Daochuan Li

A novel GSK-3 beta–C/EBP alpha–miR-122–insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma†

miR‐122 is a highly abundant, hepatocyte‐specific microRNA. The biomedical significance and regulatory mechanisms of miR‐122 remain obscure. We explored the role of miR‐122 in tumorigenesis in the context of gene regulatory network. The miR‐122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR‐122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B1 (AFB1)‐transformed cells. We characterized the −5.3 to −4.8 kb region upstream of miR‐122 precursor as miR‐122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer‐binding protein alpha (C/EBPα) binding sites C/EBPα knockdown significantly reduced miR‐122 promoter activity and endogenous miR‐122 expression; and C/EBPα directly interacted with the miR‐122 promoter in vitro and in vivo. These data suggest that C/EBPα is a transactivator for miR‐122 transcription. We further demonstrated that miR‐122 suppressed insulin‐like growth factor 1 receptor (IGF‐1R) translation and sustained glycogen synthase kinase‐3 beta (GSK‐3β) activity. The activated GSK‐3β not only repressed cell proliferation, but also activated C/EBPα, which maintained miR‐122 levels and thereby enforced IGF‐1R suppression. Interestingly, down‐regulation of miR‐122 and C/EBPα, and up‐regulation of IGF‐1R were frequently observed in HCC tissues, and decreased miR‐122 levels were associated with worse survival of HCC patients. Moreover, AFB1 exposure resulted in decreased activity in GSK‐3β, C/EBPα, and miR‐122 and increased levels of IGF‐1R, whereas restoration of miR‐122 suppressed the tumorigenicity of HCC and AFB1‐transformed cells. Conclusion: We have identified a novel GSK‐3β–C/EBPα–miR‐122–IGF‐1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR‐122s function and the mechanisms of hepatocarcinogenesis. (Hepatology 2010;52:1702‐1712)

Aberrant Expression of miR-638 Contributes to Benzo(a)pyrene-Induced Human Cell Transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G0–G1 phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST–PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.

α4 is highly expressed in carcinogen-transformed human cells and primary human cancers.

A regulator of the protein phosphatase 2A (PP2A), α4, has been implicated in a variety of functions that regulate many cellular processes. To explore the role of α4 in human cell transformation and tumorigenesis, we show that α4 is highly expressed in human cells transformed by chemical carcinogens including benzo(a)pyrene, aflatoxin B1, N-methyl-N′-nitro-N-nitrosoguanidine, nickel sulfate and in several hepatic and lung cancer cell lines. In addition, overexpression of α4 was detected in 87.5% (74/80) of primary hepatocellular carcinomas, 84.0% (21/25) of primary lung cancers and 81.8% (9/11) of primary breast cancers, indicating that α4 is ubiquitously highly expressed in human cancer. Functional studies revealed that elevated α4 expression results in an increase in cell proliferation, promotion of cell survival and decreased PP2A-attributable activity. Importantly, ectopic expression of α4 permits non-transformed human embryonic kidney cells (HEKTER) and L02R cells to form tumors in immunodeficient mice. Furthermore, we show that the highly expressed α4 in transformed cells or human tumors is not regulated by DNA hypomethylation. A microRNA, miR-34b, that suppresses the expression of α4 through specific binding to the 3′-untranslated region of α4 is downregulated in transformed or human lung tumors. Taken together, these observations identify that α4 possesses an oncogenic function. Reduction of PP2A activity due to an enhanced α4–PP2A interaction contributes directly to chemical carcinogen-induced tumorigenesis.

Development of human cell models for assessing the carcinogenic potential of chemicals.

To develop human cell models for assessing the carcinogenic potential of chemicals, we established transgenic human cell lines and tested the sensitivity of known carcinogens using a cell transformation assay. A retroviral vector encoding an oncogenic allele of H-Ras (HBER) or c-Myc (HBEM) was introduced into human bronchial epithelial cells (HBE) immortalized by SV40 large T (LT) antigen, leading to increased cell proliferation but failing to confer a transformed phenotype characterized by anchorage-independent cell growth and tumor formation of immunodeficient mice. When these pre-transformed cells were treated with nickel sulfate (NiSO4), we found that it shortened the latency of malignant transformation at least by 19 wk in HBER cells or 16 wk in HBEM cells compared to vector control cells. Similarly, the latency of cell transformation was shorter by 15 wk in HBER cells or 9 wk in HBEM cells when cells were treated with benzo(a)pyrenediol epoxide (BPDE). HBER cells appeared to be more sensitive to TPA, NiSO4 or BPDE-induced cell transformation compared to human embryonic kidney cells expressing H-Ras (HEKR), implying that cell-type specificity is one of important factors determining the effectiveness of the assay. Using AFB1 and BaP as the representative pro-carcinogens, we also compared the efficiency of three different metabolic conditions in mediating cell transformation. Low dose chemical induction seems to be a prospective system used for metabolic activation of pro-carcinogens. Our findings provided direct evidence that a genetically modified human cell transformation model can be applied to the assessment of potent carcinogens.

CpG Site–Specific Hypermethylation of p16INK4α in Peripheral Blood Lymphocytes of PAH-Exposed Workers

Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAH-associated biologic consequence. Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16INK4α methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results: Compared with the control group, PAH-exposed workers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 μg/L), p16INK4α methylation (7.95% vs. 1.14% for 22 “hot” CpG sites), and CBMN (7.28% vs. 2.92%) in PBLs. p16INK4α hypermethylation in PAH-exposed workers exhibited CpG site specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16INK4α gene. The degree of p16INK4α methylation was associated with the levels of DNA damage and internal exposure. Impact: p16INK4α hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 21(1); 182–90. ©2011 AACR.

PP2A-AMPKα-HSF1 axis regulates the metal-inducible expression of HSPs and ROS clearance.

Metals such as cadmium and arsenic are ubiquitous toxicants that cause a variety of adverse health effects. Heat shock proteins (HSPs) response to metal-induced stress and protect cells from further damage. However, the intracellular signalling pathways responsible for activation of HSPs expression are not fully understood. Here, we demonstrate that protein phosphatase 2A (PP2A) regulates expression of HSP70 and HSP27 via dephosphorylation of an AMP-activated protein kinase α subunit (AMPKα) at Thr172. Dephosphorylated AMPKα phosphorylates heat shock factor 1 (HSF1) at Ser303, leading to significant transcriptional suppression of HSP70 and HSP27 in CdCl2- or NaAsO2-treated cells. Suppression of PP2A regulatory B56δ subunit resulted in the sustained phosphorylation of AMPKα upon CdCl2 treatment, subsequent reduction in expression of HSP70 and HSP27, and thereby dramatic reduction of reactive oxygen species (ROS) clearance. We further revealed that PP2A B56δ physically interacted with AMPKα, providing evidence that PP2A B56δ-AMPKα-HSF1 signalling pathway participated in regulating the inducible expression of HSPs and ROS clearance. Taken together, we identified a novel PP2A-dependent signalling pathway involved in regulation of HSPs expression in response to metal stress.

Specific histone modification responds to arsenic-induced oxidative stress

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P<0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showed that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β=0.16; P=0.042, H3K18ac: β=-0.24; P=0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO2 treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage.

Heavy Metal-Induced Metallothionein Expression is Regulated by Specific Protein Phosphatase 2A Complexes

Background: The molecular mechanism and key signaling pathways underlying MT expression in response to metal stress remains elusive. Results: Upon metal stress, PP2A PR110 complexes bind to and dephosphorylate MTF-1 at Thr-254, leading to the transactivation of MTs. Conclusion: Specific PP2A complexes regulate metal-induced MTs expression. Significance: Delineate a novel pathway regulating metal-induced cytotoxicity and clarify the role of PP2A in cellular stress response. Induction of metallothionein (MT) expression is involved in metal homeostasis and detoxification. To identify the key pathways that regulate metal-induced cytotoxicity, we investigate how phosphorylated metal-responsive transcription factor-1 (MTF-1) contributed to induction of MT expression. Immortal human embryonic kidney cells (HEK cells) were treated with seven kinds of metals including cadmium chloride (CdCl2), zinc sulfate (ZnSO4), copper sulfate(CuSO4), lead acetate (PbAc), nickel sulfate (NiSO4), sodium arsenite (NaAsO2), and potassium bichromate (K2Cr2O7). The MT expression was induced in a dose-response and time-dependent manner upon various metal treatments. A cycle of phosphorylation and dephosphorylation was required for translocation of MTF-1 from cytoplasm to nucleus, leading to the up-regulation of MTs expression. Protein phosphatase 2A (PP2A) participated in regulating MT expression through dephosphorylation of MTF-1. A loss-of-function screen revealed that the specific PP2A complexes containing PR110 were involved in metal-induced MT expression. Suppression of PP2A PR110 in HEK cells resulted in the persistent MTF-1 phosphorylation and the disturbance of MTF-1 nuclear translocation, which was concomitant with a significant decrease of MT expression and enhanced cytotoxicity in HEK cells. Notably, MTF-1 was found in complex with specific PP2A complexes containing the PR110 subunit upon metal exposure. Furthermore, we identify that the dephosphorylation of MTF-1 at residue Thr-254 is directly regulated by PP2A PR110 complexes and responsible for MTF-1 activation. Taken together, these findings delineate a novel pathway that determines cytotoxicity in response to metal treatments and provide new insight into the role of PP2A in cellular stress response.

Modulation of DNA repair capacity by ataxia telangiectasia mutated gene polymorphisms among polycyclic aromatic hydrocarbons-exposed workers.

The purpose of this study was to address the association between the ataxia telangiectasia mutated (ATM) gene polymorphisms and susceptibility to DNA repair capacity (DRC) among polycyclic aromatic hydrocarbons (PAHs)-exposed workers. Polymorphisms of ATM were genotyped. DRC was determined by comet assay. Chromosomal damage was detected by cytokinesis-block micronucleus (CBMN) assay. Flow cytometry was used to detect the distributions of cell cycle. Expressions of ATM and rH2AX were determined by immunoblotting analysis. Luciferase assays were performed to determine the functional difference of ATM promoter region allele. Subjects carrying T allele of rs228589 had significantly lower DRC compared with those with AA genotype. Subjects carrying G allele of rs652311 had significantly lower DRC than those with zero copy number of haplotype CGGT. SH ataxia telangiectasia mutated (SHATM) cells had significantly lower DRC than SH green fluorescent protein (SHGFP) cells induced by bleomycin and higher CBMN frequencies treated by benzo(a)pyrene [B(a)P] than SHGFP cells. After B(a)P treatment, a decrease in the percentage of G1 phase cells was observed in SHATM cells compared with SHGFP cells, rH2AX expressions were increased in SHATM cells and SHGFP cells, but ATM expressions had no change in 16HBE-SHGFP cells and HEK-SHGFP cells. Luciferase expression was not different between rs228589T and rs228589A plasmid constructs. In conclusions, it is suggested that ATM polymorphisms are associated with DRC among PAHs-exposed workers and ATM plays key roles in repair of chromosomal damage and cell cycle control with the treatment of B(a)P.