Prostate Stromal Cells Express the Progesterone Receptor to Control Cancer Cell Mobility

<div><p>Background</p><p>Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.</p><p>Methods</p><p>Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.</p><p>Results</p><p>Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using <i>in vitro</i> prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.</p><p>Conclusions</p><p>Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.</p></div

Pathological Parameters of Study Patients and PSA Levels.

<p>Pathological Parameters of Study Patients and PSA Levels.</p

PR negatively regulates prostate cancer migration through a paracrine pathway.

<p>Conditioned media (CM) were collected from parental hCAFs, WPMY-1 or their derived cell lines expressing mock, PRA or PRB in the presence of vehicle or 10 nM P4. PC-3 cells were seeded in 6 well plates and incubated with CM from hCAFs (<b>A</b>) or from WPMY-1 (<b>B</b>) cells for 24 hours in wound healing assays. Representative images after 24 hour CM treatment were captured by an inverted microscope. WPMY-1 and its derived cell lines expressing mock, PRA or PRB were treated with 0, 10 nM and 100 nM of P4 for 24 hours (<b>C and E</b>) or with vehicle, 10 nM of P4 and/or 10 uM of RU486 for 24 hours (<b>D and F</b>). CM were then collected and incubated with PC-3 cells (<b>C–D</b>) and C4-2B (<b>E–F</b>) in cell migration assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092714#s2" target="_blank">material and methods</a> section. One-way ANOVA and paired student's t-test calculate the statistical significance set at P<0.05 as * and P<0.001 as ***.</p

Measurements of PR protein levels in human prostate tumor tissues.

<p>Whole mount sections of human prostate biopsies (n = 27) were immunostained with PR antibody (AbCam) or PRB antibody (Cell Signaling). Representative IHC images of PR (<b>A</b>) and PRB (<b>B</b>) from benign peripheral zones, cancer peripheral zones and transitional zones are shown. IHC staining of PR (<b>C</b>) and PRB (<b>D</b>) protein levels in paired benign <i>vs</i> cancer peripheral zones and in paired benign peripheral <i>vs</i> transition zones from 27 whole mount sections of human prostate biopsies were scored by Digital Image Hub (Leica Biosystem). HSCORE indexes were plotted as ±SE. One-way ANOVA and paired student's t-test calculate the level of significance.</p

PR negatively regulates prostate cancer invasion through a paracrine pathway.

<p>WPMY-1 and its derived cell lines expressing mock, PRA or PRB were treated with 0, 10 nM and 100 nM of P4 for 24 hours (<b>A and C</b>) or with vehicle, 10 nM of P4 and/or 10 uM of RU486 for 24 hours (<b>B and D</b>). CM were then collected and incubated with PC-3 (<b>A–B</b>) and C4-2B (<b>C–D</b>) cells in cell invasion assays. hCAFs (<b>E</b>), HPS-19I (<b>F</b>) cells and their derived cell lines expressing mock, PRA or PRB were treated with 0, 10 nM and 100 nM of P4 for 24 hours. CM were then collected and incubated with PC-3 cells in cell invasion assays. Cell invasion rate was calculated as described in Material and Method section. One-way ANOVA and paired student's t-test calculate statistical significance set at P<0.05 as * and P<0.001 as ***.</p

PR represses transcription of SDF-1 and IL-6 genes.

<p>WPMY-1 cells expressing mock, PRA or PRB were transiently transfected with control siRNA or siRNA against PR. SDF-1 (<b>A</b>) and IL-6 (<b>B</b>) mRNA levels relative to GAPDH were measured by real-time PCR. hCAFs expressing mock, PRA or PRB isoform were treated with either control or 20 ug/ml of cycloheximide for 16 hours. Real-time PCR assays measured mRNA levels of SDF-1 (<b>C</b>) and IL-6 (<b>D</b>) relative to GAPDH. (<b>E</b>) WPMY-1 cells and their derived cells expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. Chromatin immunoprecipitation assays were performed using acetyl-Histone 3 antibody. Eluted DNA fragments were subjected to measure the enrichment of acetyl-Histone 3 levels in SDF-1 and GAPDH promoter regions. One-way ANOVA and student's t-test calculated the significance set with P<0.01 as * and P<0.001 as ***.</p

PR inhibitory effects to cancer cell mobility are mediated by SDF-1 and IL-6.

<p>WPMY-1 cells expressing mock, PRA or PRB were transiently transfected with control siRNA or siRNA against PR for 24 hours. Cells were washed twice with PBS buffer and replenished with serum free DMEM medium for 48 hours. CM were collected and incubated with PC-3 (<b>A and C</b>) or C4-2B (<b>B and D</b>) cells for cell migration assays (<b>A and B</b>) and Matrigel invasion assays (<b>C and D</b>) as described in Material and Method section. (<b>E</b>) CM were collected from hCAFs in the presence of vehicle or 10 nM of P4 and then mixed with vehicle, 10 ng/ml of SDF-1 or 10 ng/ml IL-6 and incubated with PC-3 cells in Matrigel invasion assays. One-way ANOVA and paired student's t-test calculate the level of significance set at P<0.05 as * and P<0.001 as ***.</p

PR supresses IL-6 expression ligand-independently in prostate stromal cells.

<p>hCAFs and their derived cells expressing mock, PRA or PRB were treated with vehicle, 1(<b>A</b>) or vehicle, 10 nM of P4 and/or 10 uM of RU486 (<b>B</b>) for 24 hours. Real-time PCR measured mRNA levels of IL-6 relative to GAPDH. (<b>C</b>) hCAFs and their derived cells expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. CM were collected and used to measure IL-6 protein levels by ELISA. WPMY-1 cells and their derived cells expressing mock, PRA or PRB were treated with vehicle, 1 nM, 10 nM and 100 nM of P4 (<b>D</b>) or vehicle, 10 nM of P4 and/or 10 uM of RU486 (<b>E</b>) for 24 hours. Real-time PCR measured mRNA levels of IL-6 relative to GAPDH. (<b>F</b>) HPS-19I cells and their derived cells expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. Real-time PCR measured mRNA levels of IL-6 relative to GAPDH. One-way ANOVA and student's t-test calculated the significance set with P<0.01 as * and P<0.001 as ***.</p

PR supresses SDF-1 expression ligand-independently in prostate stromal cells.

<p>hCAFs and their derived cell lines expressing mock, PRA or PRB were treated with vehicle, 1(<b>A</b>) or vehicle, 10 nM of P4 and/or 10 uM of RU486 (<b>B</b>) for 24 hours. Real-time PCR measured mRNA levels of SDF-1 relative to GAPDH. (<b>C</b>) hCAFs and their derived cell lines expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. CM were collected and used to measure SDF-1 protein levels by ELISA. WPMY-1 and its derived cell lines expressing mock, PRA or PRB were treated with vehicle, 1 nM, 10 nM and 100 nM of P4 (<b>D</b>) or vehicle, 10 nM of P4 and/or 10 uM of RU486 (<b>E</b>) for 24 hours. Real-time PCR measured mRNA levels of SDF-1 relative to GAPDH. (<b>F</b>) HPS-19I cells and their derived cells expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. Real-time PCR measured mRNA levels of SDF-1 relative to GAPDH. One-way ANOVA and followed by student's t-test calculate the significance set at P<0.01 as * and P<0.001 as ***.</p

Ultrasound imaging.

<p>A. The mice were anesthetized with isoflurane and mounted on the heated imaging table with continuous monitoring of vital signs. After visualization of the bladder with the Vevo 700® small animal imaging platform the skin was perforated with a 30G needle. B. Ultrasound visualisation of normal mouse bladder in sagittal section with typical dimensions indicated (lumen dimensions 4.4×6.5 mm; wall thickness 0.25 mm).</p