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Abstract Background Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC). Methods Using multiplex-fluorescence in situ hybridization (M-FISH) and oligo array-based comparative hybridization (array-CGH), we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. Results M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134) of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM) (P = 0.004 and 0.022) and advanced stages (P = 0.004 and 0.039). Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026). Conclusions The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

Abstract Background The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

Small cell lung cancer (SCLC) is a highly aggressive disease and miRNAs may play an important role in modulating SCLC progression. We have previously screened 924 miRNAs and found that miR-886-3P was negatively associated with SCLC survival. In the current study, we further investigated the role of miR-886-3P mimic in regulating SCLC cell phenotypic alteration in vitro and xenograft tumor formation in vivo. We found that transfection of miR-886-3P mimic significantly inhibited SCLC cell proliferation, migration, and colony formation, and induced mesenchymal-epithelial transition (MET) by suppressing TGF-ß1 synthesis in vitro. Furthermore, intra-tumor injection of miR-886-3P mimic lead to necrosis and suppression of tumor invasion to the surrounding tissue in the subcutaneous xenograft tumor, and intra-vein injection of miR-886-3P mimic suppressed xenograft lung cancer growth in vivo. These findings suggested that miR-886-3P functions as a tumor suppressor in SCLC and thus, it might be a potential therapeutic molecule in the treatment of lung cancer. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. In order to identify useful biomarkers for accurately classifying prognostic risks for ESCC patients, we examined the expression of six proteins by immunohistochemistry (IHC) in 590 paraffin-embedded ESCC samples. The candidate proteins include p53, EGFR, c-KIT, TIMP1 and PI3K-p110α reported to be altered in ESCC tissues as well as another important component of PI3K, PI3K-p85α. Of the six proteins tested, p53, EGFR, c-KIT, TIMP1 and PI3K-p85α were detected with high expression in 43.0%, 36.6%, 55.9%, 70.7% and 57.1% of tumors, respectively. Significant associations were found between high expression of PI3K-p85α, EGFR and p53 and poor prognosis (P = 0.00111; 0.00001; 0.00426). Applying these three proteins as an IHC panel could divide patients into different subgroups (P<0.000001). Multivariate cox regression analysis indicated that the three-protein panel was an independent prognostic factor with very high statistical significance (HR = 2.090, 95% CI: 1.621–2.696, P = 0.00000001). The data suggest that the three-protein panel of PI3K-p85α, EGFR and p53 is an important candidate biomarker for the prognosis of patients with ESCC. [ABSTRACT FROM AUTHOR]

Aim:Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro.Methods:The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2.Results:In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive.Conclusion:Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect. [ABSTRACT FROM AUTHOR]

The obstacle of gene therapy is the shortage of efficient delivery system. The development of the gene delivery system with high transfection efficiency and low toxicity appears to be crucial. Recently, we reported that the dextran-graft-poly((2-dimethyl amino)ethyl methacrylate) (DPD) can be potentially used as efficient gene vector. Herein, DPD was systematically studied for its potential in tumor gene therapy. DPD was synthesized and characterized by agarose gel electrophoresis, particle size and zeta potential. The particle size and zeta potential of the DPD/enhanced green fluorescent protein (pEGFP-C1) plasmid complexes at various N/P ratios were 130–150 nm and about 40 mV, respectively. The results showed that DPD exhibit a higher transfection effect compared with Lipofectamine 2K (Lipo 2K), a commercialized vector. The possibility of DPD in gene therapy was evaluated using p53, a gene that has been wildly applied in the research of cancer gene therapy. DPD/pEGFP-C1–p53 complex was found to be able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis. Moreover, the tumor growth was found to be restrained when DPD/pEGFP-C1–p53 complex was used in a xenograft MCF7 tumor model in vivo. These observations indicated that DPD/pEGFP-C1–p53 complex may be considered to be an efficient delivery system for tumor gene therapy. [ABSTRACT FROM PUBLISHER]

There have been multiple lines of evidence suggesting that autophagy selectively targets signalling proteins and regulates cancer cell signalling in addition to bulk clearance of long-lived proteins and organelles. Protein degradation through autophagy requires receptor protein LC3B to sequester the substrates into the autophagosome. In the present study, we screened LC3B (light-chain 3B)-binding partners and identified autophagic substrates in cancer cells. With lung cancer NCI-H1975 and oesophageal cancer KYSE30 cell lines as models, we found that VPRBP (viral protein R-binding protein) was a novel LC3B-binding protein through GST (glutathione transferase)-LC3B pull-down combined with LC-MS/MS (liquid chromatography-tandem MS) methods. Co-immunoprecipitation assay showed that VPRBP-LC3/p62 were in the same protein complex as the two cell lines. Induction of autophagy led to a down-regulation of VPRPB, which could be rescued by the inhibition of autophagy degradation by BFA1 (bafilomycin A1) and by the disruption of autophagy through ATG5-knockdown. We also found that induction of autophagy promotes VPRBP-LC3/p62 interaction. Immunohistochemical examination of human NSCLC (non-small cell lung cancer) tissues showed that VPRBP was positively correlated with p62 and negatively correlated with LC3B. Moreover, p62 and VPRBP were associated with poor prognosis in lung ADC (adenocarcinoma) (p62, P=0.019; VPRBP, P=0.005). Patients with low expression of both p62 and VPRBP showed the best prognosis. [ABSTRACT FROM AUTHOR]

Aim:Filamin binding LIM protein 1, also known as migfilin, is a skeleton organization protein that binds to mitogen-inducible gene 2 at cell-extracellular matrix adhesions. The aim of this study was to investigate the role of migfilin in cisplatin-induced apoptosis in human glioma cells, to determine the functional domains of migfilin, and to elucidate the molecular mechanisms underlying the regulation of cisplatin-related chemosensitivity.Methods:The human glioma cell lines Hs683, H4, and U-87 MG were transfected with pEGFP-C2-migfilin to elevate the expression level of migfilin. RNA interference was used to reduce the expression of migfilin. To determine the functional domains of migfilin, U-87 MG cells were transfected with plasmids of migfilin deletion mutants. After treatment with cisplatin (40 μmol/L) for 24 h, the cell viability was assessed using the MTS assay, and the cell apoptotic was examined using the DAPI staining assay and TUNEL analysis. Expression levels of apoptosis-related proteins were detected by Western blot analysis.Results:Overexpression of migfilin significantly enhanced cisplatin-induced apoptosis in Hs683, H4, and U-87 MG cells, whereas downregulation of migfilin expression inhibited the chemosensitivity of these cell lines. The N-terminal region of migfilin alone was able to enhance the cisplatin-induced apoptosis. However, despite the existence of the N-terminal region, mutants of migfilin with any one of three LIM domains deleted led to a function loss. Furthermore, apoptotic proteins (PARP and caspase-3) and the anti-apoptotic protein Bcl-xL were modulated by the expression level of migfilin in combination with cisplatin.Conclusion:The LIM1-3 domains of migfilin play a key role in sensitizing glioma cells to cisplatin-induced apoptosis through regulation of apoptosis-related proteins. [ABSTRACT FROM AUTHOR]

Radiotherapy is one of the standard therapies for esophageal squamous cell carcinoma (ESCC), but the efficacy is far from desirable. Large scale genome sequencing reveals PI3Kα is frequently hyper-activated in ESCC. We found that ESCC cells harboring alterations in PI3K pathway were more resistant to radiation and combination of a clinical PI3Kα-selective inhibitor CYH33 and radiation synergistically inhibited cell proliferation in 14 ESCC cell lines. Radiation induced phosphorylation of FOXO1 and Akt, which sensitized ESCC cells to PI3Kα inhibitors. Both S1PR3 and DNA-PK contributed to radiation-induced Akt phosphorylation, which were revealed to be collectively dependent on PI3Kα. By contrast, constitutively active Akt abrogated the synergism between PI3Kα inhibitors and radiation. PI3Kα inhibition enhanced radiation-induced DNA damage, G2/M arrest and apoptosis. Combination of CYH33 and radiation significantly inhibited the growth of xenografts derived from ESCC patients, which was accompanied with abrogation of radiation-induced phosphorylation of Akt and filtration of M2-like macrophages. Taken together, combination of CYH33 and radiation possesses synergism in ESCC, which provides promising rationale to test this combinatorial regimen in ESCC patients. [ABSTRACT FROM AUTHOR]

Abstract: Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements. [Copyright &y& Elsevier]

Abstract: Purpose: To identify potential biomarkers for the prognosis of non-small cell lung cancer (NSCLC) patients by using bronchial brushing specimens. Methods: The expression of MCM7, Ki67 and EGFR was evaluated in 494 NSCLC tissues and 174 bronchial brushings using immunohistochemical and immunocytochemical techniques. Associations between protein expression and clinico-pathologic parameters were assessed, and the impact on overall survival (OS) was analyzed. Results: High expression of MCM7, Ki67 and EGFR was detected in 33.3%, 23.5% and 12.7% of tissues and in 52.4%, 52.7% and 20.6% of bronchial brushings, respectively. Expression of MCM7 and Ki67 was associated with squamous cell carcinoma (SCC) in both tissues and bronchial brushings (MCM7: P =0.0007, 0.00003; Ki67: P <0.00001, 0.00001). Overexpression of MCM7 in tumor tissues was detected more frequently in poorly differentiated tumors (P =0.0120) and non-bronchioloalveolar carcinomas (non-BACs) (P =0.0238). EGFR overexpression was observed in tissues of larger tumors (P =0.00004) and in bronchial brushings at later stage (P =0.0262). Kaplan–Meier curves indicated that patients with overexpression of MCM7 or Ki67 had a poorer OS compared to those with low expression for all stages (P <0.00001, 0.0233) and early-stages (P <0.00001, 0.0032). In particular, the patients with MCM7 overexpression in bronchial brushings had a poorer prognosis (P =0.0045). Multivariate Cox regression analysis showed that MCM7 was an independent prognostic indicator both in tissue samples and bronchial brushings. Conclusions: Our data suggest that MCM7 and Ki67 in tumor tissues may be potential markers of a poor prognosis for NSCLC patients. MCM7 in bronchial brushings also showed an independent prognostic value, which may be useful when biopsies are unavailable. [Copyright &y& Elsevier]

Abstract: Allelic loss on chromosome 3p occurs frequently in esophageal cancer. The human ribosomal protein L14 gene (RPL14) is located on chromosome 3p21.3. In the present study, we investigated alteration of RPL14 at both the genomic DNA and RNA levels in 129 Chinese esophageal squamous cell carcinomas (ESCC) and 17 dysplasia adjacent to tumor tissues by a combination of tissue microdissection, microsatellite analysis of the intragenic marker, reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. In the tested informative cases, loss of heterozygosity (LOH) of RPL14 was observed in 29 out of 68 (43%) tumors. Decreased expression of the gene was detected in 31 out of 49 (63%) carcinomas. No mutation was found in the remaining RPL14 allele of the tumors with LOH. We examined subsequently the allelic status of RPL14 in the dysplasia (preneoplastic lesions) between malignant tissues and histologically normal epithelia. Of 17 tested dysplasia in which the tumors showed LOH, eight (47%) displayed the same allelic loss as their corresponding tumors, seven (41%) exhibited microsatellite instability (MSI), and only two retained both the RPL14 alleles. The data suggest that alteration of RPL14 occurred frequently in ESCC and might be an earlier event in the tumorigenesis of the esophagus. Analysis to RPL14 gene may contribute to the early detection of ESCC as a potential molecular marker. [Copyright &y& Elsevier]

Metabolic activities are often accompanied by cell-cycle progression, yet known connections between these two processes remain limited. Here, we identified the isocitrate dehydrogenase 3β (IDH3β) as a novel substrate of anaphase-promoting complex/cyclosome (APC/C)-CDH1 and an important regulator of the cell cycle. In esophageal squamous cell carcinoma (ESCC), IDH3β was posttranslationally upregulated in late G 1 phase, and overexpression of IDH3β accelerated G 1 -S transition, contributing to the promotion of cell proliferation in vitro and in vivo . α-Ketoglutarate (α-KG), a crucial metabolite in tricarboxylic acid (TCA) cycle, was dependent on IDH3β level and partially accounted for IDH3β-mediated cell growth. IDH3β expression increased PFKFB3 protein levels and enhanced glucose uptake, and high expression of IDH3β correlated with poor survival in patients with ESCC, suggesting a potential application of IDH3β in prognosis. Overall, our results highlight a new molecular connection between cell-cycle regulation and the TCA cycle in ESCC. SIGNIFICANCE: These findings show that IDH3β is an APC/C-CDH1 substrate and is expressed in a cell-cycle-dependent manner, highlighting novel molecular cross-talk between the TCA cycle and cell cycle in cancer cells. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3281/F1.large.jpg., (©2019 American Association for Cancer Research.)

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort ( n = 241) for five groups of CSC + HCC tissues, i.e., EpCAM + , CD90 + , CD133 + , CD44 + , and CD24 + HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC + HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC + HCC from two independent cohorts ( n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC + HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC + HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis. SIGNIFICANCE: miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis., (©2018 American Association for Cancer Research.)

Competing Interests: Competing interests: We have read and understood BMJ policy on declaration of interests and declare that the article was funded by the World Health Organization (WHO Reference 2014/435380-0), the National Key Technology R&D Program of the Ministry of Science and Technology (2016YFC1305400), and the University of Michigan Health System-Peking University Health Science Center Joint Institute for Translational and Clinical Research (BMU20140479).

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.

Objectives: Anoctamin 1 (ANO1) has been found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous study. Herein we showed the clinical relevance of ANO1 alterations with ESCC and esophageal precancerous lesion progression., Results: ANO1 was detected in 38.1% (109/286) and 25.4% (77/303) of tumors in the two cohorts, but in none of morphologically normal operative margin tissues. ANO1 expression was significantly associated with a shorter overall survival (OS), especially in patients with moderately differentiated and stage IIA tumors. In 499 iodine-unstained biopsies from the endoscopic screening cohort in 2005-2007, all the 72 pathologically normal epithelial mucosa presented negative immunostaining, whereas ANO1 expression was observed in 3/11 tumors and 5/231 intraepithelial lesions. 7/8 ANO1-positive cases had developed unfavorable outcomes revealed by endoscopic follow-up in 2012. Analysis of another independent cohort of 148 intraepithelial lesions further confirmed the correlation between ANO1 expression and progression of precancerous lesions. 3/4 intraepithelial lesions with ANO1 expression had developed ESCC within 4-9 years after the initial endoscopic examination., Methods: Immunohistochemistry (IHC) was performed to examine ANO1 expression in surgical ESCC specimens and two independent cohorts of esophageal biopsies from endoscopic screening in high-incidence area of ESCC in northern China. Association between ANO1 expression, clinico-pathologic parameters, and the impact on overall survival was analyzed., Conclusions: Positive ANO1 is a promising biomarker to predict the unfavorable outcome for ESCC patients. More importantly, it can predict disease progression of precancerous lesions., Competing Interests: None of the authors of this manuscript have any conflicts of interest related to this work.

Purpose: We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1., Experimental Design: Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC)., Results: Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10(-5)). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025)., Conclusions: Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC., (©2014 American Association for Cancer Research.)

Purpose: Our aim was to identify frequent genomic aberrations in both esophageal squamous cell carcinoma (ESCC) and esophageal dysplasia and to discover important copy number-driving genes and microRNAs (miRNA) in ESCC., Experimental Design: We conducted array-based comparative genomic hybridization (array CGH) on 59 ESCC resection samples and 16 dysplasia biopsy samples. Expression of genes at 11q13.3 was analyzed by real-time PCR (RT-PCR) and immunohistochemistry (IHC). Integrated analysis was conducted to identify genes or miRNAs with copy number-expression correlations., Results: Array CGH identified 11 amplifications and eight homozygous deletions in ESCC. Integrated analysis of array CGH data with matched gene expression microarray data showed that 90 overexpressed genes and 24 underexpressed genes were consistent with DNA copy number changes, including 12 copy number-driving miRNAs. In esophageal dysplasia, six gains, four losses, 12 amplifications, and four homozygous deletions were detected. Amplifications of 7p11.2 and 11q13.2-11q13.3 (CCND1) and homozygous deletion at 9p21.3 (CDKN2A) were consistent genomic changes in both dysplasia and carcinoma. ANO1 at 11q13.3 was overexpressed at the mRNA and protein levels in tumors, and higher mRNA expression was correlated with the copy number increase. In particular, ANO1 expression was elevated in moderate dysplasia compared with normal esophageal epithelium. IHC revealed that ANO1 overexpression was positively correlated with lymph node metastasis and advanced clinical stage. Knockdown of ANO1 significantly inhibited the proliferation of KYSE30 and KYSE510 cells., Conclusion: Copy number aberrations in both esophageal dysplasia and ESCC may be useful as potential biomarkers for early detection. In addition, ANO1 may be a candidate target gene in esophageal tumorigenesis.

Unlabelled: Calreticulin (CRT) is a Ca(2+)-binding chaperone protein that alters cellular Ca(2+)-homeostasis in the endoplasmic reticulum (ER). Previously it was shown that CRT was overexpressed in esophageal squamous cell carcinoma (ESCC), and elevated CRT expression promoted the migration and invasion of ESCC cells. In the present study, the mechanisms underlying the role of CRT in esophageal carcinoma progression were investigated. Critically, depletion of CRT or protein-tyrosine phosphatase 1B (PTP1B) reduced ESCC cell migration and metastasis to the lung, whereas restoration of PTP1B protein levels rescued cell migration in CRT-silenced cells. Knockdown of CRT decreased PTP1B protein expression by reducing phosphorylation at the Y694 site of STAT5A, whereas knockdown of PTP1B reduced ERK1/2 phosphorylation at T204. Immunohistochemical analysis of CRT and PTP1B expression in ESCC patient tissues was strongly correlated. Importantly, PTP1B expression was associated with poor survival in patients with CRT overexpression. Overall, these data indicate a novel signaling pathway connecting CRT, STAT5A, PTP1B, and ERK1/2 in the regulation of ESCC cell migration., Implications: These findings suggest that PTP1B is a downstream effector of CRT signaling, promotes tumor progression, and can potentially be used as a new drug target for ESCC., (©2013 AACR.)

Purpose: To investigate the molecular mechanisms through which polo-like kinase-1 (PLK1) takes part in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells., Experimental Design: The role of PLK1 in cell anoikis resistance was examined by ectopic gene expression and siRNA-mediated knockdown. Glutathione S-transferase pull-down and co-immunoprecipitation assays were utilized to investigate PLK1-interacting proteins. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter gene assays were carried out to identify the transcription factors responsible for PLK1 expression during anoikis resistance., Results: We found that detachment of ESCC cells triggers the upregulation of PLK1. Elevated PLK1 expression contributes to protection against anoikis in cancer cells through the regulation of β-catenin expression. Moreover, we showed that, through direct binding to the PLK1 promoter, the NF-κB subunit RelA transcriptionally activates PLK1, which inhibits the ubiquitination and degradation of β-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/β-catenin expression. In addition, RelA gene amplification and protein overexpression was significantly correlated with PLK1 expression in ESCC tissues., Conclusions: Our findings suggest that upregulation of PLK1 triggered by cell detachment is regulated by RelA at the transcriptional level. PLK1 protects esophageal carcinoma cells from anoikis through modulation of β-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/β-catenin in anoikis resistance of ESCC cells.

Protein kinase Cι (PKCι) is an atypical PKC isoform and participates in multiple aspects of the transformed phenotype in human cancer cells. We previously reported that frequent amplification and overexpression of PKCι were correlated with lymph node metastasis in primary esophageal squamous cell carcinomas (ESCC). In the present study, short interfering RNA-mediated silencing of PKCι revealed that this enzyme was required for cell migration, invasion, and resistance to anoikis. In vivo experiments showed that PKCι suppression decreased tumor growth in esophageal cancer xenografts and lung metastases in nude mice. At the molecular level, knockdown of PKCι in suspended ESCC cells caused a decrease in S-phase kinase-associated protein 2 (SKP2) that had been reported to promote resistance to anoikis via the PI3K/AKT pathway. AKT phosphorylation was abolished after PKCι suppression, but AKT activation could be refreshed by PKCι upregulation, suggesting that PKCι enhanced cell resistance to anoikis via the PKCι-SKP2-PI3K/AKT pathway. Addition of the proteasome inhibitor MG132 prevented the decrease of SKP2 in PKCι silenced cells, and polyubiquitin-SKP2 was elevated after PKCι depletion, showing that PKCι might regulate the expression of SKP2 through the ubiquitin-proteasome pathway in suspended cells. Furthermore, overexpression of SKP2 in PKCι-downregulated cells restored cell resistance to anoikis. Most importantly, PKCι expression significantly correlated with SKP2 in 133 ESCC tissues (P = 0.031). Taken together, our data show that PKCι promotes tumorigenicity and metastasis of human esophageal cancer and that SKP2 is a candidate downstream effector of PKCι signaling in ESCC., (©2011 AACR.)

Background: Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown., Methods and Findings: Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis., Conclusion and Significance: Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.

Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis., Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8(+) T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice., Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating a life-threatening metastasis.

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G(1)-S transition, putatively through its control of several cell cycle regulator proteins including p27(kip1), p21(cip1), p57(kip2), p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway.

Background: The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression., Methods: Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5gamma2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay., Results: In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5gamma2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5gamma2 and SPARC., Conclusion: Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5gamma2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.

Gain of chromosome 11q13 is a common event in esophageal squamous cell carcinoma (ESCC). The cortactin gene (CTTN, also EMS1), located at 11q13, plays a pivotal role in coupling membrane dynamics to cortical actin assembly. This gene has been implicated in the motility of several types of cells. In the present study, we found that the amplification and overexpression of the CTTN gene was associated with lymph node metastasis in ESCC. Functional analysis by small interfering RNA-mediated silencing of CTTN revealed that in addition to the effect on cell migration, CTTN influenced cell invasiveness by anoikis resistance. In vivo assay showed that inhibition of CTTN expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, we showed for the first time that the protective role of CTTN in anoikis resistance was correlated with the activation of the phosphatidylinositol 3-kinase/Akt pathway. Overall, the data suggest that CTTN is an oncogene in the 11q13 amplicon and exerts functions on tumor metastasis in ESCC.

Aurora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma (ESCC). It has been demonstrated that cells overexpressing Aurora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Aurora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) in Aurora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A's effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.