Snail is a repressor of RKIP transcription in metastatic prostate cancer cells

Abstract

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

Figure 1. The expression of RKIP is repressed in highly metastatic cancer cells

a) Immunoblot analysis of extracts from prostate or breast cancer cells with specific Abs. b) The endogenous levels of RKIP mRNA in prostate cancer cells as measured by qRT-PCR and normalized to the level of GAPDH (left) or mATP6. Each bar represents the mean ± SEM (standard error mean) of the PCR reactions in triplicate. c-d) Immunoblot analysis of extracts from prostate cancer cells DU145 treated with c) TSA or d) 5-Aza-2-dC for 24 or 48 h as indicated. Data shown are representative of three independent experiments.

Figure 2. Snail is a direct repressor of RKIP expression

a) Comparison of RKIP, and Snail expression in prostate cancer cell lines by Western blot analysis. Triton x-100 extracted cell lysates (30 μg) were immunoblotted using polyclonal anti-RKIP, or Snail antibodies. The same membrane was re-blotted with an anti-actin serum as a loading control. b-c) Ectopic expression of Snail represses RKIP expression. b) Immunoblot analysis of extracts from cancer cells stably infected with the indicated retroviruses or EVC (empty vector control) with specific Abs. c) Comparison of RKIP mRNA levels in LNCaP cells stably infected with the indicated retroviruses. The endogenous levels of RKIP mRNA in infected cells were measured by qRT-PCR and normalized to the level of GAPDH. Each bar represents the mean ± SEM of the PCR reactions in triplicate. d) Downregulation of Snail in prostate cancer cells enhances RKIP expression. Cells were stably infected with the indicated retroviruses expressing shRNA for luciferase or Snail. The endogenous levels of RKIP, tubulin and Snail proteins in infected cells were measured by immunoblot analysis. e) Snail represses RKIP promoter reporter in cancer cells. MCF7 cells were transfected with the indicated effector and reporter plasmids. 48h after transfection cells were harvested for luciferase assay. Schematic of the RKIP-LUC reporter containing the RKIP promoter region was shown on the left. Positions of the putative E-box are indicated. TK stands for Thymidine Kinase. f) Snail associates with RKIP promoter. ChIP assays were performed with anti Flag or anti-HA Ab on LNCaP cells that stably express Flag-tagged Snail protein. DNAs that were immunoprecipitated down with the Abs were amplified with primer pair by PCR as indicated by arrows in the lower panel.

Figure 3. The expression of RKIP negatively correlates with Snail in prostate cancer samples

a) Box plots of RKIP and Snail levels in prostate cancer microarray datasets. The box represents the standard deviation of the distribution and the line through that box represents the mean of that distribution. The horizontal lines above and below the box represent the extreme values of the distribution. Ratios of prostate cancer samples referenced against a pool of normal adjacent prostate (NAP) samples from patients were log-transformed and plotted using the statistical program R. NAP N=3, BPH N=5, PCA N=10, and MET N=7. b) The log transformed expression units of RKIP or Snail were plotted for each sample. Samples were grouped as NAP, BPH, PCA and MPC as in a). c) Scatterplots of Snail versus RKIP of all samples displayed in a)