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STIM1 accelerates cell senescence in a remodeled microenvironment but enhances the epithelial-to-mesenchymal transition in prostate cancer.

Xu Y, Zhang S, Niu H, Ye Y, Hu F, Chen S, Li X, Luo X, Jiang S, Liu Y, Chen Y, Li J, Xiang R, Li N - Sci Rep (2015)

Bottom Line: The importance of store-operated Ca(2+) entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging.Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells.However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China [2] State Key Lab of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.

ABSTRACT
The importance of store-operated Ca(2+) entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging. Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells. In addition, STIM1 and ORAI1 inhibited NF-κB signaling and remodeled the tumor microenvironment by reducing the formation of M2 phenotype macrophages, possibly creating an unfavorable tumor microenvironment and inhibiting cancer development. However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways. Thus, our study revealed novel regulatory effects and the mechanisms by which STIM1 affects cell senescence, tumor migration and the tumor microenvironment, revealing that STIM1 has multiple functions in prostate cancer cells.

No MeSH data available.


Related in: MedlinePlus

Overexpression of STIM1 and/or ORAI1 in DU145 and PC3 cells remodels the tumor microenvironment.A. Transwell assay of the migration of U937 cells after incubation with the conditioned medium (CM) from DU145 and PC3 cells in the low chamber for 12 h; representative crystal violet staining of U937 cells that migrated and attached to the bottom of the transwell filter are shown. B. Statistical analysis of violet staining intensity, measured as the absorbance at 560 nm (n = 3). C. Real-time RT-PCR analysis of mRNA expression changes of MMP9, VEGFA, IL10, IL6 and CD163 in U937 cells after treatment with the CM of DU145 and PC3 for 48 h (n = 3). D. STIM1 regulates the expression of cytokines in DU145 cells. Upper panel: representative results of cytokine arrays for DU145-YFP and DU145-STIM1-YFP. Lower panel: Statistical analysis of the relevant protein expression level of cytokines in DU145 cells. E. Real-time RT-PCR analysis of mRNA expression changes of IL8, MIF, CD54, CXCL1, IFNG, IL23A and SERPINE1 in DU145 and PC3 cells with STIM1-YFP and /or ORAI1 overexpression in comparison with those in control cells. F. Dual luciferase assay for NF-κB-RE activation in DU145 and PC3 cells.
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f6: Overexpression of STIM1 and/or ORAI1 in DU145 and PC3 cells remodels the tumor microenvironment.A. Transwell assay of the migration of U937 cells after incubation with the conditioned medium (CM) from DU145 and PC3 cells in the low chamber for 12 h; representative crystal violet staining of U937 cells that migrated and attached to the bottom of the transwell filter are shown. B. Statistical analysis of violet staining intensity, measured as the absorbance at 560 nm (n = 3). C. Real-time RT-PCR analysis of mRNA expression changes of MMP9, VEGFA, IL10, IL6 and CD163 in U937 cells after treatment with the CM of DU145 and PC3 for 48 h (n = 3). D. STIM1 regulates the expression of cytokines in DU145 cells. Upper panel: representative results of cytokine arrays for DU145-YFP and DU145-STIM1-YFP. Lower panel: Statistical analysis of the relevant protein expression level of cytokines in DU145 cells. E. Real-time RT-PCR analysis of mRNA expression changes of IL8, MIF, CD54, CXCL1, IFNG, IL23A and SERPINE1 in DU145 and PC3 cells with STIM1-YFP and /or ORAI1 overexpression in comparison with those in control cells. F. Dual luciferase assay for NF-κB-RE activation in DU145 and PC3 cells.

Mentions: Stroma cells from the TME are essential for the growth, chemoresistance and metastasis of cancer cells; the transition of macrophages from the M1 to the M2 phenotype is a vital factor in modulating the TME, thus promoting tumorigenesis and metastasis41. To elucidate the function of SOCE in modulating the TME and tumor-associated macrophages (TAM, M2 phenotype), we tested the effect of STIM1 and ORAI1 on recruitment U937, a human leukemic monocyte macrophage cell line, in both DU145 and PC3 cells. Our study found that the recruitment of U937 is inhibited by the conditioned medium (CM) from DU145 and PC3 cells overexpressing either STIM1-YFP or ORAI1 or both genes (Fig. 6A,B) and increased recruitment of U937 was demonstrated by using the CM from DU145-siSTIM1 and PC3-siSTIM1 when compared with the control from transwell assay (Fig.S5). In addition, real time RT-PCR showed reduced expression levels of IL10, IL6 and CD163 in U937 cells after treatment with CM from DU145 and PC3 cells overexpressing STIM1-YFP and /or ORAI1 (Fig. 6C), indicating that U937 cells experienced a transition from the M2 to the M1 phenotype and that the recruitment of these macrophages was inhibited by the TME of prostate cells possessing enhanced SOCE activity. In addition, the transcripts of VEGFA and MMP9 in U937 were also significantly reduced after treatment with the CM from DU145 or PC3 cells overexpressing STIM1-YFP and /or ORAI1, suggesting that the TME of prostate cancer cells with enhanced SOCE activity may be unfavorable to tumor growth and metastasis. To test this hypothesis, we studied the possible effect of STIM1 on regulating the secretion of cytokines in prostate cancer cells using the cytokine array. The results showed that STIM1 overexpression reduced the secretion of IL-8, macrophage migration inhibitory factor (MIF), CD54 (also known as intercellular adhesion molecule 1), and CXC ligand 1 (CXCL1) and increased the secretion of IFN-γ, IL-23 and Serpin E1 in DU145 cells (Fig. 6D). The screening results were further verified by real time RT-PCR in both DU145 and PC3 cells overexpressing STIM1 (Fig. 6E), except that the mRNA expression of IL23A decreased in DU145-STIM1-YFP and that of IFNG reduced in PC3-STIM1-YFP. It is reported that CXCL1 boosts tumor angiogenesis and development42. IL-8 contributes to tumor progression and metastasis43. MIF regulates innate immunity44 and promotes the metastasis of colorectal cancer45. CD54 promotes prostate tumor metastasis46. IL-23 enhances the production of IFN-γ47. Serpin E1, a serine protease inhibitor, functions as the principal inhibitor of urokinase (uPA) and tissue plasminogen activator (tPA), thus playing an important role in regulating fibrinolysis. With the exceptions of the no significant change for the mRNA expression of IL8 and CD54 in DU145-ORAI1 and increase expression of these two genes in PC3 cells with ORAI1 overexpression and the decrease in the mRNA expression of SERPINE1 in DU145-ORAI1 cells, the effects of ORAI1 on regulating the expression of these cytokine genes are similar to the effects of STIM1 on the secretion of the corresponding proteins observed in DU145 cells (Fig. 6E). These findings support the hypothesis that STIM1 and ORAI1 overexpression promote the formation of an unfavorable TME, which inhibits the development of advanced prostate cancer. In addition, a dual luciferase assay revealed that STIM1 and/or ORAI1 overexpression significantly inhibited the activation of response element (RE) for NFκB (NFκB-RE) (Fig. 6F); this might represent the mechanism that alters the production of cytokines as observed in both the DU145 and PC3 cells.


STIM1 accelerates cell senescence in a remodeled microenvironment but enhances the epithelial-to-mesenchymal transition in prostate cancer.

Xu Y, Zhang S, Niu H, Ye Y, Hu F, Chen S, Li X, Luo X, Jiang S, Liu Y, Chen Y, Li J, Xiang R, Li N - Sci Rep (2015)

Overexpression of STIM1 and/or ORAI1 in DU145 and PC3 cells remodels the tumor microenvironment.A. Transwell assay of the migration of U937 cells after incubation with the conditioned medium (CM) from DU145 and PC3 cells in the low chamber for 12 h; representative crystal violet staining of U937 cells that migrated and attached to the bottom of the transwell filter are shown. B. Statistical analysis of violet staining intensity, measured as the absorbance at 560 nm (n = 3). C. Real-time RT-PCR analysis of mRNA expression changes of MMP9, VEGFA, IL10, IL6 and CD163 in U937 cells after treatment with the CM of DU145 and PC3 for 48 h (n = 3). D. STIM1 regulates the expression of cytokines in DU145 cells. Upper panel: representative results of cytokine arrays for DU145-YFP and DU145-STIM1-YFP. Lower panel: Statistical analysis of the relevant protein expression level of cytokines in DU145 cells. E. Real-time RT-PCR analysis of mRNA expression changes of IL8, MIF, CD54, CXCL1, IFNG, IL23A and SERPINE1 in DU145 and PC3 cells with STIM1-YFP and /or ORAI1 overexpression in comparison with those in control cells. F. Dual luciferase assay for NF-κB-RE activation in DU145 and PC3 cells.
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f6: Overexpression of STIM1 and/or ORAI1 in DU145 and PC3 cells remodels the tumor microenvironment.A. Transwell assay of the migration of U937 cells after incubation with the conditioned medium (CM) from DU145 and PC3 cells in the low chamber for 12 h; representative crystal violet staining of U937 cells that migrated and attached to the bottom of the transwell filter are shown. B. Statistical analysis of violet staining intensity, measured as the absorbance at 560 nm (n = 3). C. Real-time RT-PCR analysis of mRNA expression changes of MMP9, VEGFA, IL10, IL6 and CD163 in U937 cells after treatment with the CM of DU145 and PC3 for 48 h (n = 3). D. STIM1 regulates the expression of cytokines in DU145 cells. Upper panel: representative results of cytokine arrays for DU145-YFP and DU145-STIM1-YFP. Lower panel: Statistical analysis of the relevant protein expression level of cytokines in DU145 cells. E. Real-time RT-PCR analysis of mRNA expression changes of IL8, MIF, CD54, CXCL1, IFNG, IL23A and SERPINE1 in DU145 and PC3 cells with STIM1-YFP and /or ORAI1 overexpression in comparison with those in control cells. F. Dual luciferase assay for NF-κB-RE activation in DU145 and PC3 cells.
Mentions: Stroma cells from the TME are essential for the growth, chemoresistance and metastasis of cancer cells; the transition of macrophages from the M1 to the M2 phenotype is a vital factor in modulating the TME, thus promoting tumorigenesis and metastasis41. To elucidate the function of SOCE in modulating the TME and tumor-associated macrophages (TAM, M2 phenotype), we tested the effect of STIM1 and ORAI1 on recruitment U937, a human leukemic monocyte macrophage cell line, in both DU145 and PC3 cells. Our study found that the recruitment of U937 is inhibited by the conditioned medium (CM) from DU145 and PC3 cells overexpressing either STIM1-YFP or ORAI1 or both genes (Fig. 6A,B) and increased recruitment of U937 was demonstrated by using the CM from DU145-siSTIM1 and PC3-siSTIM1 when compared with the control from transwell assay (Fig.S5). In addition, real time RT-PCR showed reduced expression levels of IL10, IL6 and CD163 in U937 cells after treatment with CM from DU145 and PC3 cells overexpressing STIM1-YFP and /or ORAI1 (Fig. 6C), indicating that U937 cells experienced a transition from the M2 to the M1 phenotype and that the recruitment of these macrophages was inhibited by the TME of prostate cells possessing enhanced SOCE activity. In addition, the transcripts of VEGFA and MMP9 in U937 were also significantly reduced after treatment with the CM from DU145 or PC3 cells overexpressing STIM1-YFP and /or ORAI1, suggesting that the TME of prostate cancer cells with enhanced SOCE activity may be unfavorable to tumor growth and metastasis. To test this hypothesis, we studied the possible effect of STIM1 on regulating the secretion of cytokines in prostate cancer cells using the cytokine array. The results showed that STIM1 overexpression reduced the secretion of IL-8, macrophage migration inhibitory factor (MIF), CD54 (also known as intercellular adhesion molecule 1), and CXC ligand 1 (CXCL1) and increased the secretion of IFN-γ, IL-23 and Serpin E1 in DU145 cells (Fig. 6D). The screening results were further verified by real time RT-PCR in both DU145 and PC3 cells overexpressing STIM1 (Fig. 6E), except that the mRNA expression of IL23A decreased in DU145-STIM1-YFP and that of IFNG reduced in PC3-STIM1-YFP. It is reported that CXCL1 boosts tumor angiogenesis and development42. IL-8 contributes to tumor progression and metastasis43. MIF regulates innate immunity44 and promotes the metastasis of colorectal cancer45. CD54 promotes prostate tumor metastasis46. IL-23 enhances the production of IFN-γ47. Serpin E1, a serine protease inhibitor, functions as the principal inhibitor of urokinase (uPA) and tissue plasminogen activator (tPA), thus playing an important role in regulating fibrinolysis. With the exceptions of the no significant change for the mRNA expression of IL8 and CD54 in DU145-ORAI1 and increase expression of these two genes in PC3 cells with ORAI1 overexpression and the decrease in the mRNA expression of SERPINE1 in DU145-ORAI1 cells, the effects of ORAI1 on regulating the expression of these cytokine genes are similar to the effects of STIM1 on the secretion of the corresponding proteins observed in DU145 cells (Fig. 6E). These findings support the hypothesis that STIM1 and ORAI1 overexpression promote the formation of an unfavorable TME, which inhibits the development of advanced prostate cancer. In addition, a dual luciferase assay revealed that STIM1 and/or ORAI1 overexpression significantly inhibited the activation of response element (RE) for NFκB (NFκB-RE) (Fig. 6F); this might represent the mechanism that alters the production of cytokines as observed in both the DU145 and PC3 cells.

Bottom Line: The importance of store-operated Ca(2+) entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging.Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells.However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China [2] State Key Lab of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.

ABSTRACT
The importance of store-operated Ca(2+) entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging. Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells. In addition, STIM1 and ORAI1 inhibited NF-κB signaling and remodeled the tumor microenvironment by reducing the formation of M2 phenotype macrophages, possibly creating an unfavorable tumor microenvironment and inhibiting cancer development. However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways. Thus, our study revealed novel regulatory effects and the mechanisms by which STIM1 affects cell senescence, tumor migration and the tumor microenvironment, revealing that STIM1 has multiple functions in prostate cancer cells.

No MeSH data available.


Related in: MedlinePlus