Gene Expression Profiling Reveals Regulation of ERK Phosphorylation by Androgen-Induced Tumor Suppressor U19/EAF2 in the Mouse Prostate

Abstract

U19/EAF2 is regulated by androgens in the prostate and capable of regulating transcriptional elongation of RNA Pol II via interaction with the ELL family proteins. Inactivation of U19/EAF2 induces tumorigenesis in multiple organs; however the mechanism of U19/EAF2 tumor suppression remains unclear. To elucidate potential mechanisms of U19/EAF2 action, we performed cDNA microarray analysis and identified 164 mRNA transcripts regulated by U19/EAF2 in the mouse ventral prostate. Bioinformatics analysis indicated that U19/EAF2 knockout activates the RAS-BRAF-ERK signaling pathway, which is known to play important roles in carcinogenesis. qPCR verified increased expression of BRAF mRNA, and immunostaining and Western blot analysis demonstrated increased expression of p-ERK at the protein level suggested U19/EAF2 knockout activates this important pathway. These findings indicate that loss of EAF2 up-regulates transcription of RAS cascade genes including Grb2, PI3K, and BRAF, leading to elevated p-ERK levels, which may represent a major functional role of U19/EAF2 in the prostate. Furthermore, these observations suggest that U19/EAF2 is a key player in crosstalk between androgen receptor and the RAS-BRAF-ERK signaling pathway.

Fig. 1

HTself2 analysis of microarray data. a. Self-self empirical probability density distribution. Virtual log ratios (M) where derived from all possible pairwise combinations among 6 independent wild type experiments analyzing gene expression in the ventral prostate at age 3 month. Fold-changes greater than 2-fold (M > 1 or M < −1) have a very small probability of happening (<0.0001) due by chance alone considering the intrinsic inter-animal genetic noise. b. Interchange between statistical significance and fold-chance cutoffs. Statistical significance thresholds (p-value) can be mapped directly to equivalent fold-change cutoffs to define differentially expressed genes in the knockout vs wild type experiment. The horizontal red line represents a traditional 0.01 significance cutoff after multiple testing correction by the stringent Bonferroni method. A 2.5-fold change cutoff criteria would be equivalent to a corrected p-value smaller than 0.01. c. MA-plot showing the knockout (KO) vs wild type (WT) experiment. Virtual log ratios (M) remain stable around zero (KO/WT = 1) along all average log intensity (A) scale. Horizontal red lines represent fold-change cutoffs (KO/WT > 2.5-fold or KO/WT < 1/2.5) presenting multiple testing adjusted p-values < 0.01 and, therefore, define the differentially expressed genes

Fig. 2

Concordance of microarray and qPCR gene expression analyses. a Bar chart of qPCR data results and corresponding array signals. Data were normalized to GAPDH. b Log-fold changes from the microarray platforms are plotted versus the corresponding fold changes from qPCR. R = 0.92. Genes 1–11 represent Adipoq, Ube1l2, Abpb, CD47, Rab2, Rhou, Uroplakin iB, hnRNP, Tesin, Thbs1, and PRLR, respectively

Fig. 3

Effect of U19/EAF2 knockout on the expression of BRAF mRNA and p-ERK in the mouse model. a. qPCR analysis of the BRAF mRNA levels in anterior prostate (AP) and ventral prostate (VP) and b. lung and liver of U19/EAF2 knockout and wild-type control mice.. Data were normalized to GAPDH, (n = 3). c. Western blot analysis of phosphorylated ERK, pERK(p44/42), in U19/EAF2 knockout and wild-type mouse tissues. Images shown are representative blots for ERK, pERK and GAPDH from ventral prostates and liver lysates of individual U19/EAF2 knockout and wild-type control mice. d. Intensity of p-ERK in /EAF2 knockout and wild-type mouse tissues. Band densities in the Western blots were analyzed with IMAGEJ (National Institutes of Health) and normalized against GAPDH. Data represent the mean ± SEM; * p < 0.05. ** p < 0.01, *** p < 0.001 versus WT

Fig. 4

Effect of U19/EAF2 knockout on ERK pathway in the murine prostate. Genes up-regulated upon U19/EAF2 knockout are colored in peach. Color intensity is used to indicate the degree of up-regulation, with light peach color for less up-regulation and darker peach color for more up-regulation. Solid arrows indicate positive regulation; solid purple lines indicate protein binding