Fig.7. Loss of SIRT1 is associated with elimination of E2-induced cell survival and mammary tumorigenesis.

(A) Control and SIRT1 knockdown ZR75.1 cells were treated with or without E2 for 2 weeks and the resulting colonies were quantified with Giemsa staining. (B) Seven days before tumor induction, female athymic nude mice (6 animals per group; 3 groups) were anesthetized and 3-mm pellets containing E2, 0.18 mg/21-day release, were implanted subcutaneously in the animal’s back. Another 18 animals were used as a control, without E2 treatment. One week after pellet implantation, ZR75.1-pLKO.1 and ZR75.1-SIRT1shRNA (two independent shRNA clones shRNA#2 and shRNA#3) cells (1×10⁷ cells in 100 µl PBS) were injected s.c. in the mammary fat pad of all animals. Tumor volume was used as a measure of tumor growth at 1, 2, 3 and 4 weeks after cell injections. We compared the tumor volume of pLKO.1 and SIRT1shRNA-induced tumor in the presence and absence of E2 and the statistical significance was calculated as ***p<0.001; ** p<0.01; *p<0.05*p<0.05 in each time point (C) Control and SIRT1 knockdown ZR75.1 cells were treated with E2, TAM, 4-OH TAM and ICI182780 for 48 h. Apoptotic cell death was analyzed by FACS. We compared the apoptotic cell death in control pLKO.1 and SIRT1shRNA cells treated with E2, TAM, 4-OH TAM and the statistical significance was calculated as ***p<0.001 (D). Proposed mechanism of E2-ERα and SIRT1 complex in regulation of tumor cell immortalization and tumor development.