PDF file - 82K, 6A, The c-Src inhibitor blocked tumor necrosis factor (TNF) super family genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol. LTA (TNF super family member 1) gene was detected by real-time PCR. P<0.001, ** compared with control. 6B, LTB (TNF super family member 3) gene was detected by real-time PCR. P<0.001, ** compared with control. 6C, TNFα activated apoptotic pathways in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (H2O) and TNFα (5ng/mL) for 24 hours. Cell lysates were harvested. Pro-apoptotic pathways PARP and Caspase-9 were examined by immunoblotting with primary antibodies. Immunoblotting for beta-actin was detected for loading control. 6D, TNFα inhibited MCF-7:5C cell growth. MCF-7:5C cells were treated with vehicle (H2O) and TNFα (5ng/mL) for 7 days. Cells were harvested and total DNA was determined using a DNA fluorescence quantitation kit. P<0.001, ** compared with control. All the data shown were representative of at least three separate experiments with similar results.
ARTICLE ABSTRACT
The emergence of anti-estrogen resistance in breast cancer is an important clinical phenomenon affecting long-term survival in this disease. Identifying factors that convey cell survival in this setting may guide improvements in treatment. Estrogen (E2) can induce apoptosis in breast cancer cells that have been selected for survival after E2 deprivation for long periods (MCF-7:5C cells), but the mechanisms underlying E2-induced stress in this setting have not been elucidated. Here, we report that the c-Src kinase functions as a key adapter protein for the estrogen receptor (ER, ESR1) in its activation of stress responses induced by E2 in MCF-7:5C cells. E2 elevated phosphorylation of c-Src, which was blocked by 4-hydroxytamoxifen (4-OHT), suggesting that E2 activated c-Src through the ER. We found that E2 activated the sensors of the unfolded protein response (UPR), IRE1α (ERN1) and PERK kinase (EIF2AK3), the latter of which phosphorylates eukaryotic translation initiation factor-2α (eIF2α). E2 also dramatically increased reactive oxygen species production and upregulated expression of heme oxygenase HO-1 (HMOX1), an indicator of oxidative stress, along with the central energy sensor kinase AMPK (PRKAA2). Pharmacologic or RNA interference–mediated inhibition of c-Src abolished the phosphorylation of eIF2α and AMPK, blocked E2-induced ROS production, and inhibited E2-induced apoptosis. Together, our results establish that c-Src kinase mediates stresses generated by E2 in long-term E2-deprived cells that trigger apoptosis. This work offers a mechanistic rationale for a new approach in the treatment of endocrine-resistant breast cancer. Cancer Res; 73(14); 4510–20. ©2013 AACR.
