GSE112883
Combined Targeting of Estrogen Receptor alpha and XPO1 prevent Akt activation, remodel metabolic pathways and induce autophagy to overcome tamoxifen resistance
Majority of breast cancer specific deaths in women with ERa (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERa (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERa and XPO1, a nuclear transport protein in overcoming endocrine resistance. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using human phosphokinase array to profile kinase signaling pathways, we found that 4-OH Tamoxifen, SXR or their combination induced differential Akt phosphorylation profiles, changing the phosphorylation status and activity of the kinase. Also, our RNA sequencing data indicated that 4-OH Tamoxifen+SXR combination treatment causes gene expression changes distinct from 4-OH Tamoxifen and SXR treatments alone in tamoxifen-resistant cell lines. Since we observed dramatic changes in Akt activity and we saw a differential gene expression pattern with individual and combined 4-OH Tamoxifen and SXR treatments, we hypothesized that metabolic profile of breast cancer cells would change in the presence of 4-OH Tamoxifen and SXR.  Using Seahorse metabolic profiler and cell viability experiments in limited media conditions, we showed that tamoxifen resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source.    We demonstrated that combined targeting of XPO1 and ERa rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy.  Remodelling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERa (+) tumors, our findings show great promise for uncovering the role of ERa-XPO1 crosstalk plays in reducing cancer recurrences. Overall design: RNASeq analysis was performed as previously described [17; 18; 19]. Briefly, BT474 cells were treated with Veh (0.5% EtOH), 10-7 M 4-OH Tamoxifen (Sigma, St. Louis, MO), 10-7 M Selinexor (SXR) (Selleckchem, Houston, TX) or 4-OH Tamoxifen and SXR combination for 24 hours. Total RNA was extracted with TRIzol reagent (Life Technologies) according to manufacturer protocol and cleaned using QIAGEN clean-up kit. RNA quality was assessed using bioanalyzer. Total RNA from each sample (3 per treatment group) were sequenced at the sequencing center at UIUC, and data was generated in Fastqc file format to compare the expressions between four treatment groups.