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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 can de-conjugate SUMO2/3 from the EMT-inducing TF FOXC2(A) SUMO3 conjugates of FOXC2, determined by NI-NTA pull down assay in HEK293T cells co-transfected with EGFP-FOXC2 wild-type/mutants and RH-SUMO3. (B) SUMO3 conjugates of FOXC2, determined by Flag immunoprecipitation in HEK293T cells co-transfected with Flag-FOXC2, RH-SUMO3 and SENP3. (C,D) Endogenous SUMO2/3 conjugates of endogenous FOXC2, determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells (C) and gastric cancers (D). Tumor tissues were collected from two representative orthotopic cancer mice of each group. Arrowheads indicated SUMO3-conjugated FOXC2 in A, B, C and D. (E, F) SENP3-FOXC2 interaction determined exogenously in HEK293T cells co-transfected with two plasmids (E), and endogenously in MGC803 cells (F).
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Figure 4: SENP3 can de-conjugate SUMO2/3 from the EMT-inducing TF FOXC2(A) SUMO3 conjugates of FOXC2, determined by NI-NTA pull down assay in HEK293T cells co-transfected with EGFP-FOXC2 wild-type/mutants and RH-SUMO3. (B) SUMO3 conjugates of FOXC2, determined by Flag immunoprecipitation in HEK293T cells co-transfected with Flag-FOXC2, RH-SUMO3 and SENP3. (C,D) Endogenous SUMO2/3 conjugates of endogenous FOXC2, determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells (C) and gastric cancers (D). Tumor tissues were collected from two representative orthotopic cancer mice of each group. Arrowheads indicated SUMO3-conjugated FOXC2 in A, B, C and D. (E, F) SENP3-FOXC2 interaction determined exogenously in HEK293T cells co-transfected with two plasmids (E), and endogenously in MGC803 cells (F).

Mentions: SENP3 is a SUMO2/3 protease that is enriched in the nucleolus and accumulates in the nucleoplasm under oxidative stress, where it interacts with many nuclear proteins. We thus speculated that the effect of SENP3 to induce the EMT might depend on its enzymatic activity to directly de-conjugate SUMO2/3 from EMT-inducing TFs. We predicted the probability of SUMOylation of a few classic or newly identified EMT-inducing TFs, i.e., Snail, Twist, Slug, and FOXC2, using an open software, and found that FOXC2, but not the remaining, had SUMOylation motifs with high probability at K214, K132, K72, and K184 (Supplementary Fig. S4). We constructed plasmids of wild-type FOXC2 or the mutants in which the predicted Lys residues of the SUMOylation sites were replaced by Arg that did not allow SUMOylation. The SUMOylation of FOXC2 was then examined using NI-NTA pull-down assays in HEK293T cells. The result showed that FOXC2 was conjugated by SUMO3 at K214, as the wild-type FOXC2 was pulled-down with SUMO3 conjugates and displayed two prominent bands on the gel (arrowheads in Fig. 4A), and the K214R mutant lacked these bands; but the remaining three mutants maintained these bands (Fig. 4A). We then tested if SENP3 could remove SUMO3 conjugates from FOXC2 using a Flag IP assay. The results showed that SENP3 de-conjugated SUMO3 from FOXC2 in a dose-dependent manner (Fig. 4B). Furthermore, endogenous FOXC2 was immunoprecipitated in SENP3-absent HEK293T cells, and its SUMO2/3 conjugates were heavier than in SENP3-present cells (Fig. 4C). Endogenous SUMO2/3 modifications on endogenous FOXC2 were further analyzed in the tissues obtained from the orthotopic gastric cancers in the mouse model. Tumors derived from SENP3-overexpressed cells had FOXC2 with much fewer SUMO2/3 conjugates (Fig. 4D). The interaction between FOXC2 and SENP3 was demonstrated by co-IP assays in both exogenous (Fig. 4E, HEK293T cells) and endogenous (Fig. 4F, MGC803 cells) settings. These data confirm that SENP3 can remove the SUMO2/3 modification of FOXC2 at K214 via a direct interaction.


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 can de-conjugate SUMO2/3 from the EMT-inducing TF FOXC2(A) SUMO3 conjugates of FOXC2, determined by NI-NTA pull down assay in HEK293T cells co-transfected with EGFP-FOXC2 wild-type/mutants and RH-SUMO3. (B) SUMO3 conjugates of FOXC2, determined by Flag immunoprecipitation in HEK293T cells co-transfected with Flag-FOXC2, RH-SUMO3 and SENP3. (C,D) Endogenous SUMO2/3 conjugates of endogenous FOXC2, determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells (C) and gastric cancers (D). Tumor tissues were collected from two representative orthotopic cancer mice of each group. Arrowheads indicated SUMO3-conjugated FOXC2 in A, B, C and D. (E, F) SENP3-FOXC2 interaction determined exogenously in HEK293T cells co-transfected with two plasmids (E), and endogenously in MGC803 cells (F).
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Figure 4: SENP3 can de-conjugate SUMO2/3 from the EMT-inducing TF FOXC2(A) SUMO3 conjugates of FOXC2, determined by NI-NTA pull down assay in HEK293T cells co-transfected with EGFP-FOXC2 wild-type/mutants and RH-SUMO3. (B) SUMO3 conjugates of FOXC2, determined by Flag immunoprecipitation in HEK293T cells co-transfected with Flag-FOXC2, RH-SUMO3 and SENP3. (C,D) Endogenous SUMO2/3 conjugates of endogenous FOXC2, determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells (C) and gastric cancers (D). Tumor tissues were collected from two representative orthotopic cancer mice of each group. Arrowheads indicated SUMO3-conjugated FOXC2 in A, B, C and D. (E, F) SENP3-FOXC2 interaction determined exogenously in HEK293T cells co-transfected with two plasmids (E), and endogenously in MGC803 cells (F).
Mentions: SENP3 is a SUMO2/3 protease that is enriched in the nucleolus and accumulates in the nucleoplasm under oxidative stress, where it interacts with many nuclear proteins. We thus speculated that the effect of SENP3 to induce the EMT might depend on its enzymatic activity to directly de-conjugate SUMO2/3 from EMT-inducing TFs. We predicted the probability of SUMOylation of a few classic or newly identified EMT-inducing TFs, i.e., Snail, Twist, Slug, and FOXC2, using an open software, and found that FOXC2, but not the remaining, had SUMOylation motifs with high probability at K214, K132, K72, and K184 (Supplementary Fig. S4). We constructed plasmids of wild-type FOXC2 or the mutants in which the predicted Lys residues of the SUMOylation sites were replaced by Arg that did not allow SUMOylation. The SUMOylation of FOXC2 was then examined using NI-NTA pull-down assays in HEK293T cells. The result showed that FOXC2 was conjugated by SUMO3 at K214, as the wild-type FOXC2 was pulled-down with SUMO3 conjugates and displayed two prominent bands on the gel (arrowheads in Fig. 4A), and the K214R mutant lacked these bands; but the remaining three mutants maintained these bands (Fig. 4A). We then tested if SENP3 could remove SUMO3 conjugates from FOXC2 using a Flag IP assay. The results showed that SENP3 de-conjugated SUMO3 from FOXC2 in a dose-dependent manner (Fig. 4B). Furthermore, endogenous FOXC2 was immunoprecipitated in SENP3-absent HEK293T cells, and its SUMO2/3 conjugates were heavier than in SENP3-present cells (Fig. 4C). Endogenous SUMO2/3 modifications on endogenous FOXC2 were further analyzed in the tissues obtained from the orthotopic gastric cancers in the mouse model. Tumors derived from SENP3-overexpressed cells had FOXC2 with much fewer SUMO2/3 conjugates (Fig. 4D). The interaction between FOXC2 and SENP3 was demonstrated by co-IP assays in both exogenous (Fig. 4E, HEK293T cells) and endogenous (Fig. 4F, MGC803 cells) settings. These data confirm that SENP3 can remove the SUMO2/3 modification of FOXC2 at K214 via a direct interaction.

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