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ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells.

Plotnik JP, Budka JA, Ferris MW, Hollenhorst PC - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.

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Related in: MedlinePlus

Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.
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Figure 2: Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.

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.


ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells.

Plotnik JP, Budka JA, Ferris MW, Hollenhorst PC - Nucleic Acids Res. (2014)

Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.
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Figure 2: Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.

Show MeSH
Related in: MedlinePlus