Functional Switching of TGF-β1 Signaling in Liver Cancer via Epigenetic Modulation of a Single CpG Site in TTP Promoter

doi:10.1053/j.gastro.2009.12.044

Gastroenterology

Volume 138, Issue 5, May 2010, Pages 1898-1908.e12

https://doi.org/10.1053/j.gastro.2009.12.044 Get rights and content

Background & Aims

Acquisition of resistance to the antiproliferative effect of transforming growth factor (TGF)-β1 is crucial for the malignant progression of cancers. In this study, we sought to determine whether deregulated expression of tristetrapolin (TTP), a negative posttranscriptional regulator of c-Myc, confers resistance to the antiproliferative effects of TGF-β1 on liver cancer cells.

Methods

The epigenetics of TTP promoter regulation and its effects on TGF-β1 signaling were examined in hepatocellular carcinoma (HCC) cell lines and patient tissues.

Results

TTP was down-regulated in HCC cell lines (10/11), compared with normal liver, as well as in tumor tissues (19/24) from paired HCC specimens. Methylation of a specific single CpG site located within the TGF-β1-responsive region (TRR) of the TTP promoter was significantly associated with TTP down-regulation in both HCC cell lines and tumor tissues (r = −0.606383, P < .001). The singly methylated CpG site was specifically bound by a transcriptional repressor complex consisting of MECP2/c-Ski/DNMT3A and abolished the TGF-β1-induced as well as basal-level expression of TTP. The epigenetic inactivation of TTP led to an increased half-life of c-Myc mRNA and blocked the cytostatic effect of TGF-β1. Statistically significant correlations were observed between the single CpG site methylation and expression levels of TTP or c-Myc in clinical samples of HCC.

Conclusions

Abrogation of the post-transcriptional regulation of c-Myc via methylation of a specific single CpG site in the TTP promoter presents a novel mechanism for the gain of selective resistance to the antiproliferative signaling of TGF-β1 in HCC.

Section snippets

Quantitative Real-time Reverse-Transcription Polymerase Chain Reaction and Primers

Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed using Exicycler (Bioneer, Daejeon, Korea), and the quantity of amplified products was normalized against that of β-Actin. The primers used in this study are described in Supplementary Table 1.

Bisulfite Sequencing and Pyrosequencing

Bisulfite sequencing analyses were performed as described previously,¹⁶ using PSQ HS 96 Gold SNP reagents and pyrosequencing machine (Biotage, Uppsala, Sweden) following manufacturer's instruction.

Construction of Recombinant DNAs

Luciferase

Down-regulation of TTP via DNA Methylation in HCC Cell Lines

Quantitative real-time RT-PCR analysis of 11 HCC cell lines revealed significantly lower TTP mRNA levels compared with that in normal human liver, except in PLC/PRF/5 cells (Figure 1A). Treatment with 5-azadeoxycytidine (AzadC), a DNA methyltransferase inhibitor, led to a significant increase in TTP expression in most cell lines displaying relatively low endogenous TTP mRNA expression (Figure 1B). Subsequent dose-response experiments in SK-Hep1, Hep3B, and SNU182 cell lines disclosed a dose

Discussion

We showed that methylation of a specific single CpG site in the TTP promoter plays critical roles in the regulation of TTP expression. Transcriptional regulation by DNA methylation of CpG islands at the promoter region is a well-defined epigenetic phenomenon.²² In cancers, tumor suppressor genes are often aberrantly hypermethylated and transcriptionally repressed.²² When certain CpG sites are committed to de novo methylation, the process tends to spread progressively through the entire CpG

Acknowledgments

B.H.S. and I.Y.P. contributed equally to this report.

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Environmental insults can lead to alteration in DNA methylation of specific genes. To address the role of altered DNA methylation in prediction of polycyclic aromatic hydrocarbons (PAHs) exposure-induced genetic damage, we recruited two populations, including diesel engine exhausts (low-level) and coke oven emissions (high-level) exposed subjects. The positive correlation was observed between the internal exposure marker (1-hydroxypyrene) and the extents of DNA damage (P < 0.05). The methylation of representative genes, including TRIM36, RASSF1a, and MGMT in peripheral blood lymphocytes was quantitatively examined by bisulfite-pyrosequencing assay. The DNA methylation of these three genes in response to PAHs exposure were changed in a CpG-site-specific manner. The identified hot CpG site-specific methylation of three genes exhibited higher predictive power for DNA damage than the respective single genes in both populations. Furthermore, the dose-response relationship analysis revealed a nonlinear U-shape curve of TRIM36 or RASSF1a methylation in combined population, which led to determination of the threshold of health risk. Furthermore, we established a prediction model for genetic damage based on the unidirectional-alteration MGMT methylation levels. In conclusion, this study provides new insight into the application of multiple epi-biomarkers for health risk assessment upon PAHs exposure.
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Citation Excerpt :
However, this comes with several drawbacks: qPCR requires relatively long amplicon lengths (70–200 bp), and these amplicons should contain at least two MSRE restriction sites to reliably inhibit amplification for non-methylated samples (Šestáková et al., 2019). Therefore, it is not possible to investigate the methylation levels of single CpG dinucleotides, which has been shown to be strongly associated with the development of gastric, liver and colon cancers (Sohn et al., 2010; Zou et al., 2006). An overview of the most used current methods and their mechanism can be found in supplementary data table 1.
The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a–MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.
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Tristetraprolin (TTP) is a key post-transcriptional regulator of inflammatory and oncogenic transcripts. Accordingly, TTP was reported to act as a tumor suppressor in specific cancers. Herein, we investigated how TTP contributes to the development of liver inflammation and fibrosis, which are key drivers of hepatocarcinogenesis, as well as to the onset and progression of hepatocellular carcinoma (HCC).
TTP expression was investigated in mouse/human models of hepatic metabolic diseases and cancer. The role of TTP in nonalcoholic steatohepatitis and HCC development was further examined through in vivo/vitro approaches using liver-specific TTP knockout mice and a panel of hepatic cancer cells.
Our data demonstrate that TTP loss in vivo strongly restrains development of hepatic steatosis and inflammation/fibrosis in mice fed a methionine/choline-deficient diet, as well as HCC development induced by the carcinogen diethylnitrosamine. In contrast, low TTP expression fostered migration and invasion capacities of in vitro transformed hepatic cancer cells likely by unleashing expression of key oncogenes previously associated with these cancerous features. Consistent with these data, TTP was significantly down-regulated in high-grade human HCC, a feature further correlating with poor clinical prognosis. Finally, we uncover hepatocyte nuclear factor 4 alpha and early growth response 1, two key transcription factors lost with hepatocyte dedifferentiation, as key regulators of TTP expression.
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As mentioned previously, TGF-β could facilitate c-Myc mRNA degradation by upregulating TTP (Sohn et al., 2010). However, methylation of a specific single CpG site in the TTP promoter abolishes its responsiveness to TGF-β, contributing to a high c-Myc level in HCC (Sohn et al., 2010). Unlike that of TGF-β or Smads, the expression of TβRII is frequently reduced in HCC, attenuating TGF-β-induced cytostasis (Furuta et al., 1999; Mamiya et al., 2010).
Hepatocellular carcinoma (HCC) is the major form of primary liver cancer and one of the most prevalent and life-threatening malignancies globally. One of the hallmarks in HCC is the sustained cell survival and proliferative signals, which are determined by the balance between oncogenes and tumor suppressors. Transforming growth factor beta (TGF-β) is an effective growth inhibitor of epithelial cells including hepatocytes, through induction of cell cycle arrest, apoptosis, cellular senescence, or autophagy. The antitumorigenic effects of TGF-β are bypassed during liver tumorigenesis via multiple mechanisms. Furthermore, along with malignant progression, TGF-β switches to promote cancer cell survival and proliferation. This dichotomous nature of TGF-β is one of the barriers to therapeutic targeting in liver cancer. Thereafter, understanding the underlying molecular mechanisms is a prerequisite for discovering novel antitumor drugs that may specifically disable the growth-promoting branch of TGF-β signaling or restore its tumor-suppressive arm. This review summarizes how TGF-β inhibits or promotes liver cancer cell survival and proliferation, highlighting the functional switch mechanisms during the process.

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by grants from the 21C Frontier Human Genome Functional Analysis Project and the Korean Systems Biology Research Program of the Ministry of Science and Technology of Korea and the KRIBB Research Initiative Program.

View full text

Basic—Liver, Pancreas, and Biliary TractFunctional Switching of TGF-β1 Signaling in Liver Cancer via Epigenetic Modulation of a Single CpG Site in TTP Promoter

Background & Aims

Methods

Results

Conclusions

Section snippets

Quantitative Real-time Reverse-Transcription Polymerase Chain Reaction and Primers

Bisulfite Sequencing and Pyrosequencing

Construction of Recombinant DNAs

Down-regulation of TTP via DNA Methylation in HCC Cell Lines

Discussion

Acknowledgments

J Biol Chem

J Biol Chem

J Biol Chem

Biochim Biophys Acta

Mol Cell

J Biol Chem

Gastroenterology

Immunity

J Biol Chem

Gastroenterology

TGF-β signaling in tumor suppression and cancer progression

Nat Genet

Escaping from the TGF-β antiproliferative control

Carcinogenesis

c-MYC: more than just a matter of life and death

Nat Rev Cancer

Defective repression of c-myc in breast cancer cells: a loss at the core of the transforming growth factor β growth arrest program

Proc Natl Acad Sci U S A

Loss of c-myc repression coincides with ovarian cancer resistance to transforming growth factor β growth arrest independent of transforming growth factor β/Smad signaling

Cancer Res

Basic—Liver, Pancreas, and Biliary Tract
Functional Switching of TGF-β1 Signaling in Liver Cancer via Epigenetic Modulation of a Single CpG Site in TTP Promoter