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The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma.

Weissinger D, Tagscherer KE, Macher-Göppinger S, Haferkamp A, Wagener N, Roth W - Mol. Cancer (2013)

Bottom Line: Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation.Further, we identified a signaling pathway regulating DcR3 expression in RCC.Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Tumor-Pathology, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany. Wilfried.Roth@med.uni-heidelberg.de.

ABSTRACT

Background: Overexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown.

Methods: Modulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens.

Results: Here, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).

Conclusions: Taken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

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NFATc1 regulates DcR3 expression at a transcriptional level. (A,B) Immunoblot analysis of whole-cell lysates and quantitative real-time-PCR assaying relative DcR3 mRNA expression of ACHN and 769-P cells 24 h after treatment with cyclosporin A (CsA, 25 μM) or Tacrolimus (FK-506, 50 μM) (A); 48 h post transfection with NFATc1 or an empty vector control (neo) (B). Expression data were normalized to internal 18S rRNA expression (mean ± SEM; n=3; *p<0.05, **p<0.01, ***p<0.001; T-test). (C) Immunoblot analysis of cytoplasmic and nuclear fractions of ACHN and 769-P cells after treatment with LY294002 (50 μM), Everolimus (1 μM), or Cyclosporine A (25 μM). (D) Relative NFATc1–luciferase reporter activity of ACHN and 769-P cells 24 h post transfection with myrAkt or an empty vector control (neo) (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
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Figure 5: NFATc1 regulates DcR3 expression at a transcriptional level. (A,B) Immunoblot analysis of whole-cell lysates and quantitative real-time-PCR assaying relative DcR3 mRNA expression of ACHN and 769-P cells 24 h after treatment with cyclosporin A (CsA, 25 μM) or Tacrolimus (FK-506, 50 μM) (A); 48 h post transfection with NFATc1 or an empty vector control (neo) (B). Expression data were normalized to internal 18S rRNA expression (mean ± SEM; n=3; *p<0.05, **p<0.01, ***p<0.001; T-test). (C) Immunoblot analysis of cytoplasmic and nuclear fractions of ACHN and 769-P cells after treatment with LY294002 (50 μM), Everolimus (1 μM), or Cyclosporine A (25 μM). (D) Relative NFATc1–luciferase reporter activity of ACHN and 769-P cells 24 h post transfection with myrAkt or an empty vector control (neo) (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).

Mentions: GSK-3β and the family of FOXO transcription factors are both known to negatively regulate the transcription factor NFAT (nuclear factor of activated T-cells) [25]. Therefore, we investigated its role in the transcriptional regulation of DcR3. We treated the cells with Cyclosporine A or FK-506 (Tacrolimus) which are both immunosuppressants that inactivate calcineurin, the major activator of NFAT. Inhibition of calcineurin dramatically decreased the expression of DcR3 (Figure 5A), indicating a functional relevance of NFAT in DcR3 regulation. Accordingly, NFAT overexpression resulted in an increase in DcR3 expression level (Figure 5B). To demonstrate that modulation of the PI3K/AKT pathway affects NFAT expression, we performed nuclear and cytoplasmic fractionation and detected a shift of NFAT localization to the cytoplasm upon PI3K inhibition. A similar shift was detectable after Cyclosporine A treatment which served as a positive control. In contrast, treatment with Everolimus had no impact on NFAT localization, confirming an mTOR independent regulation (Figure 5C). In addition, the activity of NFAT was enhanced upon overexpression of a constitutively active form of AKT (Figure 5D).


The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma.

Weissinger D, Tagscherer KE, Macher-Göppinger S, Haferkamp A, Wagener N, Roth W - Mol. Cancer (2013)

NFATc1 regulates DcR3 expression at a transcriptional level. (A,B) Immunoblot analysis of whole-cell lysates and quantitative real-time-PCR assaying relative DcR3 mRNA expression of ACHN and 769-P cells 24 h after treatment with cyclosporin A (CsA, 25 μM) or Tacrolimus (FK-506, 50 μM) (A); 48 h post transfection with NFATc1 or an empty vector control (neo) (B). Expression data were normalized to internal 18S rRNA expression (mean ± SEM; n=3; *p<0.05, **p<0.01, ***p<0.001; T-test). (C) Immunoblot analysis of cytoplasmic and nuclear fractions of ACHN and 769-P cells after treatment with LY294002 (50 μM), Everolimus (1 μM), or Cyclosporine A (25 μM). (D) Relative NFATc1–luciferase reporter activity of ACHN and 769-P cells 24 h post transfection with myrAkt or an empty vector control (neo) (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
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Figure 5: NFATc1 regulates DcR3 expression at a transcriptional level. (A,B) Immunoblot analysis of whole-cell lysates and quantitative real-time-PCR assaying relative DcR3 mRNA expression of ACHN and 769-P cells 24 h after treatment with cyclosporin A (CsA, 25 μM) or Tacrolimus (FK-506, 50 μM) (A); 48 h post transfection with NFATc1 or an empty vector control (neo) (B). Expression data were normalized to internal 18S rRNA expression (mean ± SEM; n=3; *p<0.05, **p<0.01, ***p<0.001; T-test). (C) Immunoblot analysis of cytoplasmic and nuclear fractions of ACHN and 769-P cells after treatment with LY294002 (50 μM), Everolimus (1 μM), or Cyclosporine A (25 μM). (D) Relative NFATc1–luciferase reporter activity of ACHN and 769-P cells 24 h post transfection with myrAkt or an empty vector control (neo) (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
Mentions: GSK-3β and the family of FOXO transcription factors are both known to negatively regulate the transcription factor NFAT (nuclear factor of activated T-cells) [25]. Therefore, we investigated its role in the transcriptional regulation of DcR3. We treated the cells with Cyclosporine A or FK-506 (Tacrolimus) which are both immunosuppressants that inactivate calcineurin, the major activator of NFAT. Inhibition of calcineurin dramatically decreased the expression of DcR3 (Figure 5A), indicating a functional relevance of NFAT in DcR3 regulation. Accordingly, NFAT overexpression resulted in an increase in DcR3 expression level (Figure 5B). To demonstrate that modulation of the PI3K/AKT pathway affects NFAT expression, we performed nuclear and cytoplasmic fractionation and detected a shift of NFAT localization to the cytoplasm upon PI3K inhibition. A similar shift was detectable after Cyclosporine A treatment which served as a positive control. In contrast, treatment with Everolimus had no impact on NFAT localization, confirming an mTOR independent regulation (Figure 5C). In addition, the activity of NFAT was enhanced upon overexpression of a constitutively active form of AKT (Figure 5D).

Bottom Line: Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation.Further, we identified a signaling pathway regulating DcR3 expression in RCC.Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Tumor-Pathology, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany. Wilfried.Roth@med.uni-heidelberg.de.

ABSTRACT

Background: Overexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown.

Methods: Modulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens.

Results: Here, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).

Conclusions: Taken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

Show MeSH
Related in: MedlinePlus