Figure 3: Distinct ShcA-driven phosphotyrosine signalling networks differentially activate STAT3 and STAT1 signalling in breast cancer cells.

(a) Immunoblot analysis of total cell lysates from MT/ShcA^+/+, MT/Shc^2F/2F and MT/Shc^313F/313F breast cancer cells (four to five per genotype) using STAT1, pY701-STAT1, STAT3, pY705-STAT3 and Tubulin antibodies. (b) Densitometric quantification of immunoblots using ImageJ software. The data show average fold change in expression levels (as indicated)±s.d. in the individual cell lines from three independent experiments. (c) Growth curves for individual tumour MT/Shc^2F/2F (5372) mammary tumours that emerge in an immunocompetent FVB background (IFNγ^+/+). Each line describes the tumour volume (mm³) of an individual breast tumour (BT) at the indicated days post injection and is representative of two independent experiments. PD, progressive disease (dark red dot), SD, stable disease (pink dot). (d) Immunohistochemical staining of mammary tumours that emerged in IFNγ^+/+ or IFNγ^−/− mice using STAT1- and pY705-STAT3-specific antibodies (n=6–8 tumours per genotype). The mean percentage of STAT1⁺ and pY705-STAT3⁺ stained nuclei±s.e.m. is shown. MT/Shc^2F/2F tumours that displayed PD or SD phenotypes were stratified. The data are representative of two independent experiments and significance was analysed by Wilcoxon’s rank-sum test (*P<0.05 and **P<0.01). (e) Representative images of STAT1- and pY705-STAT3-stained paraffin-embedded sections. Scale bars, 50 μm. (f) Schematic diagram summarizing how altered pY239/240- and pY313-ShcA signalling affect STAT1 and STAT3 activation, both in established cell lines in vitro and in mammary tumours in vivo (induced by the tumour microenvironment, TME). The consequence of the individual ShcA tyrosine phosphorylation sites on emergence of pro- or anti-tumorigenic immune responses is also shown.