Modulation of SOCS protein expression influences the interferon responsiveness of human melanoma cells

Abstract

Background: Endogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells.

Methods: Basal and interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells. Over-expression of SOCS1 and SOCS3 proteins in melanoma cells was achieved using the PINCO retroviral vector, while siRNA were used to inhibit SOCS1 and SOCS3 expression. Tyr701-phosphorylated STAT1 (P-STAT1) was measured by intracellular flow cytometry and IFN-stimulated gene expression was measured by Real Time PCR.

Results: SOCS1 and SOCS3 proteins were expressed at basal levels in melanocytes and in all melanoma cell lines examined. Expression of the SOCS1 and SOCS3 proteins was also enhanced following stimulation of a subset of cell lines with IFN-alpha or IFN-gamma. Over-expression of SOCS proteins in melanoma cell lines led to significant inhibition of Tyr701-phosphorylated STAT1 (P-STAT1) and gene expression following stimulation with IFN-alpha (IFIT2, OAS-1, ISG-15) or IFN-gamma (IRF1). Conversely, siRNA inhibition of SOCS1 and SOCS3 expression in melanoma cells enhanced their responsiveness to interferon stimulation.

Conclusions: These data demonstrate that SOCS proteins are expressed in human melanoma cell lines and their modulation can influence the responsiveness of melanoma cells to IFN-alpha and IFN-gamma.

Figure 1

SOCS1, SOCS3 and STAT3 expression in melanoma cell lines. (a) Basal expression of SOCS1 and SOCS3 proteins in a panel of n = 10 human melanoma cell lines was measured by immunoblot analysis. β-actin served as a control for equal loading. Basal and IFN-induced SOCS1 and SOCS3 expression was evaluated in (b) human metastatic melanoma cell lines and (c) human embryonic melanocytes (HEM) or vertical (WM793b) and radial (WM1552c) human melanoma cell lines by immunoblot following treatment of human melanoma cell lines with IFN-α (4 hours; 10⁴U/mL) and IFN-γ (4 hours; 10 ng/mL). A375 cells were used as a positive control for SOCS1 and SOCS3 expression (Pos. Ctrl). Immunoblot data shown are representative from at least 3 independent experiments. (d) Constitutive phosphorylation of STAT3 was evaluated in this panel of cell lines by immunoblot analysis. Antibodies directed against STAT3 protein and β-actin were included to control for variations in STAT3 across cell lines and equal loading, respectively.

Figure 2

Retroviral mediated over-expression of SOCS1 and SOCS3. GFP expression was evaluated by flow cytometry in melanoma cells stably transduced with PINCO retroviral vectors over-expressing SOCS1 or SOCS3. A representative example is shown in (a). Over-expression of (b) SOCS1 and (c) SOCS3 at the protein level was confirmed by immunoblot analysis. Membranes were stripped and re-probed with β-actin as a control for equal loading.

Figure 3

Reduced IFN-induced P-STAT1 in melanoma cells over-expressing SOCS1 and SOCS3. Phosphorylated STAT1 was measured by flow cytometry following a 15-minute stimulation of SOCS1- and SOCS3-over-expressing cell lines with (a, b) IFN-α or (c, d) IFN-γ. Both non-transduced and empty vector transduced melanoma cell lines served as negative controls in these experiments. Error bars represent standard deviation and are derived from triplicate experiments. * Denotes p < 0.05 as compared to empty vector transduced cells.

Figure 4

Reduced IFN-stimulated gene expression in melanoma cells over-expressing SOCS1 and SOCS3. Transcript levels of the IFN-α-responsive genes (a) ISG-15, (b) OAS-1, and (c) IFIT2 were measured following a 4 hour stimulation of SOCS1- and SOCS3-over-expressing cell lines with IFN-α (10⁴ U/mL) by Real Time PCR. (d) Transcript levels of the IFN-γ-responsive gene IRF1 was also measured in the cell lines following a 4 hour stimulation with 1 ng/mL IFN-γ. All data were normalized to β-actin (housekeeping gene) and expressed relative to PBS-treated cells. * Denotes p < 0.05 as compared to empty vector transduced cells.

Figure 5

siRNA-mediated reduction of SOCS1 and SOCS3 enhances IFN-responsiveness of melanoma cells. Real Time PCR was used to validate reduced expression of (a) SOCS1 and (b) SOCS3 48 hours following transient transfection of 1259 MEL cells with empty pSilencer vector (Control) or pSilencer expressing SOCS-specific siRNA. Data were normalized to β-actin (housekeeping gene) and expressed as mean expression values relative to cells transfected with control vector. (c) P-STAT1 levels were measured following a 15 minute stimulation of melanoma cells with IFN-α (10⁴ U/mL) or IFN-γ (1 ng/mL) by flow cytometry. PBS (vehicle) treated cells served as negative controls. Data represent the mean Fsp values (± standard deviation) from n = 2 experiments with similar results. (d) IFN-stimulated gene expression was evaluated in response to a 4 hour treatment with IFN-α (10⁴ U/mL) 48 hours post-transfection with empty vector (negative control) or vector expressing SOCS1- or SOCS3-specific siRNA. Data were normalized to β-actin and expressed relative to PBS-treated cells. * Denotes p < 0.05 as compared to empty vector transfected cells.