Enhanced anti-melanoma efficacy of interferon alfa-2b via inhibition of Shp2

Abstract

Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.

Fig. 1

Shp2 knockdown enhances IFN-α2b-induced STAT1 Tyr-701 phosphorylation. A375/R0946 and A375/R1049 cells were cultured with or without dox for 6 days and stimulated with IFN (1,000 IU/ml) for the indicated times. Cell lysates were analyzed by immunoblotting to assess phosphorylation of STAT1 Tyr-701 (A), STAT2 Tyr-690 (B), and the activating, dual phosphorylation sites of Erk1/2 (C).

Fig. 2

Shp2 knockdown enhances IFN-α2b-induced ISRE-Luc reporter activity and growth inhibition. (A) ISRE-Luc reporter activity was measured as described in Materials and Methods. (B) Cells were cultured in 96-wells in medium with or without dox (3 µg/ml) and IFN-α2b (1,000 IU/ml) as indicated. Relative viable cell numbers were measured on Day 6. Data were from three independent experiments (n = 15). The mean ± SD are shown. *, p < 0.05. #, p <0.05 between the control (-Dox, -IFN) and the treated cells.

Fig. 3

SPI-112Me enhances IFN-α2b-activated STAT1 activation, inhibits Erk1/2, and increases the IFN-α2b-induced cell growth inhibition activity. A375 cells were pre-treated with 12 µM (A) or the indicated concentrations (B) of SPI-112Me or mock-treated with the vehicle overnight and then stimulated with IFN-α2b (1,000 IU/ml) for the indicated time. Cell lysates were analyzed by immunoblotting with the indicated antibodies (A, B). (C) ISRE-Luc reporter activity was determined as described in the Materials and Methods. (D) Cells were cultured in 96-well plates, treated with IFN-α2b (1,000 IU/ml), SPI-112Me (6 µM) or the vehicle. Relative cell numbers were measured on Day 6. Data were from two independent experiments (n=6). *, p < 0.05.

Fig. 4

SPI-112Me enhances IFN-α2b responses in SK-MEL-2 cells. (A) SK-MEL-2 cells were treated with SPI-112Me and/or IFN-α2b and cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) SK-MEL-2 cell proliferation was assayed in 96-well plate cultures. Experiments were performed similar to that described in Fig. 3.

Fig. 5

Effects of Shp2 knockdown on A375 melanoma tumor growth. (A, B) Cells (1 × 10⁶ cells/each) were injected s.c. on both flanks of each mouse on Day 0 (2 sites/mouse). Tumor sizes were measured on the indicated days. Each group has six mice bearing 12 tumors of similar sizes at the start of dox induction on Day 6 (A375/R0946 tumors) or Day 10 (A375/R1049 tumors) as indicated by the arrow. Arrowhead indicates the starting date of IFN-α2b treatment (Day 9 and 14, respectively as shown in the graphs). Mice were euthanized at the end points (Day 27 or Day 28 as shown in the graphs). Mean ± SEM are shown. *, p < 0.05. (C) Immunoblotting analysis of A375/R1049 tumor samples.