Zeyi Liu

JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells

Epithelial-mesenchymal transition (EMT), a key step in the early stages of cancer metastasis, is orchestrated by several signaling pathways, including IL-6/JAK/STAT3 and TGF-β/Smad signaling. However, an association between the two signaling pathways during the EMT process is largely unknown. Here, we focused on lung cancer and demonstrated that TGF-β1 induced the phosphorylation of Smad3 (p-Smad3), upregulation of Snail, a fibroblast-like morphology, and downregulation of E-cadherin as well as upregulation of vimentin in lung cancer cell lines. SIS3 (an inhibitor of Smad3) suppressed TGF-β1-induced activation of Smad3, upregulation of Snail and the EMT process. Importantly, the JAK2/STAT3-specific inhibitor AG490 blocked Stat3 phosphorylation, resulting in attenuated levels of TGF-β1-induced p-Smad3, Snail, MMP2, and Smad-mediated PAI-1 promoter reporter gene activity in A549 and H1650 cells. Subsequently, AG490 inhibited TGF-β-induced cell migration and invasion. Moreover, exogenous IL-6 treatment stimulated Stat3 activation, enhanced TGF-β-induced expression of p-Smad3 and Snail, aggravated the EMT process, and increased lung cancer cell migration and invasion induced by TGF-β1. Our findings show that the JAK/STAT3 pathway is required for TGF-β-induced EMT and cancer cell migration and invasion via upregulation of the expression of p-Smad3 and Snail, and the IL-6/JAK/STAT3 and TGF-β/Smad signaling synergistically enhance EMT in lung carcinomas. The present study suggests a novel rationale for inhibiting cancer metastasis using anti-IL-6/JAK/STAT3 and anti-TGF-β/Smad therapeutic strategies.

CpG island methylator phenotype involving tumor suppressor genes located on chromosome 3p in non-small cell lung cancer

CpG island methylator phenotype (CIMP) involving methylation abnormalities of tumor suppressor gene (TSG) on short arm of chromosome 3 (chromosome 3p) has not been so far epigenetically elucidated in non-small cell lung cancer (NSCLC). Using methylation-specific PCR (MSP) method, we examined methylation profiles for eight TSGs harbored in chromosome 3p in 60 NSCLC tissues and 60 paired normal tissues as well as 11 normal blood samples. CIMP positive is referred to having four or more than four synchronously methylated genes per sample. Consequently, 59 of 60 (98.3%) NSCLC presented promoter methylation of at least one gene while only one malignant tumor showed no methylation of any of eight genes. The frequency of promoter methylation for eight genes explored ranged from 12% for hMLH1 to 67% for RASSF1A given that of VHL (none) was not considered. Interestingly, CIMP+ was found in 56.7% (34/60) of NSCLC, and in 6.7% (4/60) of paired normal tissues and 0% (0/11) of normal blood samples, respectively; CIMP- was present in 43.3% (26/60) of NSCLC, 93.3% (56/60) of paired normal tissues, and 100% (11/11) of normal blood samples, respectively. The data suggest that CIMP status was significantly associated with NSCLC, paired normal tissues and normal blood samples (P<0.001). In addition, there appeared to be a significant association between CIMP status and survival prognosis of NSCLC (P=0.0166). In the present study, for the first time, we shed light on the presence of chromosome 3p-specific CIMP, which might play an important role in tumorigenesis of NSCLC.

CpG Island Methylator Phenotype Involving Chromosome 3p Confers an Increased Risk of Non-small Cell Lung Cancer

Purpose: This study aims to explore the association of CpG island methylator phenotype (CIMP) involving tumor suppressor genes on short arm of chromosome 3 (3p) with increased risk of non-small cell lung cancer (NSCLC). Methods and Materials: In this study, four NSCLC cell lines were cultured, and peripheral blood mononuclear cell (PBMC) specimens from 80 patients with NSCLC and 80 matched controls were collected for 3p-involved CIMP (3pCIMP) analysis. 3pCIMP was referred to as having at least three synchronously methylated genes of 3p per sample. Methylation-specific polymerase chain reaction was performed to examine the methylation status of each gene. DNA demethylation of NSCLC cell lines was achieved through the treatment with 5-aza-deoxycytidine. Logistic regression was used to assess odds ratios and 95% confidence intervals, which were adjusted for gender, age, and smoking status. Results: Demethylation experiment showed that 3pCIMP status could play an important role in NSCLC cell proliferation. A total of 97.5% of PBMC specimens from NSCLC patients presented promoter methylation of any one of six genes (hOGG1, RAR-B, SEMA3B, RASSF1A, BLU, or FHIT) on 3p. Interestingly, 3pCIMP+ was found in 43.8% of NSCLC PBMC specimens and only in 6.3% of normal PBMC samples. The data suggest that 3pCIMP status is significantly associated with NSCLC and normal PBMC samples (p < 0.001). More importantly, the results show that 3pCIMP positive carriers have a 12.8-fold increased risk of NSCLC (adjusted odds ratio, 12.8; 95% confidence interval, 4.38–37.4, p < 0.001) in Chinese population. Conclusions: This is the first evidence of an association between PBMC 3pCIMP and risk for NSCLC.

TGFBR1 Haplotypes and Risk of Non–Small-Cell Lung Cancer

Transforming growth factor beta (TGF-beta) receptors are centrally involved in TGF-beta-mediated cell growth and differentiation and are frequently inactivated in non-small-cell lung cancer (NSCLC). Constitutively decreased type I TGF-beta receptor (TGFBR1) expression is emerging as a novel tumor-predisposing phenotype. The association of TGFBR1 haplotypes with risk for NSCLC has not yet been studied. We tested the hypothesis that single-nucleotide polymorphisms (SNP) and/or TGFBR1 haplotypes are associated with risk of NSCLC. We genotyped six TGFBR1 haplotype-tagging SNPs (htSNP) by PCR-RFLP assays and one htSNP by PCR-single-strand conformation polymorphism assay in two case-control studies. Case-control study 1 included 102 NSCLC patients and 104 healthy controls from Suzhou. Case-control study 2 included 131 patients with NSCLC and 133 healthy controls from Wuxi. Individuals included in both case-control studies were Han Chinese. Haplotypes were reconstructed according to the genotyping data and linkage disequilibrium status of these seven htSNPs. None of the htSNP was associated with NSCLC risk in either study. However, a four-marker CTGC haplotype was significantly more common among controls than among cases in both studies (P = 0.014 and P = 0.010, respectively), indicating that this haplotype is associated with decreased NSCLC risk {adjusted odds ratio [OR], 0.09 [95% confidence interval (95% CI), 0.01-0.61] and 0.11 [95% CI, 0.02-0.59], respectively}. Combined analysis of both studies shows a strong association of this four-marker haplotype with decreased NSCLC risk (adjusted OR, 0.11; 95% CI, 0.03-0.39). This is the first evidence of an association between a TGFBR1 haplotype and risk for NSCLC.

No Association between TGFBR1*6A and Lung Cancer

Introduction: TGFBR1*6A, a functionally polymorphic allele in the transforming growth factor &bgr; receptor 1 gene (TGFBR1), has been hypothesized to increase risk of various cancers. However, little has been documented about connection of this variant with lung cancer. Methods: In an attempt to explore whether the TGFBR1*6A is associated with lung cancer, we performed genotyping followed by sequencing in 252 patients with lung cancer and 250 healthy controls. Results: The frequency for the heterozygote 9A/6A is 13.9% in cases compared with 12.4% in controls, and the odds ratio is 1.14 (95% confidence interval: 0.68–1.91), which is not statistically significant (p = 0.62), suggesting that TGFBR1*6A could not be a cancer susceptibility factor for Chinese patients with lung cancer. Conclusions: We have no evidence to support the hypothesis that TGFBR1*6A is associated with lung cancer.

Infrequently methylated event at sites −362 to −142 in the promoter of TGFβR1 gene in non-small cell lung cancer

IntroductionMany malignant tumor cells, including non-small cell lung cancer (NSCLC) cells, are frequently resistant to transforming growth factor β (TGF-β)-mediated signal transduction. This refractory response might be due to reduced/loss expression of the TGF-β receptor 1 (TGFβR1). However, little was known about connection between inactivation of the TGFβR1 gene and the presence of CpG methylated promoter in NSCLC.Materials and methodsTo investigate whether, there is an epigenetic mechanism underlying inactivation of TGFβR1 in NSCLC, we performed the immunohistochemical and DNA methylation analyzes of TGFβR1 in tumor and the paired normal tissues from 35 resection specimens.ConclusionAs the first report, the present study demonstrated loss or reduction of TGFβR1 expression in 11 (31.4%) of 35 NSCLC tissues, suggesting that reduced TGFβR1 expression could contribute to the development of malignant phenotype of NSCLC, even if no aberrant DNA methylated site was found at sites −362 to −142 of TGFβR1 promoter region under investigation.

Variant SNPs at the microRNA complementary site in the B7‑H1 3'‑untranslated region increase the risk of non‑small cell lung cancer

Single nucleotide polymorphisms (SNPs) in microRNA-binding sites located in the 3′-untranslated region (UTR) of target genes can have an effect on the interaction of microRNA-mediated regulation, which results in changes in the expression levels of target genes ultimately associated with cancer risk and patient prognosis. However, the role of SNPs at the 3′-UTR of B7-H1 in the susceptibility of non-small cell lung cancer (NSCLC) remains to be fully elucidated. In the present study, SNPs with a minor allele frequency >10%, which were located at the microRNA complementary site in the PD-L1 3′-UTR, were selected via bioinformatic prediction using Ensembl and miRanda 2010. A total of three SNPs were selected, s2297136, rs4143815 and rs4742098, in the 3′-UTR of B7-H1. The rs2297136 and rs4742098 SNPs exhibited significant differences between 320 patients with NSCLC and 199 healthy individuals, respectively (P<0.001 and P=0.007). For the rs2297136 SNP, the AG genotype was significantly associated with evaluation of the risk of NSCLC, compared the AA genotype [odds ratio (OR)=2.287; 95% confidence interval (95% CI)=1.558–3.358]. Similarly, for the rs4742098 SNP, the AG genotype differed from the AA genotype on evaluation of the risk of NSCLC (OR=1.599; 95% CI=1.027–2.488). Dual-luciferase reporter assays showed that rs2297136 and rs4742098 in the B7-H1 3′-UTR contributed to the occurrence of NSCLC through disrupting the interaction between miR-296-5p, miR-138 and B7-H1 mRNA. These results indicated that genetic polymorphisms affecting the expression of B7-H1 modified cancer susceptibility.

INFREQUENTLY METHYLATED EVENT AT SITES −181 TO −9 WITHIN THE 5′ CpG ISLAND OF E-CADHERIN IN NON-SMALL CELL LUNG CANCER

Epigenetic silencing of E-cadherin via aberrant methylation has been investigated in various human tumors, whereas evidence for elucidating mechanism underlying reduction of E-cadherin mRNA remains unclear in non-small cell lung cancer (NSCLC). The authors previously found that reduction of E-cadherin mRNA or protein expression has been frequently observed in NSCLC. In this study, the authors explore the contribution of E-cadherin methylation to the development and progression of NSCLC. The authors directly performed the bisulfite DNA sequencing to examine CpG methylation within the 5′ CpG island of E-cadherin in 35 tumor and paired normal tissue specimens from patients with primary NSCLC. Then, the authors measured the level of E-cadherin mRNA by real-time quantitative polymerase chain reaction (PCR) analysis. Despite of reduction in E-cadherin mRNA by 65.7% (23/35) and presence of methylation by 28.6% (10/35) in tumors, the authors found no association of reduction of E-cadherin mRNA level with methylation of 19 sites from −181 to −9 bp located upstream from the translation start of E-cadherin in NSCLC. In conclusion, the authors provide no evidence for the presence of aberrant methylation sites of E-cadherin in tumors from patients with NSCLC, which can explain decrease of E-cadherin mRNA. Decrease in E-cadherin mRNA may be regulated by methylation-independent pathways in NSCLC.

Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

BackgroundTGF-β promotes tumor invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in human cancers. However, whether and how circRNAs contribute to TGF-β-induced EMT and metastasis in NSCLC remain vague. Here, we investigated the regulation and function of Circular RNA hsa_circ_0008305 (circPTK2) in TGF-β-induced EMT and tumor metastasis, as well as a link between circPTK2 and transcriptional intermediary factor 1 γ (TIF1γ) in NSCLC.MethodsCircular RNAs were determined by human circRNA Array analysis, real-time quantitative reverse transcriptase PCR and northern blot. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were employed to test the interaction between circPTK2 and miR-429/miR-200b-3p. Ectopic overexpression and siRNA-mediated knockdown of circPTK2, TGF-β-induced EMT, Transwell migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPTK2. Transcription and prognosis analyses were done in public databases.ResultsCircPTK2 and TIF1γ were significantly down-regulated in NSCLC cells undergoing EMT induced by TGF-β. CircPTK2 overexpression augmented TIF1γ expression, inhibited TGF-β-induced EMT and NSCLC cell invasion, whereas circPTK2 knockdown had the opposite effects. CircPTK2 functions as a sponge of miR-429/miR-200b-3p, and miR-429/miR-200b-3p promote TGF-β-induced EMT and NSCLC cell invasion by targeting TIF1γ. CircPTK2 overexpression inhibited the invasion-promoting phenotype of endogenous miR-429/miR-200b-3p in NSCLC cells in response to TGF-β. CircPTK2 overexpression significantly decreased the expression of Snail, an important downstream transcriptional activator of TGF-β/Smad signaling. In an in vivo experiment of metastasis, circPTK2 overexpression suppressed NSCLC cell metastasis. Moreover, circPTK2 expression was dramatically down-regulated and positively correlated with TIF1γ expression in human NSCLC tissues. Especially, circPTK2 was significantly lower in metastatic NSCLC tissues than non-metastatic counterparts.ConclusionOur findings show that circPTK2 (hsa_circ_0008305) inhibits TGF-β-induced EMT and metastasis by controlling TIF1γ in NSCLC, revealing a novel mechanism by which circRNA regulates TGF-β-induced EMT and tumor metastasis, and suggesting that circPTK2 overexpression could provide a therapeutic strategy for advanced NSCLC.

CPNE1 is a target of miR-335-5p and plays an important role in the pathogenesis of non-small cell lung cancer

BackgroundDespite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor. There is therefore a strong need to identify potential molecular targets for the treatment of NSCLC. In the present study, we investigated the function of CPNE1 in the regulation of cell growth, migration and invasion.MethodsQuantitative real-time PCR (qRT-PCR) was used to detect the expression of CPNE1 and miR-335-5p. Western blot and immunohistochemical assays were used to investigate the levels of CPNE1 and other proteins. Flow cytometry was used to determine cell cycle stage and apoptosis. CCK-8 and clonogenic assays were used to investigate cell proliferation. Wound healing, migration and invasion assays were used to investigate the motility of cells. A lung carcinoma xenograft mouse model was used to investigate the in vivo effects of CPNE1 overexpression.ResultsWe observed that knockdown of CPNE1 and increased expression of miR-335-5p inhibits cell proliferation and motility in NSCLC cells, and found that CPNE1 was a target of miR-335-5p. In addition, our data indicated that CPNE1 inhibition could improve the clinical effects of EGFR-tyrosine kinase inhibitors.ConclusionsThe present results indicate that CPNE1 may be a promising molecular target in the treatment of NSCLC.