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Efficient TGF-β/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression.

Javelaud D, van Kempen L, Alexaki VI, Le Scolan E, Luo K, Mauviel A - Mol. Cancer (2011)

Bottom Line: There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice.TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity.Its highly labile nature makes it an unlikely target for therapeutic intervention.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Curie, University Center, Building 110, Orsay, France.

ABSTRACT

Background: SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.

Results: In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.

Conclusions: Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

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Another High SKI expression in melanoma cells does not prevent efficient transcriptional responses to TGF-β. A. 1205Lu melanoma cells were incubated for 60 min. with increasing concentrations of TGF-β. Cell extracts were then prepared and subjected to Western analysis for SKI and P-SMAD3 content. B. 1205Lu melanoma cells were transfected in triplicate dishes with (CAGA)9-MLP-luc and pRL-TK. Four hours later, TGF-β was added at the indicated concentrations and reporter activity was measured 16 h later. Results are mean+/- SD from one representative experiment. C. Three distinct human melanoma cell lines (501mel, 1205Lu and WM852) were incubated with TGF-β (10 ng/ml) for various time periods. Cell extracts were prepared and subjected to Western analysis to determine SKI content. Actin was used as an internal control. D. 1205Lu cells were incubated for 1 h in the absence (DMSO) or presence (SB) of the TβRI inhibitor SB431542 (5 μM) prior to addition of TGF-β (10 ng/ml). Protein extracts were prepared 60 min later and subjected to Western analysis for SKI content.
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Figure 2: Another High SKI expression in melanoma cells does not prevent efficient transcriptional responses to TGF-β. A. 1205Lu melanoma cells were incubated for 60 min. with increasing concentrations of TGF-β. Cell extracts were then prepared and subjected to Western analysis for SKI and P-SMAD3 content. B. 1205Lu melanoma cells were transfected in triplicate dishes with (CAGA)9-MLP-luc and pRL-TK. Four hours later, TGF-β was added at the indicated concentrations and reporter activity was measured 16 h later. Results are mean+/- SD from one representative experiment. C. Three distinct human melanoma cell lines (501mel, 1205Lu and WM852) were incubated with TGF-β (10 ng/ml) for various time periods. Cell extracts were prepared and subjected to Western analysis to determine SKI content. Actin was used as an internal control. D. 1205Lu cells were incubated for 1 h in the absence (DMSO) or presence (SB) of the TβRI inhibitor SB431542 (5 μM) prior to addition of TGF-β (10 ng/ml). Protein extracts were prepared 60 min later and subjected to Western analysis for SKI content.

Mentions: We next investigated whether high SKI levels in melanoma cells are associated with an absence of transcriptional responses to TGF-β. Incubation of 1205Lu melanoma cells with increasing concentrations of TGF-β for 30 min lead to a dose-dependent decrease in SKI protein content (Figure 2A), accompanied with an inversely correlated increase in P-SMAD3 levels. Parallel transient cell transfection experiments with SMAD3/4-specific (CAGA)9-MLP-luc reporter construct indicated dose-dependent transcriptional activation in response to TGF-β (Figure 2B).


Efficient TGF-β/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression.

Javelaud D, van Kempen L, Alexaki VI, Le Scolan E, Luo K, Mauviel A - Mol. Cancer (2011)

Another High SKI expression in melanoma cells does not prevent efficient transcriptional responses to TGF-β. A. 1205Lu melanoma cells were incubated for 60 min. with increasing concentrations of TGF-β. Cell extracts were then prepared and subjected to Western analysis for SKI and P-SMAD3 content. B. 1205Lu melanoma cells were transfected in triplicate dishes with (CAGA)9-MLP-luc and pRL-TK. Four hours later, TGF-β was added at the indicated concentrations and reporter activity was measured 16 h later. Results are mean+/- SD from one representative experiment. C. Three distinct human melanoma cell lines (501mel, 1205Lu and WM852) were incubated with TGF-β (10 ng/ml) for various time periods. Cell extracts were prepared and subjected to Western analysis to determine SKI content. Actin was used as an internal control. D. 1205Lu cells were incubated for 1 h in the absence (DMSO) or presence (SB) of the TβRI inhibitor SB431542 (5 μM) prior to addition of TGF-β (10 ng/ml). Protein extracts were prepared 60 min later and subjected to Western analysis for SKI content.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Another High SKI expression in melanoma cells does not prevent efficient transcriptional responses to TGF-β. A. 1205Lu melanoma cells were incubated for 60 min. with increasing concentrations of TGF-β. Cell extracts were then prepared and subjected to Western analysis for SKI and P-SMAD3 content. B. 1205Lu melanoma cells were transfected in triplicate dishes with (CAGA)9-MLP-luc and pRL-TK. Four hours later, TGF-β was added at the indicated concentrations and reporter activity was measured 16 h later. Results are mean+/- SD from one representative experiment. C. Three distinct human melanoma cell lines (501mel, 1205Lu and WM852) were incubated with TGF-β (10 ng/ml) for various time periods. Cell extracts were prepared and subjected to Western analysis to determine SKI content. Actin was used as an internal control. D. 1205Lu cells were incubated for 1 h in the absence (DMSO) or presence (SB) of the TβRI inhibitor SB431542 (5 μM) prior to addition of TGF-β (10 ng/ml). Protein extracts were prepared 60 min later and subjected to Western analysis for SKI content.
Mentions: We next investigated whether high SKI levels in melanoma cells are associated with an absence of transcriptional responses to TGF-β. Incubation of 1205Lu melanoma cells with increasing concentrations of TGF-β for 30 min lead to a dose-dependent decrease in SKI protein content (Figure 2A), accompanied with an inversely correlated increase in P-SMAD3 levels. Parallel transient cell transfection experiments with SMAD3/4-specific (CAGA)9-MLP-luc reporter construct indicated dose-dependent transcriptional activation in response to TGF-β (Figure 2B).

Bottom Line: There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice.TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity.Its highly labile nature makes it an unlikely target for therapeutic intervention.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Curie, University Center, Building 110, Orsay, France.

ABSTRACT

Background: SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.

Results: In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.

Conclusions: Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

Show MeSH
Related in: MedlinePlus