Snail1 Induced Suppression of Proliferation via EGR1, FOXO1, and CEPBg Creates a Vulnerability for Targeting Apoptotic and Cellular Senescence Pathways [RNA-Seq]
The annual ~36,000 prostate cancer (PCa) deaths represent a large clinical unmet need and a call for deeper understanding of PCa metastasis. Epithelial-mesenchymal-transition (EMT) has been used to model metastatic behaviors in numerous cancers including PCa. One hallmark of EMT is cell cycle suppression, but how EMT impacts PCa proliferation remains unclear primarily due to the lack of appropriate models. Methods: We transiently induced expression of Snail1 (SNAI1), an EMT driver expressed in PCa, at physiological levels in three PCa cells lines, C4-2B, 22Rv1, and DU145. We used RNA-seq, ChIP-Seq, bioinformatics, qRT-PCR, shRNA, and immunoblotting to identify mechanisms of Snail1-driven inhibition of proliferation. Results: Snail1 suppressed proliferation and the G2/M cell cycle progression, without affecting cell death. Mechanistically, Snail1 upregulated expression of CEBPg, ERG1, FOXO1, cyclin G1, p21, stress genes SESN3 and SOD3, apoptotic programmers Puma, Bax, and Noxa, and senescence-related laminB1, and downregulated Ki67, cyclins A2 and B2. ChIP-Seq data identified Snail1 direct binding to p21, cyclin B2 and G1, EGR1, and CEPBg promoters. EGR1 induced FOXO1 and EGR1 was required for Snail1-induced SOD3 and Puma, and suppression of Caspase 3 to prevent apoptosis. The EGR1/FOXO1 axis induced BAX, Noxa, and SESN3. CEBPg was required for Snail1 induction of Lamin B1 to block Snail1-induced senescence. Conclusions: We identified three new major downstream targets of Snail1 that improve our understanding of the role of EMT in limiting stress signaling, apoptosis, and senescence during cell cycle suppression to create a vulnerability for therapeutic targeting. Overall design: Prostate cancer cell line model C4-2B is stably transduced with inducible Cumate-Snail1 and tetON-shAR(androgen receptor) plasmid vectors. Cells were incubated in 2% CSS phenol-red free RPMI1640 media. Cells were pretreated for 2 days with 4.5ug/ml cumate or H2O control. After the 2 days, further treatments were further stratified to a total of 4 groups to either induce Snail1 and or AR-knockdown:  control+control, cumate+veh, cumate+dox. The described treated were done for 4/6 days. At the end of 4/6 days, cells were harvested in trizol lysis reagent for downstream total RNA isolation. Total RNA was isolated using standard alcohol precipitation. Isolated total RNA were submitted to NOVOGENE for next generation, selective for mRNA.