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. 2020 Jun 11;12(6):1540. doi: 10.3390/cancers12061540

Figure 3.

Figure 3

HSP27 directly modulates HER2 and its downstream protein kinase B (AKT) pathway to induce TZMB-resistance in HER2+ BC. (A) Direct interactions between HSP27 and HER2 were confirmed in JIMT-1 cells using immunoprecipitation assay. (B) Extent of the HSP27 and HER2 interaction was compared between BT-P and BT-TR cells. Binding between the two proteins was increased in BT-TR cells and was significantly reduced along with HSP27 knockdown. (C) HSP27-mediated upregulation of HER2 was confirmed by transiently overexpressing HSP27. The fluorescence intensity of HER2 was increased only in those cells in which overexpression of HSP27 was successfully induced. Scale bars = 20 μM. (D) Alterations in the mRNA level were assessed by silencing HSP27 in BT-TR cells and restoring HSP27 expression in BT-TR shHSP27 cells. In all cases, the mRNA level of HER2 did not change significantly (n = 3). ANOVA, *** p < 0.001. (E) The overexpression of HER2 and HSP27-mediated changes in the endogenous level of HER2 and HSP27 were evaluated. Transduction of HER2 did not change HSP27 expression. (F–H) Changes in TZMB responsiveness were assessed by silencing HSP27 in BT-TR cells and re-expressing HSP27 in BT-TR shHSP27 cells. HSP27 silencing significantly downregulated HER2 and phospho-AKT (p-AKT) ((F) 12 h treatment of TZMB), leading to short-term ((G) 48 h treatment of 10 μg/mL of TZMB) and long-term ((H) 10-day incubation with TZMB) growth inhibition in response to TZMB. Inversely, restoration of HSP27 in BT-TR shHSP27 completely rescued the TZMB-resistant phenotype. ANOVA, *** p < 0.001.