Nrf2 is repressed during cellular transformation via activation of RAS/RAF/ERK pathway. (A) Western-blot shows down-regulation of Nrf2 and downstream target NQO1 during transformation of human MSC. Activation of AKT and ERK pathways, confirmed by increased phosphorylation of AKT and ERK1/2 proteins, correlates with decreased expression of Nrf2 and NQO1 in tMSC. (B) Expression of constitutive active mutants H-RasV12 and Raf-CAAX, but not myrAKT, leads to Nrf2 and NQO1 down-regulation in non-transformed MSC4. (C) Immortal human mammary epithelial cells (HMEC1) were serially transduced with retroviral vectors encoding the genes E6 and E7 from HPV-16 (HMEC3), ST antigen from SV40 (HMEC4), and H-RasV12 (tHMEC). Top panel shows the number of colonies in soft-agarose counted in triplicate wells after two weeks in culture. ***P < 0.0005 with respect to all. Western-blot at the bottom panel shows Nrf2 and NQO1 repression during HMEC transformation. (D) qRT-PCR shows that 16 hours treatment with the ERK inhibitor U0126 induces expression of Nrf2 and its downstream gene NQO1 in MDA-MB-231 and MCF-7 cells. However, treatment with the AKT inhibitor GSK690693 or with the PI3K inhibitors LY294002 and wortmannin does not induce Nrf2 expression. DMSO served as a control. (E) Western-blots show inactivation of ERK and PI3K/AKT pathways following 16 hours treatment with inhibitors. Membranes were probed with rabbit polyclonal antibodies for Nrf2, total ERK1/2, phosphorylated ERK1/2 (p-ERK1/2 (Thr202/Tyr204)), total AKT, phosphorylated AKT (p-AKT (Ser473)), and monoclonal antibodies for NQO1. Actin and Hsp90 were used as a loading control. P values are *P < 0.05, **P < 0.005 and ***P < 0.0005.