Figure 5: ER-independent exit from metabolic dormancy and HTR disease require IL6/Notch3 upregulation.

(a) Western blot analysis of Notch3, ERα, pStat3, Stat3, HK-2, PMK-2, LDHA and Tubulin from extracts of MCF7 cells treated with oligomycin (Oligo, 1 μM) or Tam (1 μM) in the presence/absence of exogenous IL6 (10 ng ml⁻¹, 24 h, see Fig. 4e). (b) OXPHOS and glycolytic potential [AUC of OCR (pMol min⁻¹) and ECAR (mpH min⁻¹) following mitochondrial stress, see Methods] of HTR shCT and shN3 cells treated with Fulv (10 μM) with/without exogenous IL6 (10 ng ml⁻¹, 24 h). Bar graph represents the median AUC±s.d. [AUC of OCR (pMol min); three biological replicates with three technical replicates each]. (c) Number of CD133^hi cells (by Flow analysis) derived from HTR shCT and shN3 cells treated with Fulv (2 months) or MitoS media (2 months, see Methods). (d) Representative image of MS grown from HTR (shCT and shN3) cells in the presence of Fulv (10 μM, 7 days; scale bar, 100 μm). (e) Schematic representation of our experimental model describing metastatic progression in luminal breast cancer. Primary tumour cancer stem cells rely on ER expression/signalling for their self-renewal. HT leads to a reduction in ERα levels and activity promoting a dormant cancer stem cell phenotype (CD133^hi/ER^lo), characterized by increased IL6R expression and decreased mitochondrial activity. Persistent loss of ERα function promotes the survival and self-renewal of CD133^hi/Notch3^hi/ER^lo CSC, which occurs in an IL6 (autocrine)-dependent manner and an ER-independent manner. IL6/Stat3 blockade (tocilizumab) restores ERα expression in tumour cells and suppresses the generation of CD133^hi cells and disrupts the establishment of an IL6^hi/ERα^lo/Notch3^hi-positive feed-forward loop, preventing the self-renewal of HTR CD133^hi CSCs. Data are reported as mean±s.d. of three independent experiments (n=3; b,c). P values (*P<0.05, **P<0.0001) refer to t-test (c) and t-test after GLM ANOVA (b).