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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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Overexpression of DPT enhances SMMC-7721 cell adhesion and affects focal adhesions and the activity of Rho GTPase(A) DPT overexpression increased the adhesion of SMMC-7721 cells to collagenI (ColI), collagenIV (ColIV), fibronectin (FN) and laminin1 (LM1). Adherent cells were stained with crystal violet and quantified by colorimetry (***P < 0.001). (B) The distribution of paxillin (upper panel) and vinculin (lower panel) was analyzed by immunofluorescence. Red fluorescence represents paxillin or vinculin staining. F-actin is shown by green fluorescence, and the cell nuclei were stained with DAPI (blue fluorescence). Scale bars, 10μm. (C) Analysis of the active and total RhoA, Cdc42 and Rac1 in Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cell lysates by pull-down assay. Values are means ± SEM (**P < 0.01). (D) The G-LISA results of active RhoA and Rac1. Values are means ± SEM (**P < 0.01), ns means no significance.
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Figure 7: Overexpression of DPT enhances SMMC-7721 cell adhesion and affects focal adhesions and the activity of Rho GTPase(A) DPT overexpression increased the adhesion of SMMC-7721 cells to collagenI (ColI), collagenIV (ColIV), fibronectin (FN) and laminin1 (LM1). Adherent cells were stained with crystal violet and quantified by colorimetry (***P < 0.001). (B) The distribution of paxillin (upper panel) and vinculin (lower panel) was analyzed by immunofluorescence. Red fluorescence represents paxillin or vinculin staining. F-actin is shown by green fluorescence, and the cell nuclei were stained with DAPI (blue fluorescence). Scale bars, 10μm. (C) Analysis of the active and total RhoA, Cdc42 and Rac1 in Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cell lysates by pull-down assay. Values are means ± SEM (**P < 0.01). (D) The G-LISA results of active RhoA and Rac1. Values are means ± SEM (**P < 0.01), ns means no significance.

Mentions: It has been reported that DPT enhances the adhesion of fibroblasts and epidermal keratinocytes via α3β1 integrin [10, 25, 26]. Therefore, we analyzed the adhesive capacity of the Lenti-DPT/SMMC-7721 and Lenti-vector/SMMC-7721 cells and found that overexpression of DPT significantly enhanced the adhesion of SMMC-7721 cells to the most common ECM proteins, such as collagen, laminin and fibronectin (Figure 7A). Integrin-mediated cell-matrix adhesion plays an important role in the regulation of cell migration [27]. This process is always accompanied with the transformation of focal adhesions, which are composed of a variety of molecules, including regulatory molecules and adapter proteins [28]. Among these molecules, paxillin is a marker of newly formed focal adhesions, and vinculin has been demonstrated to be a key player in the maturation and stability of focal adhesions [29]. Next, we investigated focal adhesion assembly in the Lenti-DPT/SMMC-7721 and Lenti-vector/SMMC-7721 cells by immunofluorescent staining of these two adapter proteins, vinculin and paxillin. As shown in Figure 7B, compared to the Lenti-vector/SMMC-7721 cells, there were many more vinculin-containing focal adhesions in the Lenti-DPT/SMMC-7721 cells, and they were distributed not only at the periphery but also at the ventral surface of the spread cells. No obvious differences were observed in the number or localization of the paxillin-containing focal adhesions between the Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cells.


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)

Overexpression of DPT enhances SMMC-7721 cell adhesion and affects focal adhesions and the activity of Rho GTPase(A) DPT overexpression increased the adhesion of SMMC-7721 cells to collagenI (ColI), collagenIV (ColIV), fibronectin (FN) and laminin1 (LM1). Adherent cells were stained with crystal violet and quantified by colorimetry (***P < 0.001). (B) The distribution of paxillin (upper panel) and vinculin (lower panel) was analyzed by immunofluorescence. Red fluorescence represents paxillin or vinculin staining. F-actin is shown by green fluorescence, and the cell nuclei were stained with DAPI (blue fluorescence). Scale bars, 10μm. (C) Analysis of the active and total RhoA, Cdc42 and Rac1 in Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cell lysates by pull-down assay. Values are means ± SEM (**P < 0.01). (D) The G-LISA results of active RhoA and Rac1. Values are means ± SEM (**P < 0.01), ns means no significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196157&req=5

Figure 7: Overexpression of DPT enhances SMMC-7721 cell adhesion and affects focal adhesions and the activity of Rho GTPase(A) DPT overexpression increased the adhesion of SMMC-7721 cells to collagenI (ColI), collagenIV (ColIV), fibronectin (FN) and laminin1 (LM1). Adherent cells were stained with crystal violet and quantified by colorimetry (***P < 0.001). (B) The distribution of paxillin (upper panel) and vinculin (lower panel) was analyzed by immunofluorescence. Red fluorescence represents paxillin or vinculin staining. F-actin is shown by green fluorescence, and the cell nuclei were stained with DAPI (blue fluorescence). Scale bars, 10μm. (C) Analysis of the active and total RhoA, Cdc42 and Rac1 in Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cell lysates by pull-down assay. Values are means ± SEM (**P < 0.01). (D) The G-LISA results of active RhoA and Rac1. Values are means ± SEM (**P < 0.01), ns means no significance.
Mentions: It has been reported that DPT enhances the adhesion of fibroblasts and epidermal keratinocytes via α3β1 integrin [10, 25, 26]. Therefore, we analyzed the adhesive capacity of the Lenti-DPT/SMMC-7721 and Lenti-vector/SMMC-7721 cells and found that overexpression of DPT significantly enhanced the adhesion of SMMC-7721 cells to the most common ECM proteins, such as collagen, laminin and fibronectin (Figure 7A). Integrin-mediated cell-matrix adhesion plays an important role in the regulation of cell migration [27]. This process is always accompanied with the transformation of focal adhesions, which are composed of a variety of molecules, including regulatory molecules and adapter proteins [28]. Among these molecules, paxillin is a marker of newly formed focal adhesions, and vinculin has been demonstrated to be a key player in the maturation and stability of focal adhesions [29]. Next, we investigated focal adhesion assembly in the Lenti-DPT/SMMC-7721 and Lenti-vector/SMMC-7721 cells by immunofluorescent staining of these two adapter proteins, vinculin and paxillin. As shown in Figure 7B, compared to the Lenti-vector/SMMC-7721 cells, there were many more vinculin-containing focal adhesions in the Lenti-DPT/SMMC-7721 cells, and they were distributed not only at the periphery but also at the ventral surface of the spread cells. No obvious differences were observed in the number or localization of the paxillin-containing focal adhesions between the Lenti-vector/SMMC-7721 and Lenti-DPT/SMMC-7721 cells.

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