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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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DcR3 promotes cellular migration in RCC. (A) Immunoblot analysis of whole-cell lysates showing DcR3 protein levels in different human RCC cell lines and human embryonic kidney 293-T cells. (B) Immunoblot analysis of precipitated protein from supernatant showing DcR3 protein secretion in different human RCC cell lines and human embryonic kidney 293-T cells. Ponceau staining is used to demonstrate equal protein loading. (C,D) Immunoblot analysis of whole-cell lysates (left) and precipitated protein from supernatant (right) of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) (C) or stably overexpressing DcR3 or an empty control vector (neo) (D). (E) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram). Bars indicate the percentage of cell migration in relation to cells transfected with a non-specific siRNA after 24 h (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test). Representative images are shown in Additional file 1: Figure S1D. (F) Scratch motility assay of ACHN and 769-P cells stably overexpressing DcR3 or an empty control vector (neo). Migration was measured over a time course of 12 h (ACHN) or 24 h (769-P). Bars indicate the percentage of cell migration in relation to cells stably transfected with an empty control vector (mean ± SEM; n=3; *p<0.05, **p<0.01, T-test). Representative images are shown in Additional file 1: Figure S1E. (G) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) and incubated with either DcR3-containing or control supernatant (SN) of stable transfectants. Migration was measured over a time course of 24 h. Bars indicate the percentage of cell migration in relation to cells transfected with a non–specific siRNA and treated with control supernatant (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
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Figure 1: DcR3 promotes cellular migration in RCC. (A) Immunoblot analysis of whole-cell lysates showing DcR3 protein levels in different human RCC cell lines and human embryonic kidney 293-T cells. (B) Immunoblot analysis of precipitated protein from supernatant showing DcR3 protein secretion in different human RCC cell lines and human embryonic kidney 293-T cells. Ponceau staining is used to demonstrate equal protein loading. (C,D) Immunoblot analysis of whole-cell lysates (left) and precipitated protein from supernatant (right) of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) (C) or stably overexpressing DcR3 or an empty control vector (neo) (D). (E) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram). Bars indicate the percentage of cell migration in relation to cells transfected with a non-specific siRNA after 24 h (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test). Representative images are shown in Additional file 1: Figure S1D. (F) Scratch motility assay of ACHN and 769-P cells stably overexpressing DcR3 or an empty control vector (neo). Migration was measured over a time course of 12 h (ACHN) or 24 h (769-P). Bars indicate the percentage of cell migration in relation to cells stably transfected with an empty control vector (mean ± SEM; n=3; *p<0.05, **p<0.01, T-test). Representative images are shown in Additional file 1: Figure S1E. (G) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) and incubated with either DcR3-containing or control supernatant (SN) of stable transfectants. Migration was measured over a time course of 24 h. Bars indicate the percentage of cell migration in relation to cells transfected with a non–specific siRNA and treated with control supernatant (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).

Mentions: As our previous work demonstrates a clinical significance of DcR3 overexpression in RCC [19], we were interested in functionally characterizing DcR3 in RCC. To this end, we started to analyze several RCC cell lines for endogenous expression of DcR3 on mRNA and protein level by quantitative RT-PCR and immunoblot analysis. Human embryonic kidney derived 293-T cells were used as a control kidney cell line. Six out of eight RCC cell lines showed a moderate to high expression of DcR3 whereas 293T cells lacked DcR3 expression (Figure 1A; Additional file 1: Figure S1A). As DcR3 is a soluble protein, we additionally investigated its secretion by DcR3 expressing tumor cells. We detected DcR3 in the supernatant of all DcR3 expressing cell lines tested (Figure 1B).


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)

DcR3 promotes cellular migration in RCC. (A) Immunoblot analysis of whole-cell lysates showing DcR3 protein levels in different human RCC cell lines and human embryonic kidney 293-T cells. (B) Immunoblot analysis of precipitated protein from supernatant showing DcR3 protein secretion in different human RCC cell lines and human embryonic kidney 293-T cells. Ponceau staining is used to demonstrate equal protein loading. (C,D) Immunoblot analysis of whole-cell lysates (left) and precipitated protein from supernatant (right) of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) (C) or stably overexpressing DcR3 or an empty control vector (neo) (D). (E) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram). Bars indicate the percentage of cell migration in relation to cells transfected with a non-specific siRNA after 24 h (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test). Representative images are shown in Additional file 1: Figure S1D. (F) Scratch motility assay of ACHN and 769-P cells stably overexpressing DcR3 or an empty control vector (neo). Migration was measured over a time course of 12 h (ACHN) or 24 h (769-P). Bars indicate the percentage of cell migration in relation to cells stably transfected with an empty control vector (mean ± SEM; n=3; *p<0.05, **p<0.01, T-test). Representative images are shown in Additional file 1: Figure S1E. (G) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) and incubated with either DcR3-containing or control supernatant (SN) of stable transfectants. Migration was measured over a time course of 24 h. Bars indicate the percentage of cell migration in relation to cells transfected with a non–specific siRNA and treated with control supernatant (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
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Figure 1: DcR3 promotes cellular migration in RCC. (A) Immunoblot analysis of whole-cell lysates showing DcR3 protein levels in different human RCC cell lines and human embryonic kidney 293-T cells. (B) Immunoblot analysis of precipitated protein from supernatant showing DcR3 protein secretion in different human RCC cell lines and human embryonic kidney 293-T cells. Ponceau staining is used to demonstrate equal protein loading. (C,D) Immunoblot analysis of whole-cell lysates (left) and precipitated protein from supernatant (right) of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) (C) or stably overexpressing DcR3 or an empty control vector (neo) (D). (E) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram). Bars indicate the percentage of cell migration in relation to cells transfected with a non-specific siRNA after 24 h (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test). Representative images are shown in Additional file 1: Figure S1D. (F) Scratch motility assay of ACHN and 769-P cells stably overexpressing DcR3 or an empty control vector (neo). Migration was measured over a time course of 12 h (ACHN) or 24 h (769-P). Bars indicate the percentage of cell migration in relation to cells stably transfected with an empty control vector (mean ± SEM; n=3; *p<0.05, **p<0.01, T-test). Representative images are shown in Additional file 1: Figure S1E. (G) Scratch motility assay of ACHN and 769-P cells transfected with two different DcR3-specific siRNAs or a non-specific siRNA (scram) and incubated with either DcR3-containing or control supernatant (SN) of stable transfectants. Migration was measured over a time course of 24 h. Bars indicate the percentage of cell migration in relation to cells transfected with a non–specific siRNA and treated with control supernatant (mean ± SEM; n=3; *p<0.05, **p<0.01; T-test).
Mentions: As our previous work demonstrates a clinical significance of DcR3 overexpression in RCC [19], we were interested in functionally characterizing DcR3 in RCC. To this end, we started to analyze several RCC cell lines for endogenous expression of DcR3 on mRNA and protein level by quantitative RT-PCR and immunoblot analysis. Human embryonic kidney derived 293-T cells were used as a control kidney cell line. Six out of eight RCC cell lines showed a moderate to high expression of DcR3 whereas 293T cells lacked DcR3 expression (Figure 1A; Additional file 1: Figure S1A). As DcR3 is a soluble protein, we additionally investigated its secretion by DcR3 expressing tumor cells. We detected DcR3 in the supernatant of all DcR3 expressing cell lines tested (Figure 1B).

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