RESUMEN
Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels1-3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown4-7. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8-10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11-13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitina , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , Humanos , Conformación Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Binding to the receptor, CD4, drives the pretriggered, "closed" (state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCE The development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (state 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in state 1 and can be efficiently cross-linked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Detergentes , Glicoproteínas/química , Glicoproteínas/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/prevención & control , VIH-1/química , VIH-1/genética , VIH-1/inmunología , Humanos , Lisina , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail, and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: (i) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that cross-linked efficiently, and was recognized by broadly neutralizing antibodies; (ii) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and (iii) the larger population of uncleaved Env lacked complex carbohydrates, cross-linked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more "open" Env oligomers reached the cell surface in the presence of brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses toward conformationally flexible, uncleaved Env without compromising virus infectivity.IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus type 1 (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this nonfunctional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Multimerización de Proteína , Virión/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Células A549 , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Humanos , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Virión/inmunologíaRESUMEN
During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)3] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/efectos de los fármacos , VIH-1/inmunología , Piperazinas/farmacología , Internalización del Virus/efectos de los fármacos , Células A549 , Anticuerpos Neutralizantes/inmunología , Antígenos CD4/efectos de los fármacos , Antígenos CD4/metabolismo , Glicoproteínas/genética , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Ligandos , Modelos Moleculares , Conformación ProteicaRESUMEN
The bone marrow has been long known to host a unique environment amenable to colonization by metastasizing tumor cells. Yet, the underlying molecular interactions which give rise to the high incidence of bone metastasis (BM) in breast cancer patients have long remained uncharacterized. In our study, in vitro and in vivo assays demonstrated that Brachyury (Bry) could promote breast cancer BM. Bry drives epithelial-mesenchymal transition (EMT) and promotes breast cancer aggressiveness. As an EMT driver, SOX5 involves in breast cancer metastasis and the specific function in BM. Chromatin immunoprecipitation (ChIP) assays revealed SOX5 is a direct downstream target gene of Bry. ChIP analysis and reporter assays identified two Bry-binding motifs; one consistent with the classic conserved binding sequence and the other a new motif sequence. This study demonstrates for the first time that Bry promotes breast cancer cells BM through activating SOX5. In clinical practice, targeting the Bry-Sox5-EMT pathway is evolving into a promising avenue for the prevention of bone metastatic relapse, therapeutic resistance and other aspects of breast cancer progression. Brachyury directly regulates the expression of SOX5 by binding to two motifs in its promoter region. The Bry-SOX5-EMT pathway may represent a potential target to develop treatments to prevent and treat bone metastasis from breast cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Proteínas Fetales/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción SOXD/genética , Proteínas de Dominio T Box/metabolismo , Activación Transcripcional , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Proteínas Fetales/genética , Humanos , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Pronóstico , Factores de Transcripción SOXD/metabolismo , Tasa de Supervivencia , Proteínas de Dominio T Box/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/clasificación , VIH-1/efectos de los fármacos , Piridonas/farmacología , Sulfonamidas/farmacología , Internalización del Virus/efectos de los fármacos , Antivirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Piridonas/química , Receptores CCR5/genética , Receptores CCR5/metabolismo , Estereoisomerismo , Sulfonamidas/química , Replicación ViralRESUMEN
Objective: To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells. Methods: RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation. Results: Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (P<0.05). The cell proliferation was inhibited (P<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (P<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all P<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (P<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(r=0.478, P<0.01). Conclusion: Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/fisiopatología , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/fisiopatología , ARN Interferente Pequeño/farmacología , Ubiquitina-Proteína Ligasas/farmacología , Adyuvantes Farmacéuticos/farmacología , Ciclo Celular , Línea Celular Tumoral , Cisplatino/farmacología , Ciclina D1/efectos de los fármacos , Ciclina D1/genética , Proteínas de Unión al ADN/administración & dosificación , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Carcinoma de Células Escamosas de Esófago , Fluorouracilo/farmacología , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Humanos , Metafase/efectos de los fármacos , ARN Interferente Pequeño/administración & dosificación , Transfección , Ubiquitina-Proteína Ligasas/administración & dosificaciónRESUMEN
BACKGROUND/AIMS: This study investigated the clinical relevance and biological function of paired box gene 2 (PAX2) in esophageal squamous cell carcinoma (ESCC). METHODS/RESULTS: Results showed that PAX2 expression was significantly increased in tumor tissues and that its expression correlated with the ESCC stage (P = 0.001), lymph node metastasis (pN, P = 0.019) and lymphatic invasion (P = 0.005) in 120 ESCC tissue specimens by immunohistochemistry. Furthermore, PAX2 overexpression resulted in markedly reduced cell proliferation but increased metastasis capacity in ESCC TE-1 and Eca-109 cells. Knockdown of PAX2 expression with a short hairpin RNA confirmed a role in the promotion of metastasis in ESCC cells. mRNA microarray screening revealed that PAX2 overexpression affected multiple genes that function in multiple pathways. Interleukin-5 (IL-5), which was induced by PAX2 and has been shown to promote tumor metastasis, was further studied in greater detail. Two PAX2 binding sites were identified in the IL-5 promoter, and PAX2 was observed to stimulate IL-5 promoter activity and IL-5 expression in esophageal cancer cells. Chromatin immunoprecipitation (ChIP) confirmed the direct binding of PAX2 in the IL-5 promoter. The expression of PAX2 mRNA significantly correlated with that of IL-5 in normal esophageal and ESCC tissues. CONCLUSION: These findings demonstrate that PAX2 is overexpressed in esophageal carcinoma and IL-5 is identified as PAX2's effector for metastasis.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Interleucina-5/genética , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Adulto , Anciano , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago , Femenino , Humanos , Interleucina-5/metabolismo , Masculino , Persona de Mediana Edad , Regulación hacia ArribaRESUMEN
Zoledronate (ZOL) is a third-generation bisphosphonate (BP), clinically used to treat lytic bone lesions caused by malignancies or bone resorption disorders. Mechanistically, ZOL was recently shown to have direct pro-apoptotic effects on tumor cells and to inhibit cancer cell invasion, adhesion, proliferation, and angiogenesis. The molecular mechanism of ZOL-induced apoptosis remains unknown. In this study, we observed that ZOL induced apoptosis in colorectal cancer cells HCT116 and Caco-2. After HCT116 and Caco-2 cells were treated with ZOL, decreased fluorescence of JC-1 aggregates (590 nm) was seen in mitochondria. Western blotting analysis showed that cytochrome c was decreased in the mitochondria and increased in the cytosol, respectively. The effects were dependent on the concentration and treatment time by ZOL. In vivo experiments showed that ZOL inhibited the growth of xenograft tumor in mice. Hematoxylin and eosin (H&E) staining of tissue samples showed a significantly increased apoptosis body in the ZOL-treated xenografts compared to control. Taken together, our data demonstrated that ZOL inhibits growth of HCT116 cells both in vitro and in vivo and induce apoptosis through the mitochondria pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Difosfonatos/administración & dosificación , Imidazoles/administración & dosificación , Animales , Neoplasias Colorrectales/patología , Células HCT116 , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido ZoledrónicoRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancer types, with a low 5-year survival rate of ~20%. Our prior research has suggested that DNA Polymerase iota (Pol ι), a member of Y-family DNA polymerase, plays a crucial role in the invasion and metastasis of ESCC. However, the underlying mechanism is not well understood. In this study, we utilized ChIP-PCR and luciferase reporter assays to investigate the binding of HIF-1α to the promoter of the Pol ι gene. Transwell, wound healing, and mouse models were employed to assess the impact of Pol ι and HIF-1α on the motility of ESCC cells. Co-immunoprecipitation and Western blot were carried out to explore the interaction between Pol ι and HIF-1α, while qRT-PCR and Western blot were conducted to confirm the regulation of Pol ι and HIF-1α on their downstream targets. Our results demonstrate that HIF-1α activates the transcription of the Pol ι gene in ESCC cells under hypoxic conditions. Furthermore, the knockdown of Pol ι impeded HIF-1α-induced invasion and metastasis. Additionally, we found that Pol ι regulates the expression of genes involved in epithelial-mesenchymal transition (EMT) and initiates EMT through the stabilization of HIF-1α. Mechanistically, Pol ι maintains the protein stability of HIF-1α by recruiting USP7 to mediate the deubiquitination of HIF-1α, with the residues 446-578 of Pol being crucial for the interaction between Pol ι and USP7. Collectively, our findings unveil a novel feedforward molecular axis of HIF-1α- Pol ι -USP7 in ESCC that contributes to ESCC metastasis. Hence, our results present an attractive target for intervention in ESCC.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Animales , Ratones , Línea Celular Tumoral , Movimiento Celular , ADN Polimerasa iota , Transición Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Regulación Neoplásica de la Expresión Génica , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
PURPOSE: Cancer development remains the most challenging obstacle in colorectal cancer (CRC) treatment. The current study aims to identify and demonstrate novel oncogenes for CRC. METHODS: The CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database were subjected to bioinformatics analysis to identify the novel potential diagnostic and prognostic biomarkers for CRC. Immunohistochemical assay, western blot, and quantitative PCR (qPCR) were used to analyze hydroxyacylglutathione hydrolase-like (HAGHL) gene expression in CRC tissues and cultured CRC cells. D-Lactate colorimetric assay was applied to determine concentration of D-lactate in supernatants from CRC tissues and cell culture medium. Cell counting kit-8 (CCK-8) assay, flow cytometry, tumor xenografts experiment, and TUNEL staining analysis were performed to evaluate the function of HAGHL in CRC. RESULTS: We comprehensively analyzed the CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database, and identified several novel potential diagnostic and prognostic biomarkers for CRC, including HAGHL, DNTTIP1, DHX34, and AP1S3. The expression of HAGHL, the strongest oncogenic activity gene, is positively related to D-lactate levels in CRC tissues and negatively associated with patient prognosis. HAGHL downregulation suppressed the production of D-lactate and induced apoptosis, resulting in inhibition of cell proliferation in vitro. In vivo experiment showed that knockdown of HAGHL induced cell apoptosis and inhibited tumor growth. CONCLUSION: These findings suggest that HAGHL acts as a novel metabolic oncogene and demonstrate the underlying mechanism by which HAGHL regulates CRC progression, highlighting its utility as a diagnostic and prognostic factor and as a potential therapeutic target for the treatment of CRC.
Asunto(s)
Neoplasias Colorrectales , MicroARNs , Humanos , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Oncogenes , Lactatos , Biomarcadores , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
TAB182 (also named TNKS1BP1), a binding protein of tankyrase 1, has been found to participate in DNA repair. Our previous study has revealed the involvement of TAB182 in the radioresistance of esophageal squamous cell carcinoma (ESCC) cells. However, whether TAB182 contributes to the ESCC tumorigenesis and progression remains unclear. In this study, we found that highly expressed TAB182 is closely associated with a poor prognosis of patients with ESCC. TAB182 silencing reduced ESCC cell proliferation and invasion in vitro, tumorigenicity and metastasis in vivo. RNA-seq and IP-MS analysis revealed that TAB182 could affect the ß-catenin signaling pathway via interacting with ß-catenin. Furthermore, TAB182 prevented ß-catenin to be phosphorylated by GSK3ß and recruited four and a half of LIM-only protein 2 (FHL2), which thereby promoted ß-catenin nucleus translocation to result in activation of the downstream targets transcription in ESCC cells. Our findings demonstrate that TAB182 enhances tumorigenesis of esophageal cancer by promoting the activation of the ß-catenin signaling pathway, which provides new insights into the molecular mechanisms by which TAB182 accelerates progression of ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Tanquirasas , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas Musculares/genética , Tanquirasas/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Radiotherapy has recently become more common for the treatment of esophageal squamous cell carcinoma (ESCC). Radioresistance, on the other hand, continues to be a major issue because it interferes with the effectiveness of ESCC radiation. It has been demonstrated that RAD18, an E3 ubiquitin-protein ligase that regulates translesion DNA synthesis (TLS), is implicated in the regulation of genomic integrity and DNA damage response. METHODS: In the present study, immunohistochemical staining and western blotting were utilized to determine RAD18 expression in ESCC tissues and cells. ESCC cell proliferation was determined using a colony formation assay. Immunofluorescence staining, comet assay, and homologous recombination (HR)/non-homologous end-joining (NHEJ) assays were conducted to examine the effect of RAD18 on the DNA damage response in ESCC cells. RESULTS: We found that high RAD18 expression was positively associated with a poorer prognosis in patients with ESCC who received radiotherapy. Downregulation of RAD18 expression significantly increased the sensitivity of ESCC cells to irradiation. Moreover, RAD18 knockdown prolonged the repair kinetics of γH2AX foci and resulted in longer comet tails. Furthermore, loss of RAD18 expression markedly decreased non-homologous end-joining (NHEJ) activity, but it did not affect homologous recombination (HR)-mediated double-strand break repair in ESCC cells. RAD18 upregulated p-DNA-dependent protein kinase complex (p-DNA-PKc) expression in vivo and in vitro. CONCLUSIONS: These data indicated that RAD18 may regulate radioresistance by facilitating NHEJ via phosphorylation of DNA-PKcs in ESCC cells, providing a novel radiotherapy target for ESCC.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/radioterapia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/metabolismo , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Línea Celular Tumoral , Reparación del ADN , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Tolerancia a Radiación/genética , Proteínas de Unión al ADN/genéticaRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers with rapid progression and a high mortality rate. Our previous study demonstrated that DNA polymerase iota (Pol ι) is overexpressed in ESCC tumors and correlates with poor prognosis. However, its role in ESCC proliferation remains obscure. We report here that Pol ι promotes ESCC proliferation and progression through Erk- O-GlcNAc transferase (OGT) regulated Glucose-6-phosphate dehydrogenase (G6PD) overactivation. Cell clonogenic ability was assessed by colony formation assay. Cell proliferation was assessed by EdU incorporation assay. Our transcriptome data was reanalyzed by GSEA and validated by analysis of cellular metabolism, G6PD activity, and cellular NADPH concentration. The level of Pol ι, OGT, G6PD and O-GlcNAcylation in ESCC cells and patient samples were analyzed. The MEK inhibitor PD98059 was applied to confirm OGT expression regulation by the Erk signaling. The G6PD inhibitor polydatin was used to examine the role of G6PD activation in Pol ι promoted proliferation. We found that Pol ι promotes ESCC proliferation. It shunted the glucose flux towards the pentose phosphate pathway (PPP) by activating G6PD through OGT-promoted O-GlcNAcylation. The expression of OGT was positively correlated with Pol ι expression and O-GlcNAcylation. Notably, elevated O-GlcNAcylation was correlated with poor prognosis in ESCC patients. Pol ι was shown to stimulate Erk signaling to enhance OGT expression, and the G6PD inhibitor polydatin attenuated Pol ι induced tumor growth in vitro and in vivo. In conclusion, Pol ι activates G6PD through Erk-OGT-induced O-GlcNAcylation to promote the proliferation and progression of ESCC, supporting the notion that Pol ι is a potential biomarker and therapeutic target of ESCC.
RESUMEN
Esophageal squamous cell carcinoma (ESCC) is an aggressive type of cancer with high mortality rate in China, largely due to its high invasive and metastatic potential. The purposes of this study are to investigate the potential molecular mechanisms behind the aggressive nature of ESCC and search for new prognostic biomarkers. By employing the quantitative proteomic based strategy, we compared the proteomic profile between three ESCC samples and paired adjacent tissues. After bioinformatics analysis, four candidate proteins were validated in thirteen paired patient samples. Further validation of the key candidate, integrin-linked kinase (ILK), was carried out in one hundred patient samples. The specific inhibitor compound 22 (cpd22) was used to assess the influence of ILK to ESCC cell motility and invasiveness by applying wound-healing and transwell assay. Western blot analysis was performed to elucidate the signaling pathways involved in ILK-mediated ESCC invasion. Total 236 proteins were identified by proteomic analysis. Bioinformatics analysis suggested a key role of the collagen/integrin/ILK signaling pathway during ESCC progression. Further validation indicated that ILK is overexpressed in ESCC tissues and is correlated with poor patient prognosis. Inhibition of ILK kinase activity suppresses proliferation and blocks invasion and migration of ESCC cells. Signaling pathway analysis revealed that ILK regulates AKT phosphorylation on Ser473 but not GSK-3ß on Ser9 to promote proliferation and motility of ESCC cells. In conclusion, our results indicated that ILK may play a crucial role in ESCC invasion and metastasis and may serve as a prognostic biomarker and therapeutic target for ESCC.
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Increasing evidence has demonstrated that long non-coding (lnc) RNA is aberrantly expressed in numerous types of cancer. Colorectal cancer is a common malignancy; however, the role and mechanism underlying the influence of lncRNA-colon cancer associated transcript 1 (CCAT1) in colorectal cancer is yet to be elucidated. The present study revealed that CCAT1 is highly expressed in colorectal cancer tissues. Bioinformatics analysis and a dual-luciferase reporter gene assay indicated that CCAT1 and microRNA (miR)-218 had complementary binding sites. Furthermore, reverse transcription-quantitative PCR revealed that miR-218 was downregulated in colorectal cancer tissues compared with paired adjacent healthy tissues. To investigate the biological effects of CCAT1 on colorectal cancer cells, MTT and Transwell assays were performed. The results revealed that when compared with the control group, CCAT1-short hairpin (sh)RNA significantly inhibited colorectal cancer cell (SW480) viability and decreased migration and invasiveness. In addition, CCAT1-shRNA significantly reduced vascular endothelial growth factor (VEGF) expression in SW480 cells; however, these effects were partially rescued by an miR-218 inhibitor. Furthermore, it was revealed that the CCAT1-plasmid significantly promoted the viability of SW480 cells, increased cell migration and invasiveness, and significantly increased VEGF expression. However, these effects were also partially rescued by with a miR-218 mimic. Taken together, the present results identified that the CCAT1/miR-218 axis serves a key role in the regulation of colorectal cancer progression, which may be used as potential therapeutic target for the treatment of colorectal cancer.
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Lupus nephritis (LN) is the most common complication that causes mortality in patients with systemic lupus erythematosus. The B7-1/B7-2 and CD28/cytotoxic T-lymphocyte associated protein 4 co-stimulatory pathway serves a key role in autoimmune disease and organ transplantation. The aim of the present study was to generate and characterize a monoclonal antibody (mAb; clone 4E5) against human B7-1 and to investigate its potential use for the treatment of LN. The results demonstrated that the 4E5 mAb was successfully generated and able to recognize both human and mouse B7-1. After injection of this mAb into a mouse model with chronic graft-vs.-host disease (cGVHD)-induced lupus-like disease, the expression of CD21, CD23, CD80 and CD86 on B220+ B-cells in the spleen, and the concentrations of serum autoantibodies and urine protein, were decreased. Direct immunofluorescence analysis of the kidneys revealed that immunoï¬uorescence of immune complex deposits was weaker in the 4E5-treated mice and electron microscopy analyses of renal tissues indicated that pathological injury of the kidneys of 4E5-treated mice was decreased compared with that in the model control mice. The results of the present study demonstrated that inhibition of the B7-1/CD28 co-stimulatory signaling pathway with the 4E5 mAb may represent a promising strategy to decelerate the progression of LN that is induced by cGVHD with potential for use in the treatment of other autoimmune diseases.
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The E3 ubiquitin ligase RAD18 has been identified as an oncoprotein that exhibits prometastatic properties in various types of cancer; however, the role of RAD18 in cervical cancer (CC) remains unclear. In the present study, it was revealed that increased expression of RAD18 was associated with worse prognosis of patients with CC. Knockdown of endogenous RAD18 suppressed the motility and invasiveness of CC cells, as evaluated by Transwell assays. mRNA sequencing revealed that silencing RAD18 altered the expression profile of proinflammatory mediators, such as interleukin1ß (IL1ß). Furthermore, exogenous IL1ß treatment rescued RAD18mediated CC cell invasion. These findings indicated an underlying mechanism via which RAD18 promotes CC progression, suggesting that RAD18 may be a potential biomarker and therapeutic target for malignant CC.
Asunto(s)
Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Interleucina-1beta/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND AND OBJECTIVE: Dysregulation of DNA polymerase iota (Pol ι) in breast cancer might contribute to the accumulation of genomic mutations and promotes breast cancer progression. In this study we explored the clinical relevance and biological function of Pol ι in breast cancer. METHODS: qRT-PCR was used to determine the expression levels of Pol ι in 31 breast cancer tissues. Then the stable overexpression of Pol ι and knockdown of Pol ι breast cancer cell lines were constructed. Wound-healing assay and transwell assay were performed to evaluate cell migratory and invasiveness, respectively. Signaling pathway was analyzed by western blot. RESULTS: The expression levels of Pol ι is overexpressed in breast cancer tissues and significantly higher in breast cancer tissues with lymph node metastasis compared to those without lymph node metastasis. Elevated Pol ι expression promoted migratory and invasiveness of breast cancer cells. Signaling pathway analysis indicated EGFR-ERK cascade works as a mediator of Pol ι-induced EMT of breast cancer cells. CONCLUSIONS: These data demonstrate the underlying mechanism by which Pol ι promotes breast cancer progression, suggesting that Pol ι may be a potential therapeutic target against breast cancer.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , ADN Polimerasa Dirigida por ADN/metabolismo , Transición Epitelial-Mesenquimal , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Adulto , Anciano , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , ADN Polimerasa Dirigida por ADN/genética , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Metástasis Linfática , Persona de Mediana Edad , Estadificación de Neoplasias , Transducción de Señal , ADN Polimerasa iotaRESUMEN
Neoadjuvant chemoradiotherapy (nCRT) has been widely applied to improve the local control rate and survival rate in patients with locally advanced rectal cancer (LARC), yet only part of LARC patients would benefit from nCRT. Therefore, it is imperative to predict the therapeutic outcome of nCRT. Here, we showed that RAD18, an E3 ubiquitin-linked enzyme, played a fundamental role in predicting the response of LARC patients to nCRT. According to clinical data, patients with low RAD18 expression level in their pre-nCRT biopsies had a superior response to nCRT compared to those with high RAD18 expression. Inhibition of RAD18 expression in rectal cancer cells pronouncedly attenuated the proliferation and promoted apoptosis after exposing to irradiation or/and 5-fluorouracil (5-Fu). Downregulated RAD18 levels increased cell apoptosis by activating caspase-9-caspase-3-mediated apoptotic pathway, thus resulting in the enhancement of cell radiosensitivity and 5-Fu susceptibility. Furthermore, a xenograft nude mouse model showed that silencing RAD18 significantly slowed tumor growth after irradiation or/and 5-Fu in vivo. Collectively, these results implied that RAD18 could be a new biomarker to predict LARC patients who might benefit from nCRT and provide new strategies for clinical treatment of LARC.