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PURPOSE: Genetic and epigenetic alterations are involved in pituitary adenoma pathogenesis, however the molecular basis of proliferative nonfunctioning pituitary adenomas (NFPAs) remains unclear. Here, we analyzed integrated multi-omics profiling including copy number variation (CNV), DNA methylation and gene expression of 8 NFPAs. METHODS: We collected 4 highly proliferative (hpNFPA, Ki-67 ≥ 3) and 4 lowly proliferative (Ki-67 ≤ 1) NFPAs, and comprehensively assessed CNV, DNA methylation, and gene expression by Illumina HumanMethylation450 BeadChip and Affymetrix GeneChip PrimeView Human Gene Expression Array. We performed Ingenuity Pathway Analysis (IPA) for differentially expressed genes to illustrate aberrant pathways and delineated protein-protein networks of selected key genes in dysregulated pathways. RESULTS: Aberrant arm level CNV, dysregulated DNA methylation, and associated impacts on gene expressions were observed in 2 early occurring hpNFPAs. Chromosomal losses were associated with attenuated expression of DNA methyltransferases, further altering global methylation in these 2 samples. Correlation analysis between DNA methylation and gene expression in 8 NFPAs indicates methylation in promoter and gene body regions are both involved in gene regulation. IPA showed PPARα/RXRα, dopamine receptor signaling, cAMP-mediated signaling, and calcium signaling were all activated, while p38 MAPK and ERK5 signaling were inhibited in hpNFPAs. Moreover, selected key gene networks in hpNFPAs exhibited concurrent methylation status and expression levels of adenylate cyclase genes, G protein subunits, HLA genes, CXCL12, and CCL2. CONCLUSION: This study presents comprehensive multi-omics views of CNV, DNA methylation, and gene expression in 8 NFPAs. Pathway analysis and network maps of key genes provide clues to elucidate the molecular basis of hpNFPA.
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Adenoma , Neoplasias Hipofisárias , Adenoma/genética , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67 , Neoplasias Hipofisárias/genética , ProteômicaRESUMO
Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)(-/-) mice exhibited induced colon p53 levels, and cross-breeding them with Apc(min+/-) mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear ß-catenin accumulation, and increased epithelial-mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the "field change" milieu permissive for neoplastic colon growth.
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Neoplasias do Colo/metabolismo , Hormônio do Crescimento/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acromegalia/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Colo/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , PTEN Fosfo-Hidrolase/metabolismo , Receptores da Somatotropina/genética , Pele/citologia , Proteína Supressora de Tumor p53/genética , Adulto Jovem , beta Catenina/metabolismoRESUMO
Neurons are commonly organized as regular arrays within a structure, and their patterning is achieved by minimizing the proximity between like-type cells, but molecular mechanisms regulating this process have, until recently, been unexplored. We performed a forward genetic screen using recombinant inbred (RI) strains derived from two parental A/J and C57BL/6J mouse strains to identify genomic loci controlling spacing of cholinergic amacrine cells, which is a subclass of retinal interneuron. We found conspicuous variation in mosaic regularity across these strains and mapped a sizeable proportion of that variation to a locus on chromosome 11 that was subsequently validated with a chromosome substitution strain. Using a bioinformatics approach to narrow the list of potential candidate genes, we identified pituitary tumor-transforming gene 1 (Pttg1) as the most promising. Expression of Pttg1 was significantly different between the two parental strains and correlated with mosaic regularity across the RI strains. We identified a seven-nucleotide deletion in the Pttg1 promoter in the C57BL/6J mouse strain and confirmed a direct role for this motif in modulating Pttg1 expression. Analysis of Pttg1 KO mice revealed a reduction in the mosaic regularity of cholinergic amacrine cells, as well as horizontal cells, but not in two other retinal cell types. Together, these results implicate Pttg1 in the regulation of homotypic spacing between specific types of retinal neurons. The genetic variant identified creates a binding motif for the transcriptional activator protein 1 complex, which may be instrumental in driving differential expression of downstream processes that participate in neuronal spacing.
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Células Amácrinas/metabolismo , Neurônios Colinérgicos/metabolismo , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica/fisiologia , Securina/biossíntese , Células Amácrinas/citologia , Animais , Sequência de Bases , Neurônios Colinérgicos/citologia , Proteínas do Olho/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Securina/genética , Deleção de SequênciaRESUMO
Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins, chromosomal instability, or DNA damage is associated with p53/p21 activation, culminating in either senescence or apoptosis, depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH, in contrast to circulating pituitary-derived endocrine GH, has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway, with subsequent marked induction of intracellular pituitary GH in vitro. In contrast, GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo, treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland, as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence, likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary, in nonpituitary cells GH exerts antiapoptotic properties. Thus, the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence.
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Senescência Celular/fisiologia , Hormônio do Crescimento/fisiologia , Hipófise/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Etoposídeo/farmacologia , Hormônio do Crescimento/genética , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Hipófise/citologia , Hipófise/efeitos dos fármacos , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Regiões Promotoras Genéticas , Ratos , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Adrenocorticotrophin (ACTH)-secreting pituitary adenoma, also known as Cushing disease (CD), is rare and causes metabolic syndrome, cardiovascular disease and osteoporosis due to hypercortisolism. However, the molecular pathogenesis of CD is still unclear because of a lack of human cell lines and animal models. Here, we study 106 clinical characteristics and gene expression changes from 118 patients, the largest cohort of CD in a single-center. RNA deep sequencing is used to examine genotypic changes in nine paired female ACTH-secreting pituitary adenomas and adjacent nontumorous pituitary tissues (ANPT). We develop a novel analysis linking disease clinical characteristics and whole transcriptomic changes, using Pearson Correlation Coefficient to discover a molecular network mechanism. We report that osteoporosis is distinguished from the phenotype and genotype analysis. A cluster of genes involved in osteoporosis is identified using Pearson correlation coefficient analysis. Most of the genes are reported in the bone related literature, confirming the feasibility of phenotype-genotype association analysis, which could be used in the analysis of almost all diseases. Secreted phosphoprotein 1 (SPP1), collagen type I α 1 chain (COL1A1), 5'-nucleotidase ecto (NT5E), HtrA serine peptidase 1 (HTRA1) and angiopoietin 1 (ANGPT1) and their signalling pathways are shown to be involved in osteoporosis in CD patients. Our discoveries provide a molecular link for osteoporosis in CD patients, and may open new potential avenues for osteoporosis intervention and treatment.
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PURPOSE: Pituitary carcinomas are extremely rare neoplasms, and molecular events leading to malignant pituitary transformation are largely unknown. Enhanced understanding of molecular mechanisms driving malignant pituitary progression would be beneficial for pituitary carcinoma diagnosis and treatment. METHODS: Differential microRNA expression in paired primary and metastatic pituitary carcinoma specimens were detected using high-throughput human microRNA microarrays and TaqMan microRNA arrays. Three of significantly deregulated miRNAs were further confirmed using quantitative real-time PCR in the metastatic carcinoma, six atypical pituitary adenomas and eight typical pituitary adenomas. Target genes of microRNAs were bioinformatically predicated and verified in vitro by Western blotting and real-time PCR and in vivo by immunohistochemistry respectively. RESULTS: We present a case of a 50-year-old woman harboring non-functioning pituitary carcinoma with multiple intracranial metastases, and identified up-regulation of miR-20a, miR-106b and miR-17-5p in the metastatic carcinoma as compared to the primary neoplasm. Furthermore, miR-20a and miR-17-5p were increased in the metastatic carcinoma and six atypical pituitary adenomas as compared to eight typical pituitary adenomas as measured by quantitative real-time PCR. Both PTEN and TIMP2 were bioinformatically predicated and confirmed in vitro as target genes of these three microRNAs. As semi-quantified by immunohistochemistry, PTEN was absent and TIMP2 was decreased in the metastatic pituitary carcinoma as compared to pituitary adenomas. CONCLUSIONS: Our results suggest microRNA involvement in malignant pituitary progression, whereby increased miR-20a, miR-106b and miR-17-5p promote metastasis by attenuating PTEN and TIMP2 in pituitary carcinoma.
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Biomarcadores Tumorais/genética , Carcinoma/genética , MicroRNAs/genética , Neoplasias Hipofisárias/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/secundário , Carcinoma/cirurgia , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imageamento por Ressonância Magnética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/cirurgia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , TransfecçãoRESUMO
CONTEXT: About 30% of patients with active acromegaly experience paradoxically increased growth hormone (GH) secretion during the diagnostic oral glucose tolerance test (OGTT). Endogenous glucose-dependent insulinotropic polypeptide (GIP) is implicated in this paradoxical secretion. OBJECTIVE: We used the GIP receptor (GIPR) antagonist GIP(3-30)NH2 to test the hypothesis that GIP mediates this paradoxical response when GIPR is abundantly expressed in somatotropinomas. DESIGN, PATIENTS, SETTING, INTERVENTIONS: 25 treatment-naïve patients with acromegaly were enrolled. Each patient underwent one OGTT during simultaneous placebo infusion and one OGTT during a GIP(3-30)NH2 infusion. Blood samples were drawn at baseline and regularly after infusions to measure GH. We assessed pituitary adenoma size by magnetic resonance imaging and GIPR expression by immunohistochemistry on resected somatotropinomas. For mechanistic confirmation, we applied in vitro and ex vivo approaches. MAIN OUTCOME MEASURE: The effect of GIP(3-30)NH2 on paradoxical GH secretion during OGTT as a measure of GIP involvement. RESULTS: In four of seven patients with paradoxical GH secretion, GIP(3-30)NH2 infusion completely abolished the paradoxical response (P = 0.0003). Somatotrophs were available from three of four of these patients, all showing abundant GIPR expression. Adenoma size did not differ between patients with and without paradoxical GH secretion. CONCLUSIONS: Of 25 patients with acromegaly, seven had paradoxical GH secretion during OGTT, and pharmaceutical GIPR blockade abolished this secretion in four. Corresponding somatotroph adenomas abundantly expressed GIPR, suggesting a therapeutic target in this subpopulation of patients. In vitro and ex vivo analyses confirmed the role of GIP and the effects of the antagonist.
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DNA damage repair (DDR) is mediated by phosphorylating effectors ATM kinase, CHK2, p53, and γH2AX. We showed earlier that GH suppresses DDR by suppressing pATM, resulting in DNA damage accumulation. Here, we show GH acting through GH receptor (GHR) inducing wild-type p53-inducible phosphatase 1 (WIP1), which dephosphorylated ATM and its effectors in normal human colon cells and three-dimensional human intestinal organoids. Mice bearing GH-secreting xenografts exhibited induced colon WIP1 with suppressed pATM and γH2AX. WIP1 was also induced in buffy coats derived from patients with elevated GH from somatotroph adenomas. In contrast, decreased colon WIP1 was observed in GHR-/- mice. WIP1 inhibition restored ATM phosphorylation and reversed GH-induced DNA damage. We elucidated a novel GH signaling pathway activating Src/AMPK to trigger HIPK2 nuclear-cytoplasmic relocation and suppressing WIP1 ubiquitination. Concordantly, blocking either AMPK or Src abolished GH-induced WIP1. We identify WIP1 as a specific target for GH-mediated epithelial DNA damage accumulation.
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CONTEXT: The identification and biological actions of pituitary-derived exosomes remain elusive. OBJECTIVE: This work aimed to validate production of exosomes derived from human and rat pituitary and elucidate their actions. METHODS: Isolated extracellular vesicles (EVs) were analyzed by Nanoparticle Tracking Analysis (NTA) and expressed exosomal markers detected by Western blot, using nonpituitary fibroblast FR and myoblast H9C2 cells as controls. Exosome inhibitor GW4869 was employed to detect attenuated EV release. Exosomal RNA contents were characterized by RNA sequencing. In vitro and in vivo hepatocyte signaling alterations responding to GH1-derived exosomes (GH1-exo) were delineated by mRNA sequencing. GH1-exo actions on protein synthesis, cAMP (3',5'-cyclic adenosine 5'-monophosphate) response, cell motility, and metastases were assessed. RESULTS: NTA, exosomal marker detection, and GW4869 attenuated EV release, confirming the exosomal identity of pituitary EVs. Hydrocortisone increased exosome secretion in GH1 and GH3 cells, suggesting a stress-associated response. Exosomal RNA contents showed profiles distinct for pituitary cells, and rat primary hepatocytes exposed to GH1-exo exhibited transcriptomic alterations distinct from those elicited by growth hormone or prolactin. Intravenous GH1-exo injection into rats attenuated hepatic Eif2ak2 and Atf4 mRNA expression, both involved in cAMP responses and amino acid biosynthesis. GH1-exo suppressed protein synthesis and forskolin-induced cAMP levels in hepatocytes. GH1-exo-treated HCT116 cells showed dysregulated p53 and mitogen-activated protein kinase (MAPK) pathways and attenuated motility of malignant HCT116 cells, and decreased tumor metastases in nude mice harboring splenic HCT116 implants. CONCLUSION: Our findings elucidate biological actions of somatotroph-derived exosomes and implicate exosomes as nonhormonal pituitary-derived messengers.
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Adenoma/patologia , Exossomos/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Hipófise/metabolismo , Adenoma/metabolismo , Adulto , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Comunicação Celular , Técnicas de Cocultura , Feminino , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Hepatócitos , Humanos , Masculino , Hipófise/citologia , Hipófise/patologia , Cultura Primária de Células , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased beta2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.
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Células Endócrinas/fisiologia , Hipófise/fisiologia , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/fisiologia , Hormônio Adrenocorticotrópico/metabolismo , Células Cultivadas , Células Endócrinas/efeitos dos fármacos , Células Endócrinas/metabolismo , Humanos , Ligantes , Octreotida/farmacologia , Peptídeos/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/genética , Somatostatina/metabolismo , Somatostatina/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
Dopaminergic neurons in the midbrain are of particular interest due to their role in diseases such as Parkinson's disease and schizophrenia. Genetic variation between individuals can affect the integrity and function of dopaminergic neurons but the DNA variants and molecular cascades modulating dopaminergic neurons and other cells types of ventral midbrain remain poorly defined. Three genetically diverse inbred mouse strains - C57BL/6J, A/J, and DBA/2J - differ significantly in their genomes (â¼7 million variants), motor and cognitive behavior, and susceptibility to neurotoxins. To further dissect the underlying molecular networks responsible for these variable phenotypes, we generated RNA-seq and ChIP-seq data from ventral midbrains of the 3 mouse strains. We defined 1000-1200 transcripts that are differentially expressed among them. These widespread differences may be due to altered activity or expression of upstream transcription factors. Interestingly, transcription factors were significantly underrepresented among the differentially expressed genes, and only one transcription factor, Pttg1, showed significant differences between all three strains. The changes in Pttg1 expression were accompanied by consistent alterations in histone H3 lysine 4 trimethylation at Pttg1 transcription start site. The ventral midbrain transcriptome of 3-month-old C57BL/6J congenic Pttg1-/- mutants was only modestly altered, but shifted toward that of A/J and DBA/2J in 9-month-old mice. Principle component analysis (PCA) identified the genes underlying the transcriptome shift and deconvolution of these bulk RNA-seq changes using midbrain single cell RNA-seq data suggested that the changes were occurring in several different cell types, including neurons, oligodendrocytes, and astrocytes. Taken together, our results show that Pttg1 contributes to gene regulatory variation between mouse strains and influences mouse midbrain transcriptome during aging.
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Wnt/ß-catenin and Hippo pathways play essential roles in the tumorigenesis and development of colorectal cancer. We found that Celastrol, isolated from Tripterygium wilfordii plant, exerted a significant inhibitory effect on colorectal cancer cell growth in vitro and in vivo, and further unraveled the molecular mechanisms. Celastrol induced ß-catenin degradation through phosphorylation of Yes-associated protein (YAP), a major downstream effector of Hippo pathway, and also Celastrol-induced ß-catenin degradation was dependent on liver kinase B1 (LKB1). Celastrol increased the transcriptional activation of LKB1, partially through the heat shock factor 1 (HSF1). Moreover, LKB1 activated AMP-activated protein kinase α (AMPKα) and further phosphorylated YAP, which eventually promoted the degradation of ß-catenin. In addition, LKB1 deficiency promoted colorectal cancer cell growth and attenuated the inhibitory effect of Celastrol on colorectal cancer growth both in vitro and in vivo. Taken together, Celastrol inhibited colorectal cancer cell growth by promoting ß-catenin degradation via the HSF1-LKB1-AMPKα-YAP pathway. These results suggested that Celastrol may potentially serve as a future drug for colorectal cancer treatment.
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As human pituitary tumor transforming gene (hPTTG1) is upregulated in endocrine tumors, we studied regulatory mechanisms for hPTTG1 expression. We identified Oct-1-binding motifs in the hPTTG1 promoter region and show Oct-1-specific binding to the hPTTG1 promoter using chromatin immunoprecipitation. We overexpressed Oct-1 and observed approximately 2.5-fold activation of hPTTG1 promoter luciferase constructs (-2642/-1 and -1717/-1). Transcriptional activation was abrogated by co-transfection of an inactive Oct-1 form lacking the POU domain or by utilizing mutated hPTTG1 promoters or mutants devoid of two Oct-1-binding motifs (-1717/-1mut, -637/-1 or -433/-1). Using biotin-streptavidin pull-down assays, we confirmed Oct-1 binding to the two octamer motifs in the hPTTG1 promoter (-1669/-1631 and -1401/-1361). Endogenous hPTTG1 mRNA and protein increased up to approximately fourfold in Oct-1 transfectants, as measured by real-time PCR and western blot. In contrast, siRNA-mediated suppression of endogenous Oct-1 attenuated both the hPTTG1 mRNA and protein levels. Using confocal immunofluorescence imaging, Oct-1 and hPTTG1 were concordantly upregulated in pituitary (57 and 62%, n=79, P<0.01) and breast tumor specimens (57 and 42%, n=77, P<0.05) respectively. The results show that Oct-1 transactivates hPTTG1, and both proteins are concordantly overexpressed in endocrine tumors, thus offering a mechanism for endocrine tumor hPTTG1 abundance.
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Neoplasias das Glândulas Endócrinas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Fator 1 de Transcrição de Octâmero/fisiologia , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias das Glândulas Endócrinas/patologia , Humanos , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Securina , Transcrição Gênica , Transfecção , Regulação para Cima/genéticaRESUMO
Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with a poor prognosis. Although aberrant activation of beta-catenin/T-cell factor (TCF) pathway has been observed in ESCC, mechanisms underlying this phenomenon remain unknown. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1), overexpressed in some ESCC lines, is a positive regulator of beta-catenin/TCF pathway. However, little is known about the molecular relationship between FRAT1 and beta-catenin/TCF in ESCC. In this study, we analyzed freshly resected ESCC specimens and demonstrated that FRAT1 was overexpressed in approximately 74% of tumor samples compared with matched normal tissue. Overexpression of FRAT1 significantly promoted esophageal cancer cells growth, whereas suppression of FRAT1 level by RNAi markedly inhibited their growth. In addition, FRAT1 overexpression induced the nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/TCF. These effects were reversed by coexpression of GSK 3beta or DeltaN TCF4. Furthermore, accumulation of beta-catenin was correlated with FRAT1 overexpression in ESCC and the basal layer of normal esophageal epithelium. Finally, continued expression of c-Myc is necessary and sufficient for maintenance of the growth state in cells expressing FRAT1. Taken together, these results support the novel hypothesis that aberrant activation of beta-catenin/TCF pathway in esophageal cancer appears to be due to upstream events such as FRAT1 overexpression, and c-Myc may be an important element in oncogenesis of human ESCC induced by FRAT1.
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Carcinoma de Células Escamosas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Esofágicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição TCF/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Núcleo Celular/metabolismo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or DeltaN TCF4, respectively. Also, we found that EB1 affected the interaction between beta-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of beta-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the beta-catenin/TCF pathway.
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Carcinoma de Células Escamosas/metabolismo , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Transativadores/metabolismo , Sequência de Bases , Western Blotting , Carcinoma de Células Escamosas/patologia , Primers do DNA , Neoplasias Esofágicas/patologia , Genes APC , Humanos , Imuno-Histoquímica , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismo , beta CateninaRESUMO
Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason underlying difficulties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive "liquid biopsy" from the blood has been attempted to characterize tumor heterogeneity. This review focuses on cell-free circulating tumor DNA (ctDNA) in the bloodstream as a versatile biomarker. ctDNA analysis is an evolving field with many new methods being developed and optimized to be able to successfully extract and analyze ctDNA, which has vast clinical applications. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. In addition, ctDNA has the potential to accurately monitor tumor burden and treatment response, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse. There are still many challenges that need to be overcome prior to this biomarker getting wide adoption in the clinical world, including optimization, standardization, and large multicenter trials.
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Biomarcadores Tumorais/genética , DNA de Neoplasias/sangue , Neoplasias/genética , Epigênese Genética , Heterogeneidade Genética , Humanos , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , PrognósticoRESUMO
INTRODUCTION: Breast cancer is the leading cause of cancer death in women worldwide. Elevated expression of c-Myc is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against c-Myc in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumor effects elicited by a decrease in the protein level of c-Myc by RNAi and its possible mechanism of effects in MCF-7 cells. METHOD: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc to reduce its expression in MCF-7 cells. Western blot analysis was used to measure the protein level of c-Myc. We assessed the effects of c-Myc silencing on tumor growth by a growth curve, by soft agar assay and by nude mice experiments in vivo. Standard fluorescence-activated cell sorter analysis and TdT-mediated dUTP nick end labelling assay were used to determine apoptosis of the cells. RESULTS: Our data showed that plasmids expressing siRNA against c-myc markedly and durably reduced its expression in MCF-7 cells by up to 80%, decreased the growth rate of MCF-7 cells, inhibited colony formation in soft agar and significantly reduced tumor growth in nude mice. We also found that depletion of c-Myc in this manner promoted apoptosis of MCF-7 cells upon serum withdrawal. CONCLUSION: c-Myc has a pivotal function in the development of breast cancer. Our data show that decreasing the c-Myc protein level in MCF-7 cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, and imply the therapeutic potential of RNAi on the treatment of breast cancer by targeting overexpression oncogenes such as c-myc, and c-myc might be a potential therapeutic target for human breast cancer.
Assuntos
Neoplasias da Mama/genética , Genes myc , Proteínas Proto-Oncogênicas c-myc/biossíntese , Interferência de RNA , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Fenótipo , Plasmídeos , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , Células Tumorais CultivadasRESUMO
Esophageal squamous cell carcinoma ranks among one of the most frequent cause of cancer death in the world. Understanding of the molecular mechanisms involved in the pathogenesis of esophageal cancer becomes critical to develop more effective treatments. Elevated expression of survivin in esophageal carcinoma has been reported before and suppression of survivin expression leads to many tumor cells growth inhibition. We hypothesized that downregulation of survivin would inhibit the growth of human esophageal cancer cells. RNA interference directed against survivin was introduced into a human esophageal squamous cell carcinoma cell line KYSE510. Stable clones were selected and western blot analysis was performed to detect the protein level of survivin. Tumor cell growth in vitro and in vivo was assessed by trypan blue exclusion and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis and TUNEL assay were used to detect apoptosis in cell culture and in nude mice. We found that RNA interference could efficiently and stably suppress survivin expression in KYSE510 cells. Downregulation of survivin resulted in significantly inhibition of tumor growth in vitro and in vivo. The mechanism appears to be increased induction of apoptosis. Our results suggest a potential role for the targeting of survivin in the treatments of esophageal carcinoma.
Assuntos
Carcinoma/patologia , Regulação para Baixo , Neoplasias Esofágicas/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Interferência de RNA , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Proteínas Inibidoras de Apoptose , RNA Interferente Pequeno/metabolismo , Survivina , Fatores de TempoRESUMO
OBJECTIVE: To detect the expression of survivin in esophageal cancer and elucidate its function in esophageal cancer. METHODS: Expression of surviv in was detected in paired normal and tumor tissues from patients with esophageal cancer by semi-quantitative RT-PCR. A dominant-negative survivin (surT34A) was transfected into esophageal cancer EC9706 cells (EC9706surT34A). Colony formation and apoptosis of the parental and surT34A-transfected EC9706 cells were examined in soft agar and by flow cytometry, respectively. RESULTS: Survivin mRNA expression of tumor tissues was higher than normal tissues in 18/27 (66.7%) samples. The expression level of survivin mRNA in tumor tissues (2.08 +/- 1.32) was significantly higher than that in normal tissues (1.22 +/- 1.09). EC9706 surT34A cells formed fewer colonies on agar than the non-transfected ones. After serum withdrawal, EC9706surT34A had higher apoptotic ratio than control, but survivin could reduce the apoptotic ratio. CONCLUSION: Overexpression of survivin is a common eventin esophageal cancer. The dominant-negative survivin can partially inhibit the malignant phenotype of esophageal cancer.