Figure 3

Continued use of trastuzumab in HER2⁺ breast cancer with PTEN inactivation switches signaling pathway dependence from HER/AKT to IL-6/STAT3/NF-κB signaling.

(A) mRNA expression of endogenous inflammatory cytokines IL-6 and IL-8 normalized to GAPDH as identified by real time PCR. N = 4 (B) Protein expression in pg/ml of IL-6 and IL-8 secreted into culture media, measured using quantitative enzyme-linked immunosorbent assay (ELISA). N = 3 (C) Normalized mRNA expression (reads/kilobase/million reads) of IL-6 signal transducer GP130 (left) and downstream signaling node STAT3 (right) by RNA-sequencing. N = 4 (D) Western blot analysis of protein expression for GP130, mesenchymal cell marker vimentin and PTEN tumor suppressor in BT474, BT474 PTEN⁻ and BT474 PTEN⁻ LTT cell lines. (E) ELISA relative quantitation of active STAT3, phosphorylated at Tyr705. N = 3 (F) Luciferase activity of cell lines following infection of with retroviral vector inducing NF-κB driven expression of luciferase. N = 3. (G) Model of trastuzumab resistance in PTEN deficient cells, T.F.s = other transcription factors. Data shown as average ± SD. *p ≤ 0.05. #p ≤ 0.01.