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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: The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed.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 expression correlates with the EMT in gastric cancer cells(A) The expressions of EMT marker genes and SENP3 in SGC7901 and MGC803 cells were assessed by immunoblotting. (B) Representative images of wound-healing in SGC7901 and MGC803 cells were shown. The number of migrated cells within the areas of healing surpassing the red lines was calculated, and each experiment was repeated 3 times. **: P < 0.01. (C) Representative images of transwell assays (6 hours) in SGC7901 and MGC803 cells were shown. The number of migrated cells on the surfaces of membrane was calculated in 3 fields respectively, and each experiment was repeated 3 times. ***: P < 0.001.
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Figure 1: SENP3 expression correlates with the EMT in gastric cancer cells(A) The expressions of EMT marker genes and SENP3 in SGC7901 and MGC803 cells were assessed by immunoblotting. (B) Representative images of wound-healing in SGC7901 and MGC803 cells were shown. The number of migrated cells within the areas of healing surpassing the red lines was calculated, and each experiment was repeated 3 times. **: P < 0.01. (C) Representative images of transwell assays (6 hours) in SGC7901 and MGC803 cells were shown. The number of migrated cells on the surfaces of membrane was calculated in 3 fields respectively, and each experiment was repeated 3 times. ***: P < 0.001.

Mentions: We screened the SENP3 protein level in a few available human gastric cell lines, and chose two cell lines with marked differences in the level of SENP3, SGC7901, with barely detectable SENP3, and MGC803, with abundant SENP3, to examine their EMT gene expression and phenotypes. Coincidently, the expression levels of a few typical mesenchymal marker genes, N-cadherin, vimentin, and fibronectin, were significantly lower in SGC7901 cells than in MGC803 cells (Fig. 1A). The typical epithelial marker gene E-cadherin was not detectable in either cell line (data not showed). Scratch wound-healing and transwell assays showed that the cell migration ability of SGC7901 cells was lower than that of MGC803 cells, as evidenced by the quantifications of migrated cell numbers in areas of healing (Fig. 1B) and cell numbers of transwells (Fig. 1C). To exclude that the differences in migrated cell numbers might be contributed by a difference in cell proliferation, we cultured cells in a serum-free medium to slow down cell proliferation. In addition, we compared the whole viable cell numbers and found no differences between two cell lines during the given time of culture (Supplementary Fig. S1). These results suggest that SENP3 expression correlates with EMT in gastric cancer cells.


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 expression correlates with the EMT in gastric cancer cells(A) The expressions of EMT marker genes and SENP3 in SGC7901 and MGC803 cells were assessed by immunoblotting. (B) Representative images of wound-healing in SGC7901 and MGC803 cells were shown. The number of migrated cells within the areas of healing surpassing the red lines was calculated, and each experiment was repeated 3 times. **: P < 0.01. (C) Representative images of transwell assays (6 hours) in SGC7901 and MGC803 cells were shown. The number of migrated cells on the surfaces of membrane was calculated in 3 fields respectively, and each experiment was repeated 3 times. ***: P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196186&req=5

Figure 1: SENP3 expression correlates with the EMT in gastric cancer cells(A) The expressions of EMT marker genes and SENP3 in SGC7901 and MGC803 cells were assessed by immunoblotting. (B) Representative images of wound-healing in SGC7901 and MGC803 cells were shown. The number of migrated cells within the areas of healing surpassing the red lines was calculated, and each experiment was repeated 3 times. **: P < 0.01. (C) Representative images of transwell assays (6 hours) in SGC7901 and MGC803 cells were shown. The number of migrated cells on the surfaces of membrane was calculated in 3 fields respectively, and each experiment was repeated 3 times. ***: P < 0.001.
Mentions: We screened the SENP3 protein level in a few available human gastric cell lines, and chose two cell lines with marked differences in the level of SENP3, SGC7901, with barely detectable SENP3, and MGC803, with abundant SENP3, to examine their EMT gene expression and phenotypes. Coincidently, the expression levels of a few typical mesenchymal marker genes, N-cadherin, vimentin, and fibronectin, were significantly lower in SGC7901 cells than in MGC803 cells (Fig. 1A). The typical epithelial marker gene E-cadherin was not detectable in either cell line (data not showed). Scratch wound-healing and transwell assays showed that the cell migration ability of SGC7901 cells was lower than that of MGC803 cells, as evidenced by the quantifications of migrated cell numbers in areas of healing (Fig. 1B) and cell numbers of transwells (Fig. 1C). To exclude that the differences in migrated cell numbers might be contributed by a difference in cell proliferation, we cultured cells in a serum-free medium to slow down cell proliferation. In addition, we compared the whole viable cell numbers and found no differences between two cell lines during the given time of culture (Supplementary Fig. S1). These results suggest that SENP3 expression correlates with EMT in gastric cancer cells.

Bottom Line: The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed.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