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Testes-specific protease 50 promotes cell invasion and metastasis by increasing NF-kappaB-dependent matrix metalloproteinase-9 expression.

Song ZB, Ni JS, Wu P, Bao YL, Liu T, Li M, Fan C, Zhang WJ, Sun LG, Huang YX, Li YX - Cell Death Dis (2015)

Bottom Line: Mechanistic studies revealed that NF-κB signaling pathway was required for TSP50-induced cell migration and metastasis, and further results indicated that TSP50 overexpression enhanced expression and secretion of MMP9, a target gene of NF-κB signaling.In addition, knockdown of MMP9 resulted in inhibition of cell migration and invasion in vitro and lung metastasis in vivo.Furthermore, we found that some breast cancer diagnosis-associated features such as tumor size, tumor grade, estrogen receptors (ER) and progesterone receptors (PR) levels, were correlated well with TSP50/p65 and TSP50/MMP9 expression status.

View Article: PubMed Central - PubMed

Affiliation: 1] National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China [2] Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China.

ABSTRACT
The high mortality in breast cancer is often associated with metastatic progression in patients. Previously we have demonstrated that testes-specific protease 50 (TSP50), an oncogene overexpressed in breast cancer samples, could promote cell proliferation and tumorigenesis. However, whether TSP50 also has a key role in cell invasion and cancer metastasis, and the mechanism underlying the process are still unclear. Here we found that TSP50 overexpression greatly promoted cell migration, invasion, adhesion and formation of the stellate structures in 3D culture system in vitro as well as lung metastasis in vivo. Conversely, TSP50 knockdown caused the opposite changes. Mechanistic studies revealed that NF-κB signaling pathway was required for TSP50-induced cell migration and metastasis, and further results indicated that TSP50 overexpression enhanced expression and secretion of MMP9, a target gene of NF-κB signaling. In addition, knockdown of MMP9 resulted in inhibition of cell migration and invasion in vitro and lung metastasis in vivo. Most importantly, immunohistochemical staining of human breast cancer samples strongly showed that the coexpression of TSP50 and p65 as well as TSP50 and MMP9 were correlated with increased metastasis and poor survival. Furthermore, we found that some breast cancer diagnosis-associated features such as tumor size, tumor grade, estrogen receptors (ER) and progesterone receptors (PR) levels, were correlated well with TSP50/p65 and TSP50/MMP9 expression status. Taken together, this work identified the TSP50 activation of MMP9 as a novel signaling mechanism underlying human breast cancer invasion and metastasis.

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Related in: MedlinePlus

Silencing of endogenous TSP50 inhibited cell migration and invasion. (a) Top panel: western blotting assay of TSP50 expression in MDA-MB-231 cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-231 cells. Bottom panel: curve of different time intervals showing wound closure. (b) Top panel: western blotting assay of TSP50 expression in MDA-MB-435S cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-435S cells. Bottom panel: curve of different time intervals showing wound closure. (c) Knockdown of endogenous TSP50 inhibited invasion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 50 μm. (d) Knockdown of endogenous TSP50 inhibited adhesion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 150 μm. (e) Knockdown of endogenous TSP50 changed the morphology of MDA-MB-231 and MDA-MB-435S cells in three-dimensional (3D) cultures system. The arrow points out the filopodium. Scale bar, 50 μm
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fig2: Silencing of endogenous TSP50 inhibited cell migration and invasion. (a) Top panel: western blotting assay of TSP50 expression in MDA-MB-231 cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-231 cells. Bottom panel: curve of different time intervals showing wound closure. (b) Top panel: western blotting assay of TSP50 expression in MDA-MB-435S cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-435S cells. Bottom panel: curve of different time intervals showing wound closure. (c) Knockdown of endogenous TSP50 inhibited invasion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 50 μm. (d) Knockdown of endogenous TSP50 inhibited adhesion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 150 μm. (e) Knockdown of endogenous TSP50 changed the morphology of MDA-MB-231 and MDA-MB-435S cells in three-dimensional (3D) cultures system. The arrow points out the filopodium. Scale bar, 50 μm

Mentions: To further analyze whether TSP50 was required for migration and invasion of breast cancer cells, we investigated the effect of the TSP50 downregulation on the migration and invasion of MDA-MB-231 and MDA-MB-435S cells. The MDA-MB-231 cells expressing TSP50 shRNA were established in our previous studies22 and were characterized again here (Figure 2a). We observed an effective suppression of wound healing in MDA-MB-231 cells with stable silencing of TSP50 expression (Figure 2a). Similarly, knockdown of TSP50 in MDA-MB-435S cells also greatly inhibited cell migration (Figure 2b). In addition, the invasion of MDA-MB-231 and MDA-MB-435S cells was also reduced by knockdown of endogenous TSP50 (Figure 2c). Likewise, cells attached to matrigel were also significantly reduced, and the branches disappeared and cells changed to spherical shape in 3D culture system when TSP50 expression was inhibited in both MDA-MB-231 and MDA-MB-435S cells (Figures 2d and e). Altogether, these data support the hypothesis of a critical role of TSP50 in promoting cell migration and invasion.


Testes-specific protease 50 promotes cell invasion and metastasis by increasing NF-kappaB-dependent matrix metalloproteinase-9 expression.

Song ZB, Ni JS, Wu P, Bao YL, Liu T, Li M, Fan C, Zhang WJ, Sun LG, Huang YX, Li YX - Cell Death Dis (2015)

Silencing of endogenous TSP50 inhibited cell migration and invasion. (a) Top panel: western blotting assay of TSP50 expression in MDA-MB-231 cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-231 cells. Bottom panel: curve of different time intervals showing wound closure. (b) Top panel: western blotting assay of TSP50 expression in MDA-MB-435S cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-435S cells. Bottom panel: curve of different time intervals showing wound closure. (c) Knockdown of endogenous TSP50 inhibited invasion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 50 μm. (d) Knockdown of endogenous TSP50 inhibited adhesion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 150 μm. (e) Knockdown of endogenous TSP50 changed the morphology of MDA-MB-231 and MDA-MB-435S cells in three-dimensional (3D) cultures system. The arrow points out the filopodium. Scale bar, 50 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385939&req=5

fig2: Silencing of endogenous TSP50 inhibited cell migration and invasion. (a) Top panel: western blotting assay of TSP50 expression in MDA-MB-231 cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-231 cells. Bottom panel: curve of different time intervals showing wound closure. (b) Top panel: western blotting assay of TSP50 expression in MDA-MB-435S cells transfected with scrambled shRNA and TSP50 shRNA vectors. Middle panel: knockdown of endogenous TSP50 inhibited migration of MDA-MB-435S cells. Bottom panel: curve of different time intervals showing wound closure. (c) Knockdown of endogenous TSP50 inhibited invasion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 50 μm. (d) Knockdown of endogenous TSP50 inhibited adhesion of MDA-MB-231 and MDA-MB-435S cells. Scale bar, 150 μm. (e) Knockdown of endogenous TSP50 changed the morphology of MDA-MB-231 and MDA-MB-435S cells in three-dimensional (3D) cultures system. The arrow points out the filopodium. Scale bar, 50 μm
Mentions: To further analyze whether TSP50 was required for migration and invasion of breast cancer cells, we investigated the effect of the TSP50 downregulation on the migration and invasion of MDA-MB-231 and MDA-MB-435S cells. The MDA-MB-231 cells expressing TSP50 shRNA were established in our previous studies22 and were characterized again here (Figure 2a). We observed an effective suppression of wound healing in MDA-MB-231 cells with stable silencing of TSP50 expression (Figure 2a). Similarly, knockdown of TSP50 in MDA-MB-435S cells also greatly inhibited cell migration (Figure 2b). In addition, the invasion of MDA-MB-231 and MDA-MB-435S cells was also reduced by knockdown of endogenous TSP50 (Figure 2c). Likewise, cells attached to matrigel were also significantly reduced, and the branches disappeared and cells changed to spherical shape in 3D culture system when TSP50 expression was inhibited in both MDA-MB-231 and MDA-MB-435S cells (Figures 2d and e). Altogether, these data support the hypothesis of a critical role of TSP50 in promoting cell migration and invasion.

Bottom Line: Mechanistic studies revealed that NF-κB signaling pathway was required for TSP50-induced cell migration and metastasis, and further results indicated that TSP50 overexpression enhanced expression and secretion of MMP9, a target gene of NF-κB signaling.In addition, knockdown of MMP9 resulted in inhibition of cell migration and invasion in vitro and lung metastasis in vivo.Furthermore, we found that some breast cancer diagnosis-associated features such as tumor size, tumor grade, estrogen receptors (ER) and progesterone receptors (PR) levels, were correlated well with TSP50/p65 and TSP50/MMP9 expression status.

View Article: PubMed Central - PubMed

Affiliation: 1] National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China [2] Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China.

ABSTRACT
The high mortality in breast cancer is often associated with metastatic progression in patients. Previously we have demonstrated that testes-specific protease 50 (TSP50), an oncogene overexpressed in breast cancer samples, could promote cell proliferation and tumorigenesis. However, whether TSP50 also has a key role in cell invasion and cancer metastasis, and the mechanism underlying the process are still unclear. Here we found that TSP50 overexpression greatly promoted cell migration, invasion, adhesion and formation of the stellate structures in 3D culture system in vitro as well as lung metastasis in vivo. Conversely, TSP50 knockdown caused the opposite changes. Mechanistic studies revealed that NF-κB signaling pathway was required for TSP50-induced cell migration and metastasis, and further results indicated that TSP50 overexpression enhanced expression and secretion of MMP9, a target gene of NF-κB signaling. In addition, knockdown of MMP9 resulted in inhibition of cell migration and invasion in vitro and lung metastasis in vivo. Most importantly, immunohistochemical staining of human breast cancer samples strongly showed that the coexpression of TSP50 and p65 as well as TSP50 and MMP9 were correlated with increased metastasis and poor survival. Furthermore, we found that some breast cancer diagnosis-associated features such as tumor size, tumor grade, estrogen receptors (ER) and progesterone receptors (PR) levels, were correlated well with TSP50/p65 and TSP50/MMP9 expression status. Taken together, this work identified the TSP50 activation of MMP9 as a novel signaling mechanism underlying human breast cancer invasion and metastasis.

Show MeSH
Related in: MedlinePlus