Hong-Tao Zhang

Germline Allele-specific Expression of TGFBR1 Confers an Increased Risk of Colorectal Cancer

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor–β (TGF-β) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-β signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.

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.

MiR-142-3p represses TGF-β-induced growth inhibition through repression of TGFβR1 in non-small cell lung cancer

TGFβR1 plays an important role in TGF‐β signaling transduction and serves as a tumor suppressor. Our previous studies show that reduced expression of TGFβR1 is common in non‐small cell lung cancer (NSCLC) and TGFβR1 variants confer risk of NSCLC. However, the epigenetic mechanisms underlying the role of TGFβR1 in NSCLC carcinogenesis are still elusive. We investigated the function and regulation of TGF‐β signaling‐based miRNAs in NSCLC. Computational algorithms predicted that the 3′‐untranslated region (3′‐UTR) of TGFβR1 is a target of miR‐142‐3p. Here a luciferase reporter assay confirmed that miR‐142‐3p can directly bind to 3′‐UTR of TGFβR1. Overexpression of miR‐142‐3p in NSCLC A549 cells suppressed expression of TGFβR1 mRNA and protein, while knockdown of endogenous miR‐142‐3p led to increased expression of TGFβR1. On TGF‐β1 stimulation, stable overexpression of miR‐142‐3p attenuated phosphorylation of SMAD3, an indispensable downstream effector in canonical TGF‐β/Smad signaling, via repression of TGFβR1 in A549 cells. Furthermore, miR‐142‐3p‐mediated down‐regulation of TGFβR1 weakened TGF‐β‐induced growth inhibition effect, and this effect was reversed by stable knockdown of endogenous miR‐142‐3p in A549 cells. In NSCLC tissues, miR‐142‐3p expression was increased and inversely correlated with TGFβR1 expression. These data demonstrate that miR‐142‐3p influences the proliferation of NSCLC cells through repression of TGFβR1.—Lei, Z., Xu, G., Wang, L., Yang, H., Liu, X., Zhao, J., Zhang, H.‐T. MiR‐142‐3p represses TGF‐β‐induced growth inhibition through repression of TGFβR1 in non‐small cell lung cancer. FASEB J. 28, 2696–2704 (2014). www.fasebj.org

TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer

OBJECTIVES Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. MATERIALS AND METHODS Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. RESULTS Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. CONCLUSIONS We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

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.

Ski prevents TGF-β-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-β (TGF-β) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-β signaling, which interacts with SMADs to repress TGF-β signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-β-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-β stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-β1 stimulation, we did not observe any effect of Ski on TGF-β-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-β-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

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.

Melatonin inhibits proliferation and invasion via repression of miRNA-155 in glioma cells

Melatonin, an indolamine mostly synthesized in the pineal gland, exerts the anti-cancer effect by various mechanisms in glioma cells. Our previous study showed that miR-155 promoted glioma cell proliferation and invasion. However, the question of whether melatonin may inhibit glioma by regulating miRNAs has not yet been addressed. In this study, we found that melatonin (100μM, 1μM and 1nM) significantly inhibited the expression of miR-155 in human glioma cell lines U87, U373 and U251. Especially, the lowest expression of miR-155 was detected in 1μM melatonin-treated glioma cells. Melatonin (1μM) inhibits cell proliferation of U87 by promoting cell apoptosis. Nevertheless, melatonin had no effect on cell cycle distribution of U87 cells. Moreover, U87 cells treated with 1μM melatonin presented significantly lower migration and invasion ability when compared with control cells. Importantly, melatonin inhibited c-MYB expression, and c-MYB knockdown reduced miR-155 expression and migration and invasion in U87 cells. Taken together, for the first time, our findings show that melatonin inhibits miR-155 expression and thereby represses glioma cell proliferation, migration and invasion, and suggest that melatonin may downregulate the expression of miR-155 via repression of c-MYB. This will provide a theoretical basis for revealing the anti-glioma mechanisms of melatonin.

RNF111/Arkadia is regulated by DNA methylation and affects TGF-β/Smad signaling associated invasion in NSCLC cells

OBJECTIVES RNF111/Arkadia is a critical regulator of TGF-β signaling, being required for SMAD3-mediated responses such as TGF-β-induced repression of E-cadherin. Previous studies show that mutations in RNF111 in human cancers are rare and RNF111 promotes lung tumor metastasis. However, the epigenetic mechanisms underlying the role of RNF111 in non-small cell lung cancer (NSCLC) metastasis remain unknown. Here, we mainly focused on low- (95C) and high-metastatic (95D) NSCLC cell lines, which share a similar genetic background, and investigated the methylation-based regulation of RNF111 expression. MATERIALS AND METHODS Clonal bisulfite sequencing, real-time qRT-PCR, western blot analysis, luciferase reporter assays, RNA interference, chromatin immunoprecipitation (ChIP) assay and transwell migration and invasion assays were performed on human NSCLC cell lines 95C and 95D. RESULTS RNF111 was high-expressed in 95D cells, which showed low-level methylation at -459CpG site in RNF111 promoter. The opposite results were obtained in 95C cells. Cell-based and biochemical assays revealed that -459CpG methylation can inhibit RNF111 transcriptional expression by interfering with the recruitment of Sp1 to RNF111 promoter. On TGF-β stimulation, siRNA-mediated RNF111 knockdown inhibited TGF-β/Smad signaling activity and Snail (an inducer of metastasis) expression, and enhanced E-cadherin (an epithelial-to-mesenchymal transition marker) expression in 95C and 95D cells. Furthermore, demethylation-induced upregulation of RNF111 enhanced phosphorylation of SMAD3 and Snail expression, and repressed E-cadherin expression in 95C cells expressing low RNF111. CONCLUSIONS Our results suggest that -459CpG methylation in Sp1-binding site of RNF111 promoter transcriptionally decreases RNF111 expression, which inhibits TGF-β/Smad signaling associated invasion in NSCLC cells.