Figure 3: Loss of HIF-2α increases mTORC1 signalling in soft tissue sarcomas.

(a) Gene set enrichment analysis (GSEA) comparing expression data of KP ‘WT’ and KPH2 ‘Hif2a’ autochthonous UPS tumours. (b) Immunoblot assessing mTORC1 and mTORC2 activity in KP and KPH2 tumours. Phosphorylated-(p) 4E-BP1 (indicated with an *) and S6K1 were used as mTORC1 readouts, and (p)-AKT was used as an mTORC2 readout. (c) Left: representative images of immunohistochemical staining of phosphorylated-S6 (phospho-S6) on KP (n=5) and KPH2 (n=5) tumours. Right: quantification of phospho-S6⁺ cells in KP and KPH2 tumours. 10 high-powered fields per tumour were quantified. ***P<0.001. (d) Immunoblot of 4E-BP1 in cell lines derived from KP and KPH2 tumours. Cells were subjected to 1% O₂ for 16 h (H) or grown at 21% O₂ (N). (e) Expression of 4E-BP1 and S6K1 phosphorylation in LPS246 xenografts with scrambled (SCR) or HIF-2α (H2α) shRNA. (f) Left: representative images of phospho-S6 immunohistochemical staining on LPS246 xenografts with SCR (n=5) or HIF-2α (H2α) shRNA (n=5; error bars are ±s.e.m.). 10 high-powered fields per tumour were quantified. *P<0.05. (g) Quantitative reverse trascriptase-PCR validation of Ano1 mRNA expression in KP (n=4) and KPH2 (n=3) tumours used for RNA-seq *P<0.05. (h) Immunoblot of ANO1 and downstream targets p-EGFR (Y1068) and p-CaMKIIα (T268) in KP and KPH2 autochthonous tumours. (i) KP and KPH2 cells were serum starved for 24 h, then replete media with DMSO or CaCCInh-A01 (10 μM) was added for 6 h to the cells. Lysates were immunoblotted for p-CAMKIIα and mTORC1 readouts p-4E-BP1 and p-S6K1. (j) ANO1 inhibitor’s effect on KP and KPH2 cell growth. Cells were treated with DMSO or CaCCInh-A01 (10 μM) for 3 days in 21% or 1% O₂ conditions. Shown is the percentage of cells counted in the CaCCInh-A01 treated versus the respective DMSO-treated control, with each bar representing three biological triplicates. **P<0.01. (k) Left: tumour volume of KP-derived UPS allografts expressing SCR (n=5) or Ano1 shRNA (Ano1 2; n=5). Right: tumour volume of KPH2-derived UPS allografts expressing SCR (n=5) or Ano1 shRNA (n=5). **P<0.01. (l) Relative average size of KP and KPH2 tumours infected with SCR or Ano1 shRNA. The SCR shRNA average volume was normalized to 1.0 for both KP and KPH2 cohorts. **P<0.01. All error bars represent the mean±s.e.m. All P-values were calculated using a two-tailed Student’s t-test.