the efficacy of standard lung cancer therapies, especially for those subgroups of tumors harboring higher expression levels of CHK1 protein.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Quinases/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma de Células Grandes/tratamento farmacológico , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Guanina/uso terapêutico , Humanos , Neoplasias Pulmonares/metabolismo , Pemetrexede , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Tiofenos/uso terapêutico , Ureia/análogos & derivados , Ureia/uso terapêutico , GencitabinaRESUMO
The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento/genética , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Interferência de RNA , Receptores de Fatores de Crescimento/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal ß-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal ß-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized ß-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized ß-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Transformação Celular Neoplásica/patologia , Progressão da Doença , Fator de Crescimento Insulin-Like II/metabolismo , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Corticosteroides/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Animais , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/genética , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Humanos , Hiperplasia , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Camundongos Knockout , Análise Multivariada , Mutação/genética , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Estabilidade Proteica , Transporte Proteico , Regulação para Cima/genéticaRESUMO
While significant progress has been made in understanding the induction of tumor vasculature by secreted angiogenic factors, little is known regarding contact-dependent signals that promote tumor angiogenesis. Here, we report that the Notch ligand Jagged1 induced by growth factors via mitogen-activating protein kinase (MAPK) in head and neck squamous cell carcinoma (HNSCC) cells triggered Notch activation in neighboring endothelial cells (ECs) and promoted capillary-like sprout formation. Jagged1-expressing HNSCC cells significantly enhanced neovascularization and tumor growth in vivo. Moreover, the level of Jagged1 was significantly correlated with tumor blood vessel content and associated with HNSCC development. Our results elucidate a novel mechanism by which the direct interplay between tumor cells and ECs promotes angiogenesis through MAPK and Notch signaling pathways.
Assuntos
Carcinoma de Células Escamosas/patologia , Endotélio Vascular/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/patologia , Animais , Proteínas de Ligação ao Cálcio , Carcinoma de Células Escamosas/irrigação sanguínea , Ativação Enzimática , Feminino , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores Notch , Proteínas Serrate-Jagged , Transdução de SinaisRESUMO
Although B-Raf(V600E) is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-Raf(V600E) resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-Raf(V600E) cells in which either B-Raf(V600E) or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.
Assuntos
Progressão da Doença , Mutação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Trombospondina 1/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Interferência de RNA , Transdução de Sinais , Sulfonamidas/uso terapêutico , Trombospondina 1/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genéticaRESUMO
Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.
Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Proteínas de Transporte de Cátions , Hiperaldosteronismo , Masculino , Humanos , Aldosterona/genética , Adenoma Adrenocortical/genética , Hiperaldosteronismo/genética , Adenoma/genética , Adenoma/complicações , Mutação , Zinco/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas de Transporte de Cátions/genéticaRESUMO
Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/ß-catenin signaling are frequently observed, the ß-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/ß-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/ß-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/ß-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/ß-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/ß-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/ß-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/ß-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this ß-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/ß-catenin inhibitor. These results show promise for the further clinical development of Wnt/ß-catenin inhibitors in ACC and unveil a novel Wnt/ß-catenin-regulated transcriptome.
RESUMO
Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Masculino , Animais , Feminino , Carcinoma Adrenocortical/genética , Envelhecimento , Senescência Celular , Transdução de Sinais , Neoplasias do Córtex Suprarrenal/genética , Microambiente TumoralRESUMO
Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.