The endogenous cell-fate factor dachshund restrains prostate epithelial cell migration via repression of cytokine secretion via a cxcl signaling module

Abstract

Prostate cancer is the second leading form of cancer-related death in men. In a subset of prostate cancer patients, increased chemokine signaling IL8 and IL6 correlates with castrate-resistant prostate cancer (CRPC). IL8 and IL6 are produced by prostate epithelial cells and promote prostate cancer cell invasion; however, the mechanisms restraining prostate epithelial cell cytokine secretion are poorly understood. Herein, the cell-fate determinant factor DACH1 inhibited CRPC tumor growth in mice. Using Dach1(fl/fl)/Probasin-Cre bitransgenic mice, we show IL8 and IL6 secretion was altered by approximately 1,000-fold by endogenous Dach1. Endogenous Dach1 is shown to serve as a key endogenous restraint to prostate epithelial cell growth and restrains migration via CXCL signaling. DACH1 inhibited expression, transcription, and secretion of the CXCL genes (IL8 and IL6) by binding to their promoter regulatory regions in chromatin. DACH1 is thus a newly defined determinant of benign and malignant prostate epithelium cellular growth, migration, and cytokine abundance in vivo.

Figure 1. DACH1 inhibits AR-negative prostate cancer cell proliferation and contact independent growth by the DS domain

A) Treeview display of microarray gene expression studies conducted on PC3 cells stably expressing either vector control or DACH1. The cell cycle control proteins are shown with the relative abundance, demonstrated with a color scale as shown. DACH1 inhibits cyclin E1, E2, A1, A2 expression. B) Phase contrast using immunofluorescent microscopy for PC3 stable cell lines for DACH1 and DAPI as a nuclear stain. Western blot of PC3 stable cell line with antibody to the N-terminal FLAG tag of DACH1. C) The cellular proliferation rate determined by MTT assay or D) cell counting. E) Colony forming assays were conducted with PC3 stable cell lines expressing control vector, DACH1 or ΔDS with colonies stained using crystal violet and F) colony number or G) colony size determined using N>5 separate experiments. H) C4-2 cells expressing either control vector DACH1 or the DACH1 ΔDS mutant were assessed for DACH1 abundance by immuno-histochemistry. DAPI and immunofluorescence for DACH1 is shown. Western blot is shown of the cells with an antibody directed to the N-terminal FLAG tag. I) The cellular proliferation rate of C4-2 cells expressing DACH1 or mutant DS was determined by either MTT assay or J) cell counting. Data are mean ±SEM for N>5 throughout.

Figure 2. DACH1 inhibits prostate cancer tumor growth and cellular proliferation in mice

A–B) PC3 cells stably transduced with either control vector DACH1 or the DACH1 ΔDS mutant were introduced into nude mice and assessed by A) tumor size or B) tumor weight after implantation C–D) 22RV1 cells stably expressing either DACH1, DACH1 ΔDS or vector control were assessed after implantation into mice by C) the tumor size or D) the tumor weight. The data are shown as mean ±SEM for N>7 throughout. E–F) Dach1 expression is detected by Western blot in primary mouse (FVB) PEC and isogenic oncogene transformed cell lines derived from FVB PEC. H) Microarray gene expression of FVB PEC or isogenic oncogene transformed PEC with relative abundance of cyclins shown. G) PEC line tumor grown in mice and primary prostate gland from which lines were derived, were immunostained for Dach1 and the % of Dach1 positive cells determined. H) Microarray analysis of cyclin gene expression in isogenic PEC grown in tissue culture or I) grown as tumors and then extirpated from FVB mice. The changes in gene expression are shown calorimetrically. Gene names are shown to the right of the panels. Genes highlighted in red are induced in both tissue culture and in vivo and were followed for mechanistic analysis. J) Analysis of bi-transgenic mice encoding Dach1^fl/fl/Probasin-Cre by immunohistochemical staining for Ki67 in the ventral prostate shown at 10x and 40x. Data is shown for mean ±SEM N>100 cells. K) Apoptosis determined by TUNEL staining of the ventral prostate. Data shown as mean ±SEM N>100 cells. L) Immunohistochemical staining for cyclin E1 and M) cyclin A2 in the ventral prostate of bi-transgenic mice encoding Dach1^fl/fl/Probasin-Cre mice. Transgenic deletion of Dach1 in the prostate epithelial cells enhances cellular proliferation and reduces cellular apoptosis.

Figure 3. DACH1 inhibits transwell migration and cytokine gene expression via the DS domain

A) PC3 cells stably expressing either vector, DACH1 or ΔDS were stained after transwell migration. B) The relative migration is shown for N>5 separate experiments. C) The PC3 stable cell lines were analyzed by video microscopy for distance traveled. Representative examples of individual cell tracking are shown for stable PC3 cells co-incubated with the media from cells expressing either DACH1, or DACH1 ΔDS. D) The cellular migratory velocity for the PC3 cells is shown as mean ±SEM for three separate experiments. E) Microarray analysis of PC3 cells stably expressing either DACH1, DACH1 ΔDS or vector control were analyzed for functional pathways using KEGG. The GO terms, the enrichment score, and number of gene enhanced for each GO term are shown. F) The relative abundance of genes significantly altered by DACH1 via the DS domain in the cytokine-cytokine receptor interaction categories is shown. DACH1 inhibits CXCL gene expression as shown by the blue color indicating repression in the presence of DACH1 expression. Genes highlighted in red are repressed by DACH1 expression via the DACH1 DS domain and used for further analysis.

Figure 4. Dach1 expression is reduced in oncogenic transformed PEC and inversely co-related with cytokine production

A) Microarray based gene expression of cytokines/chemokines in isogenic PEC in tissue culture or B) from tumors grown in FVB mice. The relative expression levels from the tumors in vivo are shown in C-E. F) Relative DACH1 mRNA levels were determined from Oncomine in either prostate cancer samples or benign prostate disease. DACH1 levels are significantly reduced in metastatic prostate cancer samples. G–H) mRNA abundance for DACH1 and either IL-6 or IL-8 in adjacent normal prostate tissue vs prostate cancer samples for individual patients was determined. The relative changes in expression for normal adjacent prostate tissue vs cancer is shown. Samples with a decrease in DACH1 mRNA abundance and increase in IL-6 or (H) IL-8 abundance are shown in the left upper quadrant. This quadrant expressing a decrease in DACH1 mRNA and corresponding increase in mRNA levels in individual patient cancer samples vs adjacent normal prostate epithelium was significantly enriched. I) The relative luciferase reporter activity of the IL-6 and J) IL-8 promoter was normalized to 1 for the vector control. The luciferase promoter reporters were transfected into PC3 cells, together with equimolar amounts of either vector, DACH1 expression vector or the ΔDS expression vector. Data are mean ± SEM for N>5 separate experiments. K) Western blot of transfected PC3 cells showing relative abundance of the exogenous DACH1 assessed by FLAG epitope. Vinculin is shown as a protein loading control. L) ChIP of the IL-6 and IL-8 promoter with oligonucleotide primers directed to the regulatory region of the genes as shown.

Figure 5. Dach1 is a dominant endogenous restraint of prostate epithelial cell cytokine production and thereby cellular migration

The Dach1^fl/fl Probasin-Cre prostate epithelial cells were cultured and comparison was made to Dach1^+/+. A) ELISA was used to determine the relative abundance of cytokines in the supernatant (ventral prostate epithelial cells) in culture for IL-6 and B) KC in pg/mL (Note: Log scale). C) Analysis of migratory cell velocity for prostate epithelial cells derived from Dach1^+/+ or Dach1^−/− PECs. Cells were co-incubated with media derived from either Dach1^+/+ or Dach1^−/−, incubated with IL-6 or anti-IL-6 antibody. KC or anti-KC antibody is indicated in the Figure. P value is indicated (significance <0.05). Data is shown as mean ± SEM of three separate experiments. D) ELISA was used to determine the relative abundance for cytokines in the Dach1 wild-type vs. knockout mice in circulating serum of IL-6 and E) KC in pg/mL (Note: Log scale). F) Schematic representation of Dach1 as key determinant of prostate cellular cytokine secretion and cellular proliferation.