ErbB3 binding protein 1 represses metastasis-promoting gene anterior gradient protein 2 in prostate cancer - PubMed (original) (raw)

ErbB3 binding protein 1 represses metastasis-promoting gene anterior gradient protein 2 in prostate cancer

Yuexing Zhang et al. Cancer Res. 2010.

Abstract

Dysregulation of the developmental gene anterior gradient protein 2 (AGR2) has been associated with a metastatic phenotype, but its mechanism of action and control in prostate cancers is unknown. In this study, we show that overexpression of AGR2 promotes the motility and invasiveness of nonmetastatic LNCaP tumor cells, whereas silencing of AGR2 in the metastatic derivative C4-2B blocks invasive behavior. ErbB3 binding protein 1 (EBP1), a putative repressor of AGR2, is attenuated in prostate cancer. We show that the anti-invasive effect of EBP1 occurs, at least in part, through its ability to inhibit AGR2 expression. Mechanistic investigations indicate that EBP1 downregulates Foxa1- and Foxa2-stimulated AGR2 transcription and decreases metastatic behavior. In contrast, EBP1 ablation upregulates AGR2 via Foxa1- and Foxa2-stimulated AGR2 promoter activity and increases metastatic behavior. In both prostate cell lines and primary tumors, we documented an inverse correlation between EBP1 and AGR2 levels. Collectively, our results reveal an EBP1-Foxa-AGR2 signaling circuit with functional significance in metastatic prostate cancer.

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Figures

Figure 1

Figure 1. EBP1 levels inversely correlate with AGR2 expression in prostate cancer cell lines

A & B, Immunoblots detecting AGR2 (A) and EBP1 (B) expression in prostate cancer cell lines. Values on top of the bands represent relative densities normalized to actin.

Figure 2

Figure 2. AGR2 promotes non-metastatic prostate cancer cell migration and invasion

A, Immunoblotting analysis of LNCaP cells stably transfected with pcDNA3 (LNCaP-pcDNA3) or pcDNA3-hAGR2 (LN-AGR2 or LNCaP-AGR2) using antibodies as indicated. Values under panel represent relative band densities normalized to actin. B, Migration ability was estimated using a Modified Boyden chamber assay. Left panel, representative image of three independent experiments showing cells that migrated through the transwell filters coated with collagen and fibronectin. LNCaP is the untransfected parental cell line. Right graph: Number of migrated cells in representative areas of each membrane that were examined microscopically at 10X magnification. Columns, mean number of cells migrated in three representative fields per well. Bars, SD (*, P<0.01 versus control, t test). Shown as one of three independent experiments**. C,** left panel, representative photomicrograph of invasion assay from three comparable experiments showing cells invading through Matrigel-coated membranes. Right graph, cells that had invaded in representative areas of each membrane were examined microscopically at 20X magnification. Columns, mean number of cells invaded in three representative fields per well. Bars, SD (*, P<0.01 versus control, t test). Shown as one of three independent experiments**. D,** left top panel, Western blot analysis of AGR2 expression in cells transduced with lentiviruses targeted to different portions of the AGR2 coding sequence. AGR2 was silenced in stable clones 1–1 and 1–2 using a viruses targeting XXXX of the coding sequence. UT-untransduced cells; shCON=empty lentivirus. Left lower panel, representative micrographs of effects of stable silencing of AGR2 on the invasiveness of C4–2B cells as described in Fig. 2B. Cells from clones 1–1 and 1–2 were pooled for the migration and invasion assays (shAGR2). Right panel, columns, mean number of cells migrated in three representative fields per well. Bars, SD (*, P<0.01 versus control, t test). Shown as one of three independent experiments.

Figure 3

Figure 3. EBP1 modulates the expression of AGR2 accompanied by changes in motility and invasiveness in prostate cancer cells

A, C4-2B cells stably expressing GFP(C4-2BG) or GFP-EBP1 fusion proteins(C4-2BE) were visualized by fluorescence microscopy(left panel) and confirmed by Western blotting assay using GFP antibody (right upper panel). AGR2 and Foxa1 levels were also analyzed in these cells and Actin was used as loading control (lower 3 panels). B, Left panel: Photomicrographs of Boyden Chamber membranes after migration assays showing that many C4-2BG but few C4-2BE cells traversed a coated membrane (left panel). Right panel: Quantification of the effects of EBP1 and AGR2 expression on migration. Columns= mean number of cells migrated in three representative fields per well. Bars=SD. Shown as one of three independent experiments. C. Invasion assay was described in Fig2. D & E, left panel, micrographs of Boyden chamber membranes after migration assays with C81V and C81E, A16 and C13. Right panel: relative quantification of the effects of EBP1 and AGR2 expression on the migration. Columns, mean number of cells migrated in three representative fields per well. Bars, SD (*, P<0.01 versus control, t test). Shown as one of three independent experiments.

Figure 4

Figure 4. EBP1 suppresses the promoter activity of the AGR2 gene by interfering with Foxa transcription factors and binds Foxa proteins

A, B & C, LNCaP or its subline EBP-null C13 and control A16 cells were transfected with an AGR2 reporter (pGL3B-AGR2, 0.25μg/well) and other indicated plasmids. Forty-eight hours later, cells were harvested for luciferase measurement. Each bar represents mean±s.e of triplicate wells. Representative of three independent experiments. D, The interaction of Foxa with the AGR2 promoter sequence was assessed by DNA affinity precipitation as previously described(28) using LNCaP cell extracts and biotinylated olig A containing a Foxa consensus sequence or Olig B lacking potential Foxa binding sites, all derived from the AGR2 promoter as described in Materials and Methods. E, Association of EBP1 and Foxa proteins in vitro (three upper panels) and in vivo (lower panel). Upper three panels: Equal amounts of GST-EBP1 or GST proteins were incubated with lysates of LNCaP, C81, or C4-2B cells. Associated proteins were analyzed by Western blot analysis with the antibodies indicated. Lower panel: Lysates of stably expressing GFP (C4-2BG) or GFP-EBP1 (C4-2BE) cells were incubated with GFP agarose and immunopreciptation performed using a GFP antibody. Associated proteins were analyzed by Western blotting using Foxa1 and Foxa2 antibodies. F. Western blot analysis of Foxa1 expression in panel of prostate cancer cell lines.

Figure 5

Figure 5. Expression of AGR2 increases with prostate cancer progression and inversely correlates with EBP1 expression

A, Left panel, box plots of immunohistochemical staining intensity of AGR2 in normal prostate tissue (n=42) or tissues from patients with prostate cancer (n=153). For all panels, the box represents the s.d. of the distribution, and the line through that box represents the mean of the distribution. The horizontal lines above and below the box represent the extreme values of the distribution. Middle panel, Correlation of AGR2 staining intensity with EBP1 staining intensity. Right panel, Correlation of AGR2 staining intensity with EBP1 proportion score (PS) (percentages of cells stained with EBP1: 1,<5%; 2, 5–30%; 3,31–70%; 4, >70%). B, representative staining of AGR2 in benign tissues (left panel), moderately malignant carcinomas (middle panel, Gleason 3+3) and highly malignant carcinomas (right panel, Gleason 4+5). C, Left panel, a public dataset ???? shows the average gene expression level at three different stages(benign n=23, primary n=64 and metastatic n=250) of prostate cancer. Right panel, scatter-plots of AGR2 versus EBP1 expression units in all these samples.

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