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1.
Aging (Albany NY) ; 12(2): 1332-1365, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31962291

RESUMO

Aberrant DNA methylation leads to abnormal gene expression, making it a significant regulator in the progression of cancer and leading to the requirement for integration of gene expression with DNA methylation. Here, we identified 120 genes demonstrating an inverse correlation between DNA methylation and mRNA expression in esophageal squamous cell carcinoma (ESCC). Sixteen key genes, such as SIX4, CRABP2, and EHD3, were obtained by filtering 10 datasets and verified in paired ESCC samples by qRT-PCR. 5-Aza-dC as a DNA methyltransferase (DNMT) inhibitor could recover their expression and inhibit clonal growth of cancer cells in seven ESCC cell lines. Furthermore, 11 of the 16 genes were correlated with OS (overall survival) and DFS (disease-free survival) in 125 ESCC patients. ChIP-Seq data and WGBS data showed that DNA methylation and H3K27ac histone modification of these key genes displayed inverse trends, suggesting that there was collaboration between DNA methylation and histone modification in ESCC. Our findings illustrate that the integrated multi-omics data (transcriptome and epigenomics) can accurately obtain potential prognostic biomarkers, which may provide important insight for the effective treatment of cancers.

2.
Front Genet ; 10: 915, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31636653

RESUMO

Esophageal squamous cell carcinoma is a leading cause of cancer death. Mapping the transcriptional landscapes such as isoforms, fusion transcripts, as well as long noncoding RNAs have played a central role to understand the regulating mechanism during malignant processes. However, canonical methods such as short-read RNA-seq are difficult to define the entire polyadenylated RNA molecules. Here, we combined single-molecule real-time sequencing with RNA-seq to generate high-quality long reads and to survey the transcriptional program in esophageal squamous cells. Compared with the recent annotations of human transcriptome (Ensembl 38 release 91), single-molecule real-time data identified many unannotated transcripts, novel isoforms of known genes and an expanding repository of long intergenic noncoding RNAs (lincRNAs). By integrating with annotation of lincRNA catalog, 1,521 esophageal-cancer-specific lincRNAs were defined from single-molecule real-time reads. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these lincRNAs and their target genes are involved in a variety of cancer signaling pathways. Isoform usage analysis revealed the shifted alternative splicing patterns, which can be recaptured from clinical samples or supported by previous studies. Utilizing vigorous searching criteria, we also detected multiple transcript fusions, which are not documented in current gene fusion database or readily identified from RNA-seq reads. Two novel fusion transcripts were verified based on real-time PCR and Sanger sequencing. Overall, our long-read single-molecule sequencing largely expands current understanding of full-length transcriptome in esophageal cells and provides novel insights on the transcriptional diversity during oncogenic transformation.

3.
Cancer Res ; 79(19): 4951-4964, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31409639

RESUMO

Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family and its nonsecreted, catalytically dead spliced isoform L2Δ13, enhance cell migration and invasion, stimulate filopodia formation, modulate the expression of cytoskeletal genes, and promote tumor development and metastasis in vivo. We previously showed that LOXL2 reorganizes the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC) cells, however, the underlying molecular mechanisms were not identified. Here, using interactome analysis, we identified ezrin (EZR), fascin (FSCN1), heat shock protein beta-1 (HSPB1), and tropomodulin-3 (TMOD3) as actin-binding proteins that associate with cytoplasmic LOXL2, as well as with its L2Δ13 variant. High levels of LOXL2 and L2Δ13 and their cytoskeletal partners correlated with poor clinical outcome in patients with ESCC. To better understand the significance of these interactions, we focused on the interaction of LOXL2 with ezrin. Phosphorylation of ezrin at T567 was greatly reduced following depletion of LOXL2 and was enhanced following LOXL2/L2Δ13 reexpression. Furthermore, LOXL2 depletion inhibited the ability of ezrin to promote tumor progression. These results suggest that LOXL2-induced ezrin phosphorylation, which also requires PKCα, is critical for LOXL2-induced cytoskeletal reorganization that subsequently promotes tumor cell invasion and metastasis in ESCC. In summary, we have characterized a novel molecular mechanism that mediates, in part, the protumorigenic activity of LOXL2. These findings may enable the future development of therapeutic agents targeting cytoplasmic LOXL2. SIGNIFICANCE: LOXL2 and its spliced isoform L2Δ13 promote cytoskeletal reorganization and invasion of esophageal cancer cells by interacting with cytoplasmic actin-binding proteins such as ezrin.

4.
Int J Biochem Cell Biol ; 112: 79-87, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31082616

RESUMO

Ezrin plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC), providing a link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and protrusions. However, the mechanism by which ezrin is activated still remains unknown in ESCC. Here, we identify a novel interaction between ezrin and heat shock protein family B (small) member 1 (HSPB1) in ESCC cells by mass spectroscopy and co-immunoprecipitation. HSPB1 only interacts with inactive ezrin and binds to the α-helical coiled coil region of ezrin. Knockdown of HSPB1 resulted to the decline of phosphorylation at ezrin Thr567, markedly suppressing the ability of ezrin to bind to the actin cytoskeleton and migration of ESCC cells. Furthermore, neither the constitutively active phosphomimetic ezrin T567D, nor inactivated ezrin T567A could restore cell migration following HSPB1 knockdown. Low HSPB1 expression was associated with favorable overall survival of ESCC patients. Taken together, HSPB1, as an important partner, participates in the activation of ezrin and merits further evaluation as a novel therapeutic target against human ESCC.

5.
Cell Biochem Funct ; 36(8): 398-407, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30484863

RESUMO

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Panobinostat/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
Biomed Res Int ; 2018: 2049313, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30327774

RESUMO

Invasion and metastasis are critical pathological and mortal processes in esophageal squamous cell carcinoma (ESCC). Novel drugs, targeting the two cancer migration stages, will augment the treatment options for ESCC therapy and improve overall survival. A novel natural macrolide F806 specifically promotes apoptosis of various ESCC cells. However, whether F806 can inhibit metastasis of ESCC cells needs further evaluation. Here, our data showed that F806 inhibits dynamic F-actin assembly and then suppresses the migration of ESCC cells in vitro and their invasion and metastasis in vivo. The correlation between cancer migration and actin cytoskeleton assembly was consistent with the ability of F806 to prevent the aggregation of Paxillin, an essential protein for focal adhesion formation through binding to the ends of actin filaments. Furthermore, F806 downregulated the expression and activity of the Rho family proteins cell division cycle 42 (CDC42), RAC family small GTPase 1 (RAC1), and RAS homolog family member A (RHOA). Taken together, these results suggest that F806 can suppress cancer invasion and metastasis via interrupting the assembly of migration components involving F-actin.


Assuntos
Actinas/metabolismo , Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/genética , Animais , Linhagem Celular Tumoral , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas rho de Ligação ao GTP/genética
7.
Nat Commun ; 9(1): 3619, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190462

RESUMO

Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that master transcription factors (TFs) TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. However, functional consequence of their frequent co-localization at super-enhancers remains incompletely understood. Here, epigenomic profilings of different types of SCCs reveal that TP63 and SOX2 cooperatively and lineage-specifically regulate long non-coding RNA (lncRNA) CCAT1 expression, through activation of its super-enhancers and promoter. Silencing of CCAT1 substantially reduces cellular growth both in vitro and in vivo, phenotyping the effect of inhibiting either TP63 or SOX2. ChIRP analysis shows that CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR expression by binding to the super-enhancers of EGFR, thereby activating both MEK/ERK1/2 and PI3K/AKT signaling pathways. These results together identify a SCC-specific DNA/RNA/protein complex which activates TP63/SOX2-CCAT1-EGFR cascade and promotes SCC tumorigenesis, advancing our understanding of transcription dysregulation in cancer biology mediated by master TFs and super-enhancers.


Assuntos
Carcinoma de Células Escamosas/genética , Elementos Facilitadores Genéticos , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos NOD , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Nutr ; 148(6): 834-843, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29741716

RESUMO

Background: Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. Objective: The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. Methods: HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Results: Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Conclusion: Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.


Assuntos
Carcinogênese/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Riboflavina/metabolismo , Riboflavina/farmacologia , Animais , Ciclo Celular/fisiologia , Proliferação de Células , Células HEK293 , Humanos , Camundongos , Células NIH 3T3
9.
Cell Mol Life Sci ; 75(14): 2643-2661, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29428966

RESUMO

The human riboflavin transporter-3 (encoded by SLC52A3) plays a prominent role in riboflavin absorption. Interestingly, abnormal expression patterns of SLC52A3 in multiple types of human cancers have been recently noted. However, the molecular mechanisms underlying its dysregulation remain unclear. In this study, we find that SLC52A3 has two transcript variants that differ in the transcriptional start site, and encode different proteins: SLC52A3a and SLC52A3b. Importantly, aberrant expressions of SLC52A3 are associated with stepwise development of esophageal squamous cell carcinoma (ESCC) as well as the survival rates of ESCC patients. Functionally, SLC52A3a, but not SLC52A3b, strongly promotes the proliferation and colony formation of ESCC cells. Furthermore, SLC52A3 5'-flanking regions contain NF-κB p65/Rel-B-binding sites, which are crucial for mediating SLC52A3 transcriptional activity in ESCC cells. Chromatin immunoprecipitation and electrophoretic mobility shift assay reveal that p65/Rel-B bind to 5'-flanking regions of SLC52A3. Accordingly, NF-κB signaling upregulates SLC52A3 transcription upon TNFα stimulation. Taken together, these results elucidate the mechanisms underlying SLC52A3 overexpression in ESCC. More importantly, our findings identify SLC52A3 as both a predictive and prognostic biomarker for this deadly cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelB/metabolismo , Região 5'-Flanqueadora/genética , Adulto , Idoso , Sequência de Bases , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sobrevida
10.
Nucleic Acids Res ; 46(4): 1793-1809, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29253179

RESUMO

EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration and cancer. SMYD3, a histone H3-lysine 4 (H3-K4)-specific methyltransferase, regulates EZR gene transcription, but the molecular mechanisms of epigenetic regulation remain ill-defined. Here, we show that antisense lncRNA EZR-AS1 was positively correlated with EZR expression in both human esophageal squamous cell carcinoma (ESCC) tissues and cell lines. Both in vivo and in vitro studies revealed that EZR-AS1 promoted cell migration through up-regulation of EZR expression. Mechanistically, antisense lncRNA EZR-AS1 formed a complex with RNA polymerase II to activate the transcription of EZR. Moreover, EZR-AS1 could recruit SMYD3 to a binding site, present in a GC-rich region downstream of the EZR promoter, causing the binding of SMYD3 and local enrichment of H3K4me3. Finally, the interaction of EZR-AS1 with SMYD3 further enhanced EZR transcription and expression. Our findings suggest that antisense lncRNA EZR-AS1, as a member of an RNA polymerase complex and through enhanced SMYD3-dependent H3K4 methylation, plays an important role in enhancing transcription of the EZR gene to promote the mobility and invasiveness of human cancer cells.


Assuntos
Proteínas do Citoesqueleto/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto/biossíntese , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Masculino , Camundongos Nus , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima
11.
Oncol Rep ; 38(6): 3608-3618, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039594

RESUMO

Stathmin 1 (STMN1) is a microtubule-regulated protein that plays an important role in tumour cell proliferation and migration. Overexpression of STMN1 is associated with clinicopathological characteristics in many human cancers. The aim of the present study was to investigate STMN1 expression, its correlation with clinicopathological characteristics, and its exact biological function in oesophageal squamous cell carcinoma (ESCC). STMN1 levels were measured in the ESCC tissue specimens of 276 patients by immunohistochemistry (IHC) to assess the prognostic efficacy of STMN1. IHC showed that patients with overexpression of STMN1 had a poorer prognosis compared with those with low expression, both in regards to 5-year overall survival (OS; 21.2 vs. 53.7%, P<0.001) and disease-free survival (DFS; 20.6 vs. 50.9%, P<0.001). STMN1 overexpression was associated with lower cell differentiation in tumour grade (correlation coefficient: 0.127, P=0.037). In multivariate analysis, STMN1 expression was found to be an independent prognostic factor for both OS (P<0.001; 95% CI, 1.555-2.970) and DFS (P=0.001; 95% CI, 1.978-2.444). Compared with the control, STMN1 downregulation significantly decreased cell migration, invasion and proliferation, whereas these were increased by STMN1 upregulation. STMN1 expression was significantly associated with prognosis and tumour differentiation in ESCC, indicating that STMN1 expression is an independent prognostic factor for ESCC and could be a potential biomarker. Regulating the expression of STMN1 could influence tumour cell motility, invasion and proliferation.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Prognóstico , Estatmina/genética , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Intervalo Livre de Doença , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética
12.
J Mol Med (Berl) ; 95(12): 1355-1368, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28939985

RESUMO

L1 cell adhesion molecule (L1CAM) is highly expressed in various types of human cancers, displaying yet unknown molecular mechanisms underlying their oncogenic potential. Here, we found that L1CAM expression was significantly increased in esophageal squamous cell carcinoma (ESCC; n = 157) lesions compared with non-cancerous tissues. High tumorous L1CAM expression significantly correlated with reduced overall survival. Experimentally, L1CAM knockdown led to decreased cell growth, migration, and invasiveness in vitro, whereas overexpression of L1CAM showed the opposite effect. In nude mice, L1CAM depletion attenuated tumorigenesis and ability to penetrate the tissues surrounding ESCC cells. Gene set enrichment analysis (GSEA) and SubpathwayMiner analysis on gene expression profiles (microarray data on ESCC tissues, GSE53625; cDNA microarray data on L1CAM-knockdown ESCC cell line, GSE86268) suggested that L1CAM-co-expression genes were related to cell motility, cell proliferation, and regulation of actin cytoskeleton, validating the above experimental findings. Further mechanistical analysis showed that L1CAM upregulated the expression of the cytoskeletal protein ezrin via activating integrin ß1/MAPK/ERK/AP1 signaling and thus led to the malignant phenotypes of ESCC cells. Together, our findings suggest that L1CAM may be employed as a valuable prognosis marker and a therapeutic target for ESCC patients and that L1CAM promotes ESCC tumorigenicity by upregulating ezrin expression. KEY MESSAGES: L1CAM promotes growth and invasiveness of ESCC cells in vitro and in vivo. L1CAM upregulates the expression of ezrin by integrin α5ß1/MAPK/ERK/AP1 pathway. Ezrin is a key downstream effector in the L1CAM-promoted malignant phenotypes. High expression levels of both L1CAM and ezrin significantly correlated with reduced overall survival. Nuclear L1CAM is an independent prognosis marker for esophageal squamous cell carcinoma.


Assuntos
Carcinogênese/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Proteínas do Citoesqueleto/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transcrição Genética , Animais , Sequência de Bases , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteínas do Citoesqueleto/metabolismo , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Fenótipo , Prognóstico , Transdução de Sinais/genética , Frações Subcelulares/metabolismo , Regulação para Cima/genética
13.
Int J Biochem Cell Biol ; 90: 59-67, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28754317

RESUMO

LncRNAs play a vital role in alternative splicing of target genes. However, the mechanisms underlying lncRNAs involvement in splicing are poorly understood. In the present study, we identified a previously uncharacterized lncRNA, which is denoted as TPM1-AS, is reverse-transcribed from the fourth intronic region of the tropomyosin I (TPM1). In situ hybridization and RNA immunoprecipitation assays demonstrated that TPM1-AS was located in the nucleus and interacted with RNA-binding motif protein 4 (RBM4) in human esophageal cancer cells. TPM1-AS overexpression or RBM4 knockdown decreased endogenous exon 2a expression of TPM1, resulting in specifically down-regulation of TPM1variant V2 and V7 in human esophageal cancer cells. Mechanismly, the interaction of TPM1-AS with RBM4 hindered binding of RBM4 to TPM1 pre-mRNA and inhibited RBM4 to promote endogenous exon 2a inclusion of TPM1. Importantly, overexpression of TPM1-AS inhibited migration and filopodium formation, whereas TPM1variant V2 and V7 promoted these behaviors of human esophageal cancer cells. Taken together, the results suggest that a natural antisense TPM1-AS regulates the alternative splicing of TPM1 through an interaction with RBM4 and involves in TPM1-mediated filopodium formation and migration of cancer cells.


Assuntos
Processamento Alternativo , RNA Antissenso/genética , Proteínas de Ligação a RNA/metabolismo , Tropomiosina/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Esofágicas/patologia , Éxons/genética , Humanos , Ligação Proteica , Pseudópodes/metabolismo
14.
Hum Pathol ; 66: 115-125, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28603065

RESUMO

Our previous studies have highlighted the importance of ezrin in esophageal squamous cell carcinoma (ESCC). Here our objective was to explore the clinical significance of ezrin-interacting proteins, which would provide a theoretical basis for understanding the function of ezrin and potential therapeutic targets for ESCC. We used affinity purification and mass spectrometry to identify PDIA3, CNPY2, and STMN1 as potential ezrin-interacting proteins. Confocal microscopy and coimmunoprecipitation analysis further confirmed the colocalization and interaction of ezrin with PDIA3, CNPY2, and STMN1. Tissue microarray data of ESCC samples (n=263) showed that the 5-year overall survival (OS) and disease-free survival (DFS) were significantly lower for the CNPY2 (OS, P=.003; DFS, P=.011) and STMN1 (OS, P=.010; DFS, P=.002) high-expression groups compared with the low-expression groups. By contrast, overexpression of PDIA3 was significantly correlated with favorable survival (OS, P<.001; DFS, P=.001). Cox regression demonstrated the prognostic value of PDIA3, CNPY2, and STMN1 in ESCC. Furthermore, decision tree analysis revealed that the resulting classifier of both ezrin and its interacting proteins could be used to better predict OS and DFS of patients with ESCC. In conclusion, a signature of ezrin-interacting proteins accurately predicts ESCC patient survival or tumor recurrence.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Técnicas de Apoio para a Decisão , Árvores de Decisões , Neoplasias Esofágicas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Estatmina/metabolismo , Biópsia , Western Blotting , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas do Esôfago , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Estimativa de Kaplan-Meier , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fosforilação , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Ligação Proteica , Estudos Retrospectivos , Fatores de Tempo , Análise Serial de Tecidos
15.
Cancer Med ; 6(7): 1707-1719, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28556501

RESUMO

Current staging is inadequate for predicting clinical outcome of esophageal squamous cell carcinoma (ESCC). Aberrant expression of LOXL2 and actin-related proteins plays important roles in ESCC. Here, we aimed to develop a novel molecular signature that exceeds the power of the current staging system in predicting ESCC prognosis. We found that LOXL2 colocalized with filamentous actin in ESCC cells, and gene set enrichment analysis (GSEA) showed that LOXL2 is related to the actin cytoskeleton. An ESCC-specific protein-protein interaction (PPI) network involving LOXL2 and actin-related proteins was generated based on genome-wide RNA-seq in 15 paired ESCC samples, and the prognostic significance of 14 core genes was analyzed. Using risk score calculation, a three-gene signature comprising LOXL2, CDH1, and FN1 was derived from transcriptome data of patients with ESCC. The high-risk three-gene signature strongly correlated with poor prognosis in a training cohort of 60 patients (P = 0.003). In mRNA and protein levels, the prognostic values of this signature were further validated in 243 patients from a testing cohort (P = 0.001) and two validation cohorts (P = 0.021, P = 0.007). Furthermore, Cox regression analysis revealed that the signature was an independent prognostic factor. Compared with using the signature or TNM stage alone, the combined model significantly enhanced the accuracy in evaluating ESCC prognosis. In conclusion, our data reveal that the tumor-promoting role of LOXL2 in ESCC is mediated by perturbing the architecture of actin cytoskeleton through its PPIs. We generated a novel three-gene signature (PPI interfaces) that robustly predicts poor clinical outcome in ESCC patients.


Assuntos
Actinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Mapas de Interação de Proteínas , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Biomarcadores Tumorais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Biologia Computacional/métodos , Citoesqueleto , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Estadiamento de Neoplasias , Prognóstico , Mapeamento de Interação de Proteínas , Curva ROC , Reprodutibilidade dos Testes
16.
Int J Biochem Cell Biol ; 88: 162-171, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28504189

RESUMO

BACKGROUND: Ezrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear. METHODS: Distribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis. RESULTS: Ezrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo. CONCLUSION: Ezrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.


Assuntos
Carcinoma de Células Escamosas/patologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Neoplasias Esofágicas/patologia , Pseudópodes/patologia , Serina/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/genética , Carcinoma de Células Escamosas do Esôfago , Humanos , Metástase Linfática , Mutação , Invasividade Neoplásica , Fosforilação , Transporte Proteico
17.
Amino Acids ; 49(5): 943-955, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28251354

RESUMO

Filopodia are dynamic membrane extensions generated by F-actin bundling and are involved in cancer cell migration, invasion and metastasis. Fascin is the crucial actin-bundling protein in filopodia, with phosphorylation at fascin serine 39 being well characterized to regulate fascin-mediated actin bundling in filopodia. However, increasing evidence indicates that fascin is phosphorylated at a number of sites. Whether phosphorylation at other sites also regulates fascin function is unknown. In this study, we show that four potential phosphorylation sites in fascin, specifically tyrosine 23, serine 38, serine 39 and serine 274, regulate cell behavior and filopodia formation in esophageal squamous cancer cells. Expression of non-phosphorylatable mutations at each of the four sites promoted anchorage-independent growth, cell motility and filopodia formation, whereas phosphomimetic mutations at each of these sites inhibited these cell behaviors, implying that fascin function in esophageal squamous cancer is regulated by fascin phosphorylation at multiple sites. Furthermore, phosphorylation at S38 and S39 cooperatively regulated cell behavior and filopodia formation, with dual dephosphorylation at both S38 and S39 residues maximally enhancing cell proliferation, migration and filopodia formation, and phosphorylation at any of the two phosphorylatable sites resulting in reduced enhancement. Taken together, our results reveal that phosphorylation at fascin amino acids Y23, S38, S39 and S274, in combination, downregulates the extent of anchorage-independent growth, cell migration and filopodia formation in esophageal squamous cancer cells.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Processamento de Proteína Pós-Traducional , Pseudópodes/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/patologia , Esôfago/metabolismo , Esôfago/patologia , Humanos , Proteínas dos Microfilamentos/genética , Mutação , Fosforilação , Pseudópodes/patologia , Pseudópodes/ultraestrutura
18.
Clin Proteomics ; 14: 6, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28184180

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a major head and neck cancer with high occurrence in Southeast Asia and southern China. We aimed to identify autoantibodies that may contribute to early detection of NPC. METHODS: We used serological proteome analysis to identify candidate autoantibodies against tumor-associated antigens. Levels of autoantibodies and Epstein-Barr virus capsid antigen-IgA (VCA-IgA) were measured by ELISA in 129 patients with NPC and 100 normal controls. We employed receiver operating characteristics to calculate diagnostic accuracy. RESULTS: Sera from patients with NPC yielded multiple spots, two of which were identified as PRDX2 and PRDX3. Levels of serum autoantibodies against PRDX2 and PRDX3 were significantly higher for patients with NPC than for normal controls (P < 0.01), respectively. Combined detection of autoantibodies against PRDX2 and PRDX3 and VCA-IgA provided a high diagnostic accuracy in NPC (an area under the curve (AUC) of 0.811 (95% CI 0.753-0.869), 66.7% sensitivity, and 95.0% specificity). This combination maintained diagnostic performance for early NPC with AUC value of 0.754 (95% CI 0.652-0.857), 50.0% sensitivity, and 95.0% specificity. CONCLUSIONS: This study reports autoantibodies against PRDX2 and PRDX3 identified by a proteomic approach in sera from NPC patients. Our findings suggest that autoantibodies against PRDX2 and PRDX3 may serve as supplementary biomarkers to VCA-IgA for the screening and diagnosis of NPC.

19.
Mol Med Rep ; 14(5): 4802-4810, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27748861

RESUMO

The key molecular events that contribute to tumorigenesis are incompletely understood. The aim of the present study was to characterize and compare the biological phenotypes of three human telomerase reverse transcriptase (hTERT) and/or human papillomavirus 16 E6 and E7­immortalized esophageal epithelial cell lines, NE2­hTERT (NE2), NE3­E6E7­hTERT (NE3) and NEcA6­E6E7­hTERT (NEcA6). The present study used soft­agar colony formation assays, tumorigenicity assays in nude mice, and cell proliferation, adhesion and migration assays to identify the biological characteristics of NE2, NE3 and NEcA6 cells. NE2 and NE3 cells exhibited characteristics of benign cells, such as the inability to grow in soft agar or form tumors in nude mice. By contrast, NEcA6 cells had undergone transformation, as demonstrated by the ability to grow in soft agar and form tumors in nude mice. In addition, NEcA6 cells exhibited increased migration and adhesion capabilities when compared with NE2 and NE3 cells. In order to identify mechanism(s) that may contribute to the altered biological phenotypes exhibited by these cells, the expression of three proteins involved in modulating cell migration [fascin, ezrin/radixin/moesin family proteins and phosphorylated­focal adhesion kinase (Tyr 397)], as well as the expression status and subcellular localization of three key focal adhesions components (paxillin, talin and kindlin­2) were examined. Paxillin, talin and kindlin­2 were localized to adhesive sites that connect F­actin with the extracellular matrix in transformed NEcA6 cells, but were distributed in a diffuse manner in NE2 and NE3 cells. Knockdown of kindlin­2 in NE3 and NEcA6 cells decreased cell adhesion, however, NEcA6 cells demonstrated a greater sensitivity to knockdown of kindlin­2. No significant differences were observed in the protein expression levels of fascin, exrin/radixin/moesin and p­FAK in the three cell lines. In conclusion, these results demonstrate that these three focal adhesion components, particularly kindlin­2, may contribute to the carcinogenesis of esophageal squamous cells.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Animais , Biomarcadores , Adesão Celular , Linhagem Celular Transformada , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Esôfago/metabolismo , Esôfago/patologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , RNA Interferente Pequeno/genética
20.
Int J Biochem Cell Biol ; 75: 85-98, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27063404

RESUMO

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family, which plays an important role in extracellular matrix protein biosynthesis and tumor progression. In the present study, we identified a novel splice variant, LOXL2Δ72, which encodes a peptide having the same N- and C-termini as wild-type LOXL2 (LOXL2WT), but lacks 72 nucleotides encoding 24 amino acids. LOXL2Δ72 had dramatically reduced enzymatic activity, and was no longer secreted. However, LOXL2Δ72 promoted greater cell migration and invasion than LOXL2WT. Furthermore, a dual luciferase reporter assay indicated that LOXL2Δ72 activates distinct signal transduction pathways compared to LOXL2WT, consistent with cDNA microarray data showing different expression levels of cell migration- and invasion-related genes induced following over-expression of each LOXL2 isoform. In particular, LOXL2Δ72 distinctly promoted esophageal squamous cell carcinoma (ESCC) cell migration via up-regulating the C-C motif chemokine ligand 28 (CCL28). Our results suggest that the new LOXL2 splice variant contributes to tumor progression by novel molecular mechanisms different from LOXL2WT.


Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Carcinoma de Células Escamosas/patologia , Movimento Celular/genética , Neoplasias Esofágicas/patologia , Deleção de Sequência , Sequência de Bases , Linhagem Celular Tumoral , Citoplasma/metabolismo , Carcinoma de Células Escamosas do Esôfago , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Invasividade Neoplásica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico
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