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DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

Fu Y, Feng MX, Yu J, Ma MZ, Liu XJ, Li J, Yang XM, Wang YH, Zhang YL, Ao JP, Xue F, Qin W, Gu J, Xia Q, Zhang ZG - Oncotarget (2014)

Bottom Line: We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis.And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling.Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. These authors contributed equally to this work.

ABSTRACT
Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

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DPT is down-regulated by hypermethylation of DNA promoter(A) The reexpression of DPT was evaluated by qPCR in the HCC cell lines and THLE-2 cell line treated with no drug, DAC, TSA or DAC plus TSA. 18s RNA was used as a loading control. Values are means ± SEM (*P < 0.05, **P < 0.01), ns means no significance. (B) Schematic representation of the location of DPT and CpG island within the promoter in chromosome and the primers designed for bisulfite sequencing. (C) Bisulfite-sequencing results of three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H), THLE-2 cell line, 2 pairs of HCC and their paracancerous liver (PCL) tissues and 3 normal liver (NL) tissues. All 13 CpG sites were sequenced. Open circles indicate unmethylated and solid circles represent methylated CpG dinucleotides. (D) The total methylation rates of DPT promoter in the three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H) and THLE-2 cell line, and in HCC, PCL, NL tissues. Values are means ± SEM (**P < 0.01, *** P < 0.001).
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Figure 3: DPT is down-regulated by hypermethylation of DNA promoter(A) The reexpression of DPT was evaluated by qPCR in the HCC cell lines and THLE-2 cell line treated with no drug, DAC, TSA or DAC plus TSA. 18s RNA was used as a loading control. Values are means ± SEM (*P < 0.05, **P < 0.01), ns means no significance. (B) Schematic representation of the location of DPT and CpG island within the promoter in chromosome and the primers designed for bisulfite sequencing. (C) Bisulfite-sequencing results of three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H), THLE-2 cell line, 2 pairs of HCC and their paracancerous liver (PCL) tissues and 3 normal liver (NL) tissues. All 13 CpG sites were sequenced. Open circles indicate unmethylated and solid circles represent methylated CpG dinucleotides. (D) The total methylation rates of DPT promoter in the three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H) and THLE-2 cell line, and in HCC, PCL, NL tissues. Values are means ± SEM (**P < 0.01, *** P < 0.001).

Mentions: During our preliminary studies, we found that DPT was significantly down-regulated in HCC. Similarly, DPT expression level has a prominent decrease in human oral squamous cell carcinoma (OSCC) [23]. However, the regulatory mechanism underlying DPT down-regulation in HCC is still unclear. Epigenetic modification including DNA methylation and histone modification are considered as the main regulatory mechanisms involved gene aberrant expression [24]. To explore the epigenetic regulation of DPT expression in HCC, three HCC cell lines SMMC-7721, Huh7 and MHCC-97H and an immortalized human liver cell line THLE-2, were treated with a demethylating agent 5-aza-2′-deoxycytidine (DAC) and a histone deacetylase inhibitor trichostatin A (TSA) both separately and in conjunction. The results showed that the expression level of DPT in HCC cells was significantly increased by treatment with DAC in all tested HCC cell lines, but not by treatment with TSA. While the expression level of DPT in THLE-2 cells was only slightly increased by treatment with DAC (Figure 3A).


DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

Fu Y, Feng MX, Yu J, Ma MZ, Liu XJ, Li J, Yang XM, Wang YH, Zhang YL, Ao JP, Xue F, Qin W, Gu J, Xia Q, Zhang ZG - Oncotarget (2014)

DPT is down-regulated by hypermethylation of DNA promoter(A) The reexpression of DPT was evaluated by qPCR in the HCC cell lines and THLE-2 cell line treated with no drug, DAC, TSA or DAC plus TSA. 18s RNA was used as a loading control. Values are means ± SEM (*P < 0.05, **P < 0.01), ns means no significance. (B) Schematic representation of the location of DPT and CpG island within the promoter in chromosome and the primers designed for bisulfite sequencing. (C) Bisulfite-sequencing results of three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H), THLE-2 cell line, 2 pairs of HCC and their paracancerous liver (PCL) tissues and 3 normal liver (NL) tissues. All 13 CpG sites were sequenced. Open circles indicate unmethylated and solid circles represent methylated CpG dinucleotides. (D) The total methylation rates of DPT promoter in the three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H) and THLE-2 cell line, and in HCC, PCL, NL tissues. Values are means ± SEM (**P < 0.01, *** P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196157&req=5

Figure 3: DPT is down-regulated by hypermethylation of DNA promoter(A) The reexpression of DPT was evaluated by qPCR in the HCC cell lines and THLE-2 cell line treated with no drug, DAC, TSA or DAC plus TSA. 18s RNA was used as a loading control. Values are means ± SEM (*P < 0.05, **P < 0.01), ns means no significance. (B) Schematic representation of the location of DPT and CpG island within the promoter in chromosome and the primers designed for bisulfite sequencing. (C) Bisulfite-sequencing results of three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H), THLE-2 cell line, 2 pairs of HCC and their paracancerous liver (PCL) tissues and 3 normal liver (NL) tissues. All 13 CpG sites were sequenced. Open circles indicate unmethylated and solid circles represent methylated CpG dinucleotides. (D) The total methylation rates of DPT promoter in the three HCC cell lines (SMMC-7721, Huh7 and MHCC-97H) and THLE-2 cell line, and in HCC, PCL, NL tissues. Values are means ± SEM (**P < 0.01, *** P < 0.001).
Mentions: During our preliminary studies, we found that DPT was significantly down-regulated in HCC. Similarly, DPT expression level has a prominent decrease in human oral squamous cell carcinoma (OSCC) [23]. However, the regulatory mechanism underlying DPT down-regulation in HCC is still unclear. Epigenetic modification including DNA methylation and histone modification are considered as the main regulatory mechanisms involved gene aberrant expression [24]. To explore the epigenetic regulation of DPT expression in HCC, three HCC cell lines SMMC-7721, Huh7 and MHCC-97H and an immortalized human liver cell line THLE-2, were treated with a demethylating agent 5-aza-2′-deoxycytidine (DAC) and a histone deacetylase inhibitor trichostatin A (TSA) both separately and in conjunction. The results showed that the expression level of DPT in HCC cells was significantly increased by treatment with DAC in all tested HCC cell lines, but not by treatment with TSA. While the expression level of DPT in THLE-2 cells was only slightly increased by treatment with DAC (Figure 3A).

Bottom Line: We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis.And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling.Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. These authors contributed equally to this work.

ABSTRACT
Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

Show MeSH
Related in: MedlinePlus