RESUMO
Epigenetic alterations are increasingly recognized as important contributors to the development and progression of pancreatic ductal adenocarcinoma. 5-hydroxymethylcytosine (5hmC) is an epigenetic DNA mark generated through the ten-eleven translocation (TET) enzyme-mediated pathway and is closely linked to gene activation. However, the timing of alterations in epigenetic regulation in the progression of pancreatic neoplasia is not well understood. In this study, we hypothesized that aberrant expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and subsequent global 5hmC alteration are linked to early tumorigenesis in the pancreas. Therefore, we evaluated alterations of 5hmC and TET1 levels using immunohistochemistry in pancreatic neoplasms (n = 380) and normal ducts (n = 118). The study cohort included representation of the full spectrum of precancerous lesions from low- and high-grade pancreatic intraepithelial neoplasia (n = 95), intraductal papillary mucinous neoplasms (all subtypes, n = 129), intraductal oncocytic papillary neoplasms (n = 12), and mucinous cystic neoplasms (n = 144). 5hmC and TET1 were significantly downregulated in all types of precancerous lesion and associated invasive pancreatic ductal adenocarcinomas compared with normal ductal epithelium (all p < 0.001), and expression of 5hmC positively correlated with expression of TET1. Importantly, downregulation of both 5hmC and TET1 was observed in most low-grade precancerous lesions. There were no clear associations between 5hmC levels and clinicopathological factors, thereby suggesting a common epigenetic abnormality across precancerous lesions. We conclude that downregulation of 5hmC and TET1 is an early event in pancreatic tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
5-Metilcitosina/análogos & derivados , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pancreáticas/metabolismo , 5-Metilcitosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/patologia , Carcinoma Ductal Pancreático/patologia , Regulação para Baixo , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxigenases de Função Mista/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.
Assuntos
Antivirais/farmacologia , Artemisininas/farmacologia , Citomegalovirus/efeitos dos fármacos , Vimentina/antagonistas & inibidores , Antivirais/química , Antivirais/metabolismo , Artemisininas/química , Artemisininas/metabolismo , Artesunato/farmacologia , Sítios de Ligação , Calpaína/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/metabolismo , Reposicionamento de Medicamentos , Humanos , Espectrometria de Massas , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Vimentina/genética , Vimentina/metabolismo , Replicação Viral/efeitos dos fármacos , Vitanolídeos/farmacologiaRESUMO
Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.
Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Compostos Heterocíclicos com 1 Anel/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Compostos de Espiro/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/sangue , Antivirais/química , Antivirais/farmacocinética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/virologia , Ganciclovir/farmacologia , Regulação da Expressão Gênica , Compostos Heterocíclicos com 1 Anel/sangue , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Compostos de Espiro/sangue , Compostos de Espiro/química , Compostos de Espiro/farmacocinéticaRESUMO
Induction of nucleotide-binding oligomerization domain 2 (NOD2) and downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2) by human cytomegalovirus (HCMV) is known to up-regulate antiviral responses and suppress virus replication. We investigated the role of nucleotide-binding oligomerization domain 1 (NOD1), which also signals through RIPK2, in HCMV control. NOD1 activation by Tri-DAP (NOD1 agonist) suppressed HCMV and induced IFN-ß. Mouse CMV was also inhibited through NOD1 activation. NOD1 knockdown (KD) or inhibition of its activity with small molecule ML130 enhanced HCMV replication in vitro. NOD1 mutations displayed differential effects on HCMV replication and antiviral responses. In cells overexpressing the E56K mutation in the caspase activation and recruitment domain, virus replication was enhanced, but in cells overexpressing the E266K mutation in the nucleotide-binding domain or the wild-type NOD1, HCMV was inhibited, changes that correlated with IFN-ß expression. The interaction of NOD1 and RIPK2 determined the outcome of virus replication, as evidenced by enhanced virus growth in NOD1 E56K mutant cells (which failed to interact with RIPK2). NOD1 activities were executed through IFN-ß, given that IFN-ß KD reduced the inhibitory effect of Tri-DAP on HCMV. Signaling through NOD1 resulting in HCMV suppression was IKKα-dependent and correlated with nuclear translocation and phosphorylation of IRF3. Finally, NOD1 polymorphisms were significantly associated with the risk of HCMV infection in women who were infected with HCMV during participation in a glycoprotein B vaccine trial. Collectively, our data indicate a role for NOD1 in HCMV control via RIPK2- IKKα-IRF3 and suggest that its polymorphisms predict the risk of infection.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Proteína Adaptadora de Sinalização NOD1/fisiologia , Animais , Células Cultivadas , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Feminino , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Camundongos Endogâmicos BALB C , Proteína Adaptadora de Sinalização NOD2/fisiologia , Polimorfismo de Nucleotídeo Único , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 µM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.
Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus , Citomegalovirus/efeitos dos fármacos , Emetina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Reação em Cadeia da Polimerase , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVES: Although uterine cancer is the fourth most common cause for cancer death in women worldwide, the molecular underpinnings of tumor progression remain poorly understood. The High Mobility Group A1 (HMGA1) gene is overexpressed in aggressive cancers and high levels portend adverse outcomes in diverse tumors. We previously reported that Hmga1a transgenic mice develop uterine tumors with complete penetrance. Because HMGA1 drives tumor progression by inducing MatrixMetalloproteinase (MMP) and other genes involved in invasion, we explored the HMGA1-MMP-2 pathway in uterine cancer. METHODS: To investigate MMP-2 in uterine tumors driven by HMGA1, we used a genetic approach with mouse models. Next, we assessed HMGA1 and MMP-2 expression in primary human uterine tumors, including low-grade carcinomas (endometrial endometrioid) and more aggressive tumors (endometrial serous carcinomas, uterine carcinosarcomas/malignant mesodermal mixed tumors). RESULTS: Here, we report for the first time that uterine tumor growth is impaired in Hmga1a transgenic mice crossed on to an Mmp-2 deficient background. In human tumors, we discovered that HMGA1 is highest in aggressive carcinosarcomas and serous carcinomas, with lower levels in the more indolent endometrioid carcinomas. Moreover, HMGA1 and MMP-2 were positively correlated, but only in a subset of carcinosarcomas. HMGA1 also occupies the MMP-2 promoter in human carcinosarcoma cells. CONCLUSIONS: Together, our studies define a novel HMGA1-MMP-2 pathway involved in a subset of human carcinosarcomas and tumor progression in murine models. Our work also suggests that targeting HMGA1 could be effective adjuvant therapy for more aggressive uterine cancers and provides compelling data for further preclinical studies.
Assuntos
Carcinossarcoma/genética , Cistadenocarcinoma Seroso/genética , Proteína HMGA1a/genética , Metaloproteinase 2 da Matriz/genética , Neoplasias Uterinas/genética , Animais , Carcinossarcoma/metabolismo , Imunoprecipitação da Cromatina , Cistadenocarcinoma Seroso/metabolismo , Feminino , Inativação Gênica , Proteína HMGA1a/biossíntese , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Camundongos Transgênicos , Regiões Promotoras Genéticas , Regulação para Cima , Neoplasias Uterinas/metabolismoRESUMO
Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.
Assuntos
Antimaláricos/farmacologia , Antivirais/farmacologia , Artemisininas/farmacologia , Ciclo Celular/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Células Cultivadas , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/biossíntese , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/biossíntese , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/biossíntese , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Fibroblastos/virologia , Fase G1/efeitos dos fármacos , Ganciclovir/farmacologia , Humanos , Fosforilação , Purinas/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Roscovitina , Replicação Viral/efeitos dos fármacosRESUMO
Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.
Assuntos
Neoplasias da Mama , Creatina Quinase Mitocondrial , Espécies Reativas de Oxigênio , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Creatina Quinase , Creatina Quinase Mitocondrial/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fibroblast activation protein (FAP) is a serine protease upregulated at sites of tissue remodeling and cancer that represents a promising therapeutic and molecular imaging target. In prostate cancer, studies of FAP expression using tissue microarrays are conflicting, such that its clinical potential is unclear. Furthermore, little is known regarding FAP expression in benign prostatic tissues. Here we demonstrated, using a novel iterative multiplex IHC assay in standard tissue sections, that FAP was nearly absent in normal regions, but was increased consistently in regions of proliferative inflammatory atrophy (PIA). In carcinoma, FAP was expressed in all cases, but was highly heterogeneous. High FAP levels were associated with increased pathological stage and cribriform morphology. We verified that FAP levels in cancer correlated with CD163+ M2 macrophage density. In this first report to quantify FAP protein in benign prostate and primary tumors, using standard large tissue sections, we clarify that FAP is present in all primary prostatic carcinomas, supporting its potential clinical relevance. The finding of high levels of FAP within PIA supports the injury/regeneration model for its pathogenesis and suggests that it harbors a protumorigenic stroma. Yet, high levels of FAP in benign regions could lead to false positive FAP-based molecular imaging results in clinically localized prostate cancer.
RESUMO
Introduction: Plasticity is an inherent property of the normal gastrointestinal tract allowing for appropriate response to injury and healing. However, the aberrancy of adaptable responses is also beginning to be recognized as a driver during cancer development and progression. Gastric and esophageal malignancies remain leading causes of cancer-related death globally as there are limited early disease diagnostic tools and paucity of new effective treatments. Gastric and esophageal adenocarcinomas share intestinal metaplasia as a key precancerous precursor lesion. Methods: Here, we utilize an upper GI tract patient-derived tissue microarray that encompasses the sequential development of cancer from normal tissues to illustrate the expression of a set of metaplastic markers. Results: We report that in contrast to gastric intestinal metaplasia, which has traits of both incomplete and complete intestinal metaplasia, Barrett's esophagus (i.e., esophageal intestinal metaplasia) demonstrates hallmarks of incomplete intestinal metaplasia. Specifically, this prevalent incomplete intestinal metaplasia seen in Barrett's esophagus manifests as concurrent development and expression of both gastric and intestinal traits. Additionally, many gastric and esophageal cancers display a loss of or a decrease in these characteristic differentiated cell properties, demonstrating the plasticity of molecular pathways associated with the development of these cancers. Discussion: Further understanding of the commonalities and differences governing the development of upper GI tract intestinal metaplasias and their progression to cancer will lead to improved diagnostic and therapeutic avenues.
RESUMO
Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell-activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.
Assuntos
Baço , Viroses , Animais , Antígenos Ly/genética , Humanos , Inflamação/genética , Linfócitos , Proteínas de Membrana/genética , Camundongos , Família Multigênica , Viroses/genéticaRESUMO
Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the major H/ACA core protein and pseudouridine synthase NAP57 (aka dyskerin) but precedes the assembly role of NAF1 at nascent H/ACA RNAs because the interaction of SHQ1 with NAP57 in vivo and in vitro precludes that of NAF1 and of the other H/ACA core proteins that are present at the sites of H/ACA RNA transcription. The N-terminal heat shock protein 20-like CS domain of SHQ1 is dispensable for NAP57 binding. Consistent with its role as an assembly factor, SHQ1 localizes to the nucleoplasm and is excluded from nucleoli and Cajal bodies, the sites of mature H/ACA RNPs. In an in vitro assembly system of functional H/ACA RNPs that is dependent on NAF1, excess recombinant SHQ1 interferes with assembly. Importantly, knockdown of cellular SHQ1 prevents accumulation of a newly synthesized H/ACA reporter RNA and generally reduces the levels of endogenous H/ACA RNAs including telomerase RNA. In summary, the sequential action of SHQ1 and NAF1 is required for functional assembly of H/ACA RNPs in vivo and in vitro. This step-wise process could serve as an efficient means of quality control during H/ACA RNP assembly.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Telomerase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , RNA/metabolismo , TransfecçãoRESUMO
Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.
Assuntos
Proteínas de Ligação a DNA/fisiologia , RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Transcrição Gênica , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismoRESUMO
Prostate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. In order to improve patient outcomes, it is critical to clinically separate aggressive PCa (AG) from non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with aggressive PCa, it is still challenging to separate AG from NAG. To better understand the functional consequences of PCa progression and the unique features of the AG subtype, we studied the proteomic signatures of primary AG, NAG and metastatic PCa. 39 PCa and 10 benign prostate controls in a discovery cohort and 57 PCa in a validation cohort were analyzed using a data-independent acquisition (DIA) SWATH-MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using a validation cohort; and was also compared with a TCGA mRNA expression dataset and confirmed by immunohistochemistry (IHC) using PCa tissue microarray (TMA). 4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed reduced expressions of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates and the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using an independently-collected tumor cohort, correlated with a TCGA mRNA dataset, and confirmed by immunohistochemical stains of PCa tumor microarray (TMA). Our study is the first large-scale proteomics analysis of PCa tissue using a DIA SWATH-MS platform. It provides not only an interrogative proteomic signature of PCa subtypes, but also indicates the critical roles played by certain proteinases during tumor progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in PCa and for the development of a clinical assay to distinguish aggressive from indolent PCa.
Assuntos
Carboxipeptidase H/metabolismo , Dipeptidil Peptidase 4/metabolismo , Regulação Neoplásica da Expressão Gênica , Calicreínas/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Conjuntos de Dados como Assunto , Seguimentos , Perfilação da Expressão Gênica , Humanos , Masculino , Gradação de Tumores , Próstata/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Proteômica/estatística & dados numéricos , Análise Serial de TecidosRESUMO
Human cytomegalovirus (HCMV) was reported to downregulate the Wnt/ß-catenin pathway. Induction of Axin1, the negative regulator of the Wnt pathway, has been reported as an important mechanism for inhibition of ß-catenin. Since Tankyrase (TNKS) negatively regulates Axin1, we investigated the effect of HCMV on TNKS expression and poly-ADP ribose polymerase (PARsylation) activity, during virus replication. Starting at 24 h post infection, HCMV stabilized the expression of TNKS and reduced its PARsylation activity, resulting in accumulation of Axin1 and reduction in its PARsylation as well. General PARsylation was not changed in HCMV-infected cells, suggesting specific inhibition of TNKS PARsylation. Similarly, treatment with XAV939, a chemical inhibitor of TNKS' activity, resulted in the accumulation of TNKS in both non-infected and HCMV-infected cell lines. Reduction of TNKS activity or knockdown of TNKS was beneficial for HCMV, evidenced by its improved growth in fibroblasts. Our results suggest that HCMV modulates the activity of TNKS to induce Axin1, resulting in inhibition of the ß-catenin pathway. Since HCMV replication is facilitated by TNKS knockdown or inhibition of its activity, TNKS may serve as an important virus target for control of a variety of cellular processes.
Assuntos
Infecções por Citomegalovirus/enzimologia , Citomegalovirus/fisiologia , Tanquirases/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Tanquirases/genética , Replicação ViralRESUMO
Emerging evidence suggests that tumor cells metastasize by co-opting stem cell transcriptional networks, although the molecular underpinnings of this process are poorly understood. Here, we show for the first time that the high mobility group A1 (HMGA1) gene drives metastatic progression in triple negative breast cancer cells (MDA-MB-231, Hs578T) by reprogramming cancer cells to a stem-like state. Silencing HMGA1 expression in invasive, aggressive breast cancer cells dramatically halts cell growth and results in striking morphologic changes from mesenchymal-like, spindle-shaped cells to cuboidal, epithelial-like cells. Mesenchymal genes (Vimentin, Snail) are repressed, while E-cadherin is induced in the knock-down cells. Silencing HMGA1 also blocks oncogenic properties, including proliferation, migration, invasion, and orthotopic tumorigenesis. Metastatic progression following mammary implantation is almost completely abrogated in the HMGA1 knock-down cells. Moreover, silencing HMGA1 inhibits the stem cell property of three-dimensional mammosphere formation, including primary, secondary, and tertiary spheres. In addition, knock-down of HMGA1 depletes cancer initiator/cancer stem cells and prevents tumorigenesis at limiting dilutions. We also discovered an HMGA1 signature in triple negative breast cancer cells that is highly enriched in embryonic stem cells. Together, these findings indicate that HMGA1 is a master regulator of tumor progression in breast cancer by reprogramming cancer cells through stem cell transcriptional networks. Future studies are needed to determine how to target HMGA1 in therapy.
Assuntos
Progressão da Doença , Proteína HMGA1a/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Inativação Gênica , Proteína HMGA1a/deficiência , Proteína HMGA1a/genética , Humanos , Mesoderma/patologia , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia with high relapse rates compared to B-lineage ALL. We previously showed that HMGA1a transgenic mice develop aggressive T-ALL, indicating that HMGA1 causes leukemic transformation in vivo. HMGA1 is also highly expressed in embryonic stem cells, hematopoietic stem cells and diverse, refractory human cancers. Disruption of the CDKN2A tumor suppressor locus occurs in most cases of T-ALL and is thought to contribute to leukemic transformation. To determine whether loss of function of CDKN2A cooperates with HMGA1 in T-ALL, we crossed HMGA1a transgenics onto a Cdkn2a null background. We discovered that T-ALL is markedly accelerated in HMGA1a transgenic Cdkn2a null mice. In addition, these mice recapitulate salient clinical and pathologic features of human T-ALL. HMGA1 is also highly overexpressed in human T-ALL. These findings suggest that HMGA1 plays a causative role in T-ALL and could represent a rational therapeutic target.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Epistasia Genética , Inativação Gênica , Proteína HMGA1a/genética , Leucemia de Células T/genética , Animais , Modelos Animais de Doenças , Feminino , Expressão Gênica , Loci Gênicos , Humanos , Imunofenotipagem , Leucemia de Células T/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
Mammalian H/ACA RNPs are essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. To form mature RNA-protein complexes, one H/ACA RNA associates with four core proteins. In the cell, this process is assisted by at least one nuclear assembly factor, NAF1. Here we report several unanticipated dynamic aspects of H/ACA RNP proteins. First, when overexpressed, NAF1 delocalizes to the cytoplasm. However, its nucleocytoplasmic shuttling properties remain unaffected. These observations demonstrate a subtle equilibrium between NAF1 expression levels and the availability of NAF1 nuclear binding sites. Second, although NAF1 is excluded from mature RNPs in nucleoli and Cajal bodies, NAF1 associates with mature H/ACA RNA in cell lysates. This association occurs post-lysis because it is observed even when NAF1 and the H/ACA RNA are expressed in separate cells. This documents a protein-RNP association in cell lysates that is absent from intact cells. Third, in similar experiments, all H/ACA core proteins, except NAP57, exchange with their exogenous counterparts, portraying an unexpected dynamic picture of H/ACA RNPs. Finally, the irreversible association of only NAP57 with H/ACA RNA and the conundrum that only NAP57 is mutated in X-linked dyskeratosis congenita (even though most core proteins are required for maintaining H/ACA RNAs) may be more than a coincidence.