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De-SUMOylation of FOXC2 by SENP3 promotes the epithelial-mesenchymal transition in gastric cancer cells.

Ren YH, Liu KJ, Wang M, Yu YN, Yang K, Chen Q, Yu B, Wang W, Li QW, Wang J, Hou ZY, Fang JY, Yeh ET, Yang J, Yi J - Oncotarget (2014)

Bottom Line: Biochemical assays identify FOXC2 as a substrate of SENP3.Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2.Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Institute of Neuroscience, Wenzhou Medical University, School of Medicine, Zhejiang, China. These authors contribute equally to the work.

ABSTRACT
The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.

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SENP3 mediates ROS-induced FOXC2 de-SUMOylation(A) ROS level determined by DCFH-DA staining and flow cytometric analysis in SGC7901 and MGC803 cells. The results were from three independent experiments. **: P<0.01. (B) The increase of SENP3 protein in SGC7901 and MGC803 cells exposed to 200μM H202 in the presence or absence of 5 mM NAC for the indicated time. (C) Live cell imaging of MGC803 cells co-transfected with EGFP-FOXC2 and DsRed-SENP3, and exposed to H2O2 (200 μM) for 30 minutes. (D) Endogenous SUMO2/3 conjugates of endogenous FOXC2 determined by co-IP in the lysates of MGC803-sh-NC and MGC803-sh-SENP3 cells exposed to 200 μM H2O2 for 30 min. (E) A model illustrating the molecular mechanism proposed by the present study for the role of SENP3 in the EMT.
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Figure 6: SENP3 mediates ROS-induced FOXC2 de-SUMOylation(A) ROS level determined by DCFH-DA staining and flow cytometric analysis in SGC7901 and MGC803 cells. The results were from three independent experiments. **: P<0.01. (B) The increase of SENP3 protein in SGC7901 and MGC803 cells exposed to 200μM H202 in the presence or absence of 5 mM NAC for the indicated time. (C) Live cell imaging of MGC803 cells co-transfected with EGFP-FOXC2 and DsRed-SENP3, and exposed to H2O2 (200 μM) for 30 minutes. (D) Endogenous SUMO2/3 conjugates of endogenous FOXC2 determined by co-IP in the lysates of MGC803-sh-NC and MGC803-sh-SENP3 cells exposed to 200 μM H2O2 for 30 min. (E) A model illustrating the molecular mechanism proposed by the present study for the role of SENP3 in the EMT.

Mentions: Our previous studies have shown that SENP3 is a redox-sensitive molecule. We detected ROS level of the two gastric cancer cell lines, and found that MGC803, which had a higher level of SENP3 (Fig. 1A), also had a higher level of ROS, in contrast to SGC7901 (Fig. 6A). ROS induction of the SENP3 protein was observed when SGC7901 and MGC803 cells were exposed to 200 μM H2O2 for 5 to 30 min, and this time-dependent SENP3 increase could be blocked by the anti-oxidant N-acetyl cysteine (NAC) (Fig. 6B). Live cell imaging under a time-lapse microscope showed that SENP3, which was normally enriched in the nucleolus and modestly distributed in the nucleoplasm, was markedly increased in the nucleoplasm after the H2O2 treatment during 30 min, leading to enhanced SENP3-FOXC2 co-localization (Fig. 6C). Moreover, after MGC803-sh-SENP3 cells and the control were exposed to H2O2, the result of IP showed that ROS-induced decrease of the SUMO2/3 modification of the FOXC2 was dependent on SENP3 (Fig. 6D). These results show that ROS can increase the level of SENP3, co-localization of SENP3 with FOXC2, and de-SUMOylation of FOXC2 in gastric cells. Taken togher, these data suggest that SENP3, which is induced by ROS in gastric cancer cells, may mediate the ROS-induced EMT process. SENP3 de-SUMOylating FOXC2 leads to an enhanced transcriptional activity of FOXC2. Consequently, the mesenchymal marker genes, e.g., N-cadherin, are transcriptionally upregulated (Fig. 6E). This mechanism may explain the role of SENP3 in the metastasis of gastric cancers, although other ROS-dependent EMT-inducing signaling may also be contributive (dash line in Fig. 6E).


De-SUMOylation of FOXC2 by SENP3 promotes the epithelial-mesenchymal transition in gastric cancer cells.

Ren YH, Liu KJ, Wang M, Yu YN, Yang K, Chen Q, Yu B, Wang W, Li QW, Wang J, Hou ZY, Fang JY, Yeh ET, Yang J, Yi J - Oncotarget (2014)

SENP3 mediates ROS-induced FOXC2 de-SUMOylation(A) ROS level determined by DCFH-DA staining and flow cytometric analysis in SGC7901 and MGC803 cells. The results were from three independent experiments. **: P<0.01. (B) The increase of SENP3 protein in SGC7901 and MGC803 cells exposed to 200μM H202 in the presence or absence of 5 mM NAC for the indicated time. (C) Live cell imaging of MGC803 cells co-transfected with EGFP-FOXC2 and DsRed-SENP3, and exposed to H2O2 (200 μM) for 30 minutes. (D) Endogenous SUMO2/3 conjugates of endogenous FOXC2 determined by co-IP in the lysates of MGC803-sh-NC and MGC803-sh-SENP3 cells exposed to 200 μM H2O2 for 30 min. (E) A model illustrating the molecular mechanism proposed by the present study for the role of SENP3 in the EMT.
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Related In: Results  -  Collection

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Figure 6: SENP3 mediates ROS-induced FOXC2 de-SUMOylation(A) ROS level determined by DCFH-DA staining and flow cytometric analysis in SGC7901 and MGC803 cells. The results were from three independent experiments. **: P<0.01. (B) The increase of SENP3 protein in SGC7901 and MGC803 cells exposed to 200μM H202 in the presence or absence of 5 mM NAC for the indicated time. (C) Live cell imaging of MGC803 cells co-transfected with EGFP-FOXC2 and DsRed-SENP3, and exposed to H2O2 (200 μM) for 30 minutes. (D) Endogenous SUMO2/3 conjugates of endogenous FOXC2 determined by co-IP in the lysates of MGC803-sh-NC and MGC803-sh-SENP3 cells exposed to 200 μM H2O2 for 30 min. (E) A model illustrating the molecular mechanism proposed by the present study for the role of SENP3 in the EMT.
Mentions: Our previous studies have shown that SENP3 is a redox-sensitive molecule. We detected ROS level of the two gastric cancer cell lines, and found that MGC803, which had a higher level of SENP3 (Fig. 1A), also had a higher level of ROS, in contrast to SGC7901 (Fig. 6A). ROS induction of the SENP3 protein was observed when SGC7901 and MGC803 cells were exposed to 200 μM H2O2 for 5 to 30 min, and this time-dependent SENP3 increase could be blocked by the anti-oxidant N-acetyl cysteine (NAC) (Fig. 6B). Live cell imaging under a time-lapse microscope showed that SENP3, which was normally enriched in the nucleolus and modestly distributed in the nucleoplasm, was markedly increased in the nucleoplasm after the H2O2 treatment during 30 min, leading to enhanced SENP3-FOXC2 co-localization (Fig. 6C). Moreover, after MGC803-sh-SENP3 cells and the control were exposed to H2O2, the result of IP showed that ROS-induced decrease of the SUMO2/3 modification of the FOXC2 was dependent on SENP3 (Fig. 6D). These results show that ROS can increase the level of SENP3, co-localization of SENP3 with FOXC2, and de-SUMOylation of FOXC2 in gastric cells. Taken togher, these data suggest that SENP3, which is induced by ROS in gastric cancer cells, may mediate the ROS-induced EMT process. SENP3 de-SUMOylating FOXC2 leads to an enhanced transcriptional activity of FOXC2. Consequently, the mesenchymal marker genes, e.g., N-cadherin, are transcriptionally upregulated (Fig. 6E). This mechanism may explain the role of SENP3 in the metastasis of gastric cancers, although other ROS-dependent EMT-inducing signaling may also be contributive (dash line in Fig. 6E).

Bottom Line: Biochemical assays identify FOXC2 as a substrate of SENP3.Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2.Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Institute of Neuroscience, Wenzhou Medical University, School of Medicine, Zhejiang, China. These authors contribute equally to the work.

ABSTRACT
The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.

Show MeSH
Related in: MedlinePlus