Fig. 3: ASH2L induces tamoxifen resistance in ER-positive breast cancer.

a Cell viability assay was assessed in the specified cell lines treated with 4-hydroxytamoxifen (4-OHT) after 5 days in MCF7, MCF7-TamR, and ZR-75-1 cells, and after 7 days in T47D cells. CON, control (empty vector); ASH2L, ASH2L overexpression; shCON, short hairpin RNA control; shASH2L, ASH2L knockdown. Data are shown as the mean ± SD from n = 3 technical replicates. p-values by RM ANOVA (MCF7 and T47D) or RM ANOVA with a post-hoc LSD test (MCF7-TamR and ZR-75-1). b Immunoblots showing caspase 7 and full length and cleaved PARP in the indicated cells. CON, control (empty vector); ASH2L, ASH2L overexpression; shCON, short hairpin RNA control; shASH2L, ASH2L knockdown. c Apoptotic cell populations were analysed by Annexin V/PI staining and flow cytometry in the indicated cell lines that had ASH2L overexpression (ASH2L) or were temporarily knocked down (siASH2L) by ASH2L siRNA. Data represents the mean ± SD from n = 3 biological replicates. P-values from one-way ANOVA with a post-hoc LSD test. d Colony formation assay was performed in the specified cell lines treated with 4-hydroxytamoxifen (4-OHT) for 15 days. CON, control (empty vector); ASH2L, ASH2L overexpression; shCON, short hairpin RNA control; shASH2L, ASH2L knockdown. Data are shown as the mean ± SD from n = 3 technical replicates. p-values by RM ANOVA (MCF7 and T47D) or RM ANOVA with a post-hoc LSD test (MCF7-TamR and ZR-75-1). e Quantification of colony formation in the specified cell lines using ImageJ software after 4-hydroxytamoxifen (4-OHT) treatment for 15 days. Data are shown as mean ± SD from n = 3 technical replicates. p-values by RM ANOVA (MCF7 and T47D) or RM ANOVA with a post-hoc LSD test (MCF7-TamR and ZR-75-1). f, g The effect of ASH2L on the response of ER-positive breast cancer cells to tamoxifen in vivo. NOD/SCID mice were orthotopically injected with the control (CON) or ASH2L-overexpressing (ASH2L) MCF7 cells (f) and the control (shCON) or ASH2L-knockdown (shASH2L) ZR-75-1 cells (g) after implantation of 17β-estradiol (E2) pellets. After the tumour volume reached ~100 mm³, the mice were treated with a placebo (E2) or tamoxifen pellets (E2 + Tam), and the tumour sizes of each group were measured for 2–3 weeks. Data are presented as mean [SEM] : CON (E2), 212.22 [16.01]mm³; CON (E2 + Tam), 100.35 [21.14]mm³; ASH2L (E2), 376.47 [16.38]mm³; ASH2L (E2 + Tam) = 366.55 [13.99]mm³; shCON (E2), 145.00 [9.60]mm³; shCON (E2 + Tam), 140.67 [4.24]mm³; shASH2L (E2), 123.60 [5.53]mm³; shASH2L (E2 + Tam), 78.79 [4.42]mm³). Scale bars = 5 mm. Mean ± SEM (n = 8 per group). p-values by RM ANOVA with a post-hoc LSD test. Waterfall plots of the percentage change in tumour volumes of individual tumour-bearing mice treated with a placebo (E2) or tamoxifen pellets (E2 + Tam) for 14 days (MCF7 xenograft) (h) or 25 days (ZR-75-1 xenograft) (i).