ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells.
Bottom Line: Genome-wide expression analysis showed that ETS1 was required for activation of RAS-regulated cell migration genes, but also identified a surprising role for ETS1 in the repression of genes such as DUSP4, DUSP6 and SPRY4 that provide negative feedback to the RAS/ERK pathway.Consistently, ETS1 was required for robust RAS/ERK pathway activation.Therefore, ETS1 has dual roles in mediating epithelial-specific RAS/ERK transcriptional functions.
Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.Show MeSH
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Mentions: In a subset of prostate cancers, aberrantly high expression of an oncogenic ETS protein (ERG, ETV1 or ETV4) leads to ETS/AP-1 occupancy and activation of cell migration in backgrounds of low RAS/ERK signaling (18,21). However, normal epithelial cells, and many carcinomas, do not express high levels of these oncogenic ETS proteins, but instead can activate ETS/AP-1 reporter genes and migrate in response to high levels of RAS/ERK signaling (21). The ETS protein that binds ETS/AP-1 sequences across the genome and mediates this RAS/ERK response has not been identified. We used a shRNA knockdown approach to test cell migration roles of five candidate ETS proteins in DU145, a prostate cancer cell line that does not express high levels of oncogenic ETS proteins (21), but has an active RAS/ERK pathway due to a chromosomal rearrangement of KRAS (32). A lentiviral vector was used to make stable lines with shRNA-mediated depletion of ETS1, ETS2, ELF1 or GABPA (Figure 2A). Despite very low ETV4 protein levels in this cell line (21), we were also able to deplete and test ETV4. In each case, lowering the level of one ETS protein did not affect the levels of the others (Figure 2A). A transwell assay tested the migration of each knockdown cell line in comparison to a control (luciferase) knockdown. Loss of ETS1, and no other ETS protein, resulted in a dramatic decrease in cell migration (Figure 2B and Supplementary Figure S2A). A second shRNA targeting ETS1 had a similar effect (Supplementary Figure S2B). To verify that this was not due to cell death, or reduced cell growth, the proliferation rate of ETS1 knockdown cells was tested. ETS1-depleted cells proliferated at a similar rate to control knockdown cells (Figure 2C). While depletion of ELF1, GABPA and ETV4 had no effect on cell migration, knockdown of ETS2, a close homolog of ETS1, actually increased cell migration (Figure 2B), without affecting proliferation (Supplementary Figure S2C), indicating a possible attenuating function for this factor.
Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.