Fig. 6

Effect of knockdown of ATF2 on ER and ER-regulated genes and proposed model of resistance. a–f MCF7 (left bars) and TAMR (right bars) cells were transfected with ATF2 siRNA and qRT-PCR was carried for ATF2 (a), ERα (ESR1) (b), TFF1 (c), GREB1 (d), PGR (e) and NCOA3 (f) using TaqMan primers. GAPDH was used as the housekeeping gene and changes in mRNA levels after ATF2 knockdown were calculated relative to the control. Asterisks indicate the genes that were significantly changed after ATF2 knockdown (*p < 0.05, **p < 0.005). g Protein lysates were also prepared in triplicates of three independent experiments and immunoblotting was carried out using the antibodies indicated. ATF2 knockdown had no effect on the ER and ER-regulated genes on the tamoxifen-sensitive MCF7 cells but affected their expression both on the mRNA and protein levels on the tamoxifen-resistant TAMRs. The following model of resistance is proposed: h In endocrine-sensitive cells, gene transcription is ER-dependent and endocrine therapy is able to stop their growth and proliferation. However, endocrine-resistant cells have a shift from ER-dependent to an ER-independent ATF2-dependent transcriptional program and therefore, they are not responding to endocrine treatment. i Targeting the ATF-2 transcription factor in endocrine-resistant cells represents a new mechanism to revert resistance and enhance endocrine sensitivity. The genes shown in this model (h, i) such as TFF1, GREB1 and NCOA3 are examples of resistance candidates only, since their biological function to endocrine resistance has not been validated in animal models in this study