Fig 2: AKTIP loss promotes tumorigenic phenotypes selectively in luminal breast cancer cells(A) AKTIP mRNA levels between matched normal breast tissues and tumor samples of all subtypes (n = 111 pairs) or luminal subtype only (n = 84 pairs) in TCGA cohort. p values of Wilcoxon signed rank test are shown.(B) Relapse-free survival of patients with ERa-, progesterone receptor (PR)-, and HER2-positive (top) and ERa-, PR-, and HER2-negative (bottom) breast cancer with a lower tertile of AKTIP mRNA level as cutoff. The analysis was generated by KM plotter using expression data obtained from Gene Expression Omnibus datasets. Hazard ratio (HR), 95% confidence interval, and log rank p values are shown.(C and D) Cells were transfected with AKTIP siRNA for 24 h before seeding into 96-well plate or culture insert.(C) Cell viability of ERa-positive MCF7 and MDA-MB-361 cells (left), HER2-enriched SKBR3 and HCC1954 cells (middle), and basal-like MDA-MB-231 and MDA-MB-468 cells (right) was measured over 7 days. Day 0 was the day of cell seeding. Data show mean ± SD of triplicates and one representative of three independent experiments. **p < 0.01; ***p < 0.001; ns, no significant difference by two-way ANOVA with Sidak’s multiple comparison test.(D) Cells were allowed to migrate or invade for 16 h. Mean numbers (top) and representative images (bottom) of migrated or invaded MCF7, MDA-MB-361, SKBR3, or HCC1954 cells of five fields at a magnification of 100× (MCF7 and HCC1954), 200× (MDA-MB-361), or 20× (SKBR3) are shown. Scale bar, 200 µm. Data shown are one of three independent experiments.(E) MCF7 cells stably expressing AKTIP shRNA or vector were subcutaneously injected into nude mice (n = 10) for 6 weeks. Image of tumor nodules extracted (left) and tumor weight and volume (right) are shown. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference compared with mock or vector using t test.

Fig 3: ERa protein levels are increased in AKTIP-depleted luminal breast cancer cells(A) Lysates of MCF7 cells transfected with AKTIP siRNA or mock for 72 h were harvested for reverse-phase protein array. The heatmap shows proteins with >20% change in levels in AKTIP-depleted cells normalized to mock-transfected cells.(B and C) Lysates of MCF7 cells stably expressing AKTIP shRNA and MDA-MB-361 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting for proteins of the (B) AKT and (C) ERa pathways. ERK2 was loading control.(D) Cells were transfected with siRNA targeting the 3' UTR of AKTIP. Eight h later, lentivirus for AKTIP overexpression (OX) was added to the culture for another 72 h prior to protein harvest for western blotting. ERK2 was loading control.(E) Representative immunohistochemical images of AKTIP-depleted and vector xenograft tumor sections stained with anti-AKTIP, anti-ERa, or anti-Ki67 antibody. Scale bar, 100 µm.(F) Human breast tumor tissue samples were subjected to immunohistochemical staining using anti-AKTIP or anti-ERa antibody. Top, representative immunohistochemical images. The boxes depict magnified areas. Scale bar, 200 µm. Bottom, heatmap illustrating the correlation between ERa and AKTIP staining intensities. p value of Pearson correlation analysis is shown.(G) Lysates of MCF7 cells stably expressing AKTIP shRNA and MDA-MB-361 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting for ERß levels. The western blots shown are representatives of three independent experiments.

Fig 4: JAK2 or STAT3 inhibition resensitizes AKTIP-depleted cells to ERa antagonists in vitro(A) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serially diluted concentrations of fulvestrant for 72 h or 4-OH-Tam for 6 days prior to cell viability assay. Lysates of cells treated with the indicated concentration of fulvestrant were harvested for western blotting (right). ERK2 was loading control.(B) Relapse-free survival of patients with ERa-positive breast cancer with high or low AKTIP mRNA levels stratified at the lower quartile. Cohorts of patients with ERa-positive breast cancer treated with adjuvant tamoxifen (n = 158) were selected for the analysis in the KM plotter. Log rank p value is shown.(C) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serial concentrations of AZD1480 for 72 h.(D and E) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serial concentrations of (D, top) fulvestrant (Ful) with or without 2 µM AZD1480 (AZD) for 72 h; (D, bottom) 4-OH-Tam with or without 2 µM AZD; or (E) 4-OH-Tam with or without 2 µM C188–9 for 6 days. The plots show mean ± SD of triplicates and one representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference by two-way ANOVA with Sidak’s multiple comparison test.

Fig 5: Loss of AKTIP activates JAK2/STAT3 signaling in ERa-positive breast cancer cells(A) MCF7 cells stably expressing AKTIP shRNA or vector were transfected with ESR1 siRNA for 24 h before cells were seeded for cell viability assay, which was measured over 7 days (top), or lysates were harvested for western blotting 8 days after siRNA transfection (bottom). ERK2 was loading control. Day 0 was the day of cell seeding. Bar graphs are mean ± SD of triplicates and one representative of three independent experiments.(B) Lysates of MCF7 cells stably expressing AKTIP shRNA, MDA-MB-361, SKBR3, and MDA-MB-453 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting.(C–F) MCF7 stable cells were (C) treated with 10 nM fulvestrant or 1 µM 4-hydroxytamoxifen (4-OH-Tam) for 48 h; (D) SGK3 siRNA for 72 h; (E) 2 µM AZD1480 for 24 h or STAT3 siRNA for 72 h; or (F) 0.5 µM BBI608 for 2 h before lysates were harvested for western blotting. *p < 0.05; ***p < 0.001; ns, no significant difference compared with vector using t test. The western blots shown are representatives of three independent experiments.