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. 2018 May;9(5-6):198-214.
doi: 10.18632/genesandcancer.182.

Common ELF1 deletion in prostate cancer bolsters oncogenic ETS function, inhibits senescence and promotes docetaxel resistance

Justin A Budka  1 , Mary W Ferris  1 , Matthew J Capone  1 , Peter C Hollenhorst  1
Affiliations

Common ELF1 deletion in prostate cancer bolsters oncogenic ETS function, inhibits senescence and promotes docetaxel resistance

Justin A Budka et al. Genes Cancer. 2018 May.

Abstract

ETS family transcription factors play major roles in prostate tumorigenesis with some acting as oncogenes and others as tumor suppressors. ETS factors can compete for binding at some cis-regulatory sequences, but display specific binding at others. Therefore, changes in expression of ETS family members during tumorigenesis can have complex, multimodal effects. Here we show that ELF1 was the most commonly down-regulated ETS factor in primary prostate tumors, and expression decreased further in metastatic disease. Genome-wide mapping in cell lines indicated that ELF1 has two distinct tumor suppressive roles mediated by distinct cis-regulatory sequences. First, ELF1 inhibited cell migration and epithelial-mesenchymal transition by interfering with oncogenic ETS functions at ETS/AP-1 cis-regulatory motifs. Second, ELF1 uniquely targeted and activated genes that promote senescence. Furthermore, knockdown of ELF1 increased docetaxel resistance, indicating that the genomic deletions found in metastatic prostate tumors may promote therapeutic resistance through loss of both RB1 and ELF1.

Keywords: ELF1; ETS; chemotherapy resistance; prostate cancer; tumor suppressor.

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Conflict of interest statement

CONFLICTS OF INTEREST None

Figures

Figure 1
Figure 1. ELF1 is the most commonly downregulated ETS factor in prostate cancer
A. Heatmap representation of mRNA expression of the ETS family in 498 prostate cancer samples relative to the background distribution of expression from 52 normal prostate samples. The values displayed represent the individual Z-score for each normalized read count as compared to the normal prostate background distribution. B. Boxplot representation of ELF1 normalized read counts in 52 normal prostate samples (Blue) paired with matched tumor samples (Red) (p-value >0.001, paired t-test). C. Column graph of log2 (median-centered ratio) expression of ELF1 in a microarray experiment comparing normal prostate samples with benign, primary, and metastatic prostate samples [39]. D. Table detailing the copy number alterations in ELF1 and RB1 from the TCGA data set using GISTIC 2.0. E. Boxplot representation of ELF1 normalized read counts in 52 normal prostate samples (Blue) compared with ELF1 expression of all prostate cancer samples that have a normal ELF1 copy number (Red) (p-value < 0.001, Welch Two Sample t-test). F. A boxplot representation of ELF1 levels in TCGA prostate tumors that are classified based on biochemical recurrence. P-values (* <0.05, ** < 0.01, *** < 0.001) are calculated with the Welch Two Sample t-test. G. RPKM expression values for ELF1 in the indicated prostate cell lines were obtained from Prensner et al. [38]. H. Immunoblot with the indicated antibodies (left) in a subset of the prostate cancer cell lines. Tubulin serves as a loading control.
Figure 2
Figure 2. ELF1 represses oncogenic ETS mediated phenotypes in prostate cells
A. Boxplot representation of transwell migration of RWPE-1, PC3, and RWPE-ERG cells expressing a shRNA targeting ELF1 (shELF1), or negative control shRNA targeting luciferase (shLuc) and/or expressing exogenous ERG or empty vector control. B. Boxplot representation of scratch assays measured as a decrease in the wound area in RWPE-1 and PC3 cells with a shRNA control (shLuc) or ELF1 shRNA knockdown (shELF1), with or without ERG. C. Immunoblots with the indicated antibodies. Tubulin is a loading control. D. Expression of indicated mRNA in the indicated cell lines relative to cells expressing shLuc alone. Values shown as mean and SEM (n = 3). E. Boxplot representation of clonogenic survival assays of cells expressing shELF1 or shLuc in RWPE-1, PC3, and RWPE-ERG cells measured as the number of colonies. All P-values (* <0.05, ** < 0.01, *** < 0.001) were calculated with the Welch Two Sample t-test.
Figure 3
Figure 3. ELF1 can bind to two distinct sets of cis-regulatory sequences
A. Heatmap representation of the ChIP enrichment of ELF1 in RWPE-1, DU145, and PC3 cells, centered on all called ELF1 bound regions in RWPE-1 cells. Bound regions are indicated between the “5′ end” and “3′ end” indicators with an extended view that includes the surrounding 7 kb on either side of the bound region. The heatmaps were generated using NGSplot [50]. B. Three most enriched motifs at ELF1 binding sites in RWPE-1 cells as determined by RSAT peak-motifs algorithms C. Heatmap representation of the ChIP enrichment of ELF1 in RWPE-1 cells and ERG in RWPE-ERG cells, centered on all called ELF1 bound regions. Bound regions are indicated between the “5′ end” and “3′ end” indicators with an extended view that includes 7 kb on either side of the bound region. Heatmaps were generated by NGSplot [50]. D. Three most enriched motifs at ELF1 bound enhancers (>500 bp from TSS) in two different categories, ELF1 enhancers that are bound by ERG and ELF1, and enhancers with only background ERG signal (ELF1 only). E. Relative luciferase reporter activity for two firefly luciferase reporter constructs (synthetic 3xETS/AP-1 sites and a fragment of a FHL3 enhancer) in RWPE-ERG cells with ELF1 shRNA knockdowns F. Immunoblot with the indicated antibodies (left) in the same cell lines as (G). Tubulin serves as a loading control. G. Relative luciferase reporter activity for the 3xETS/AP-1 firefly luciferase reporter constructs in PC3 cells with ELF1 shRNA or overexpression as indicated. Luciferase values are the ratio of firefly luciferase to minimal promoter controlled renilla luciferase signal, and this ratio was then normalized to shLuc control.
Figure 4
Figure 4. ELF1 represses EMT, but activates cellular senescence
A. Gene Set Enrichment Analysis (GSEA) was used to compare RNA-seq of RWPE-ERG and RWPE-ERG shELF1 for the indicated gene sets from MSigDB Hallmarks or Reactome. The fpkm values were provided for the three biological replicates in each condition and the Signal2Noise ratio was used to rank genes. B. Immunoblot with the displayed antibodies (left) in PC3 cells with a control shRNA (shLuc) or ELF1 shRNA knockdown (shELF1), or a retroviral overexpression of ELF1 (ELF1 OvExp). C. Microscopic images PC3 cells from (B). Images were converted to grayscale and the saturation was adjusted to provide a clearer outline of the cells. The black arrows indicate cells that appear to be undergoing senescence. D. Quantification of β-galactosidase positive staining in PC3 cells expressing indicated constructs relative to shLuc (n = 3). P-values determined by one-way ANOVA with Tukey's Test as the post-hoc analysis (* <0.05, ** < 0.01, *** <0.001).
Figure 5
Figure 5. ELF1 sensitizes prostate cells to chemotherapy
A. Cell viability analysis of RWPE-1 or RWPE-ERG cells with a lentiviral luciferase knockdown (shLuc) or ELF1 knockdown (shELF1) (n = 3). B. Barplot representation of the Mean and SEM IC50 value for analysis in (A). IC50 values were calculated using a nonlinear fit of the log(inhibitor) vs. response - Variable slope (four parameters) in Prism. P-values were determined using a one-way ANOVA with Tukey's Test as the post-hoc analysis (* <0.05, ** < 0.01, *** <0.001).
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