BIRC6 Protein, an Inhibitor of Apoptosis: Role in Survival of Human Prostate Cancer Cells

<div><h3>Background</h3><p>BIRC6 is a member of the Inhibitors of Apoptosis Protein (IAP) family which is thought to protect a variety of cancer cells from apoptosis. The main objective of the present study was to investigate whether BIRC6 plays a role in prostate cancer and could be useful as a novel therapeutic target.</p> <h3>Methods</h3><p>BIRC6 expression in cell lines was assessed using Western blot analysis and in clinical samples using immunohistochemistry of tissue microarrays. The biological significance of BIRC6 was determined by siRNA-induced reduction of <em>BIRC6</em> expression in LNCaP cells followed by functional assays.</p> <h3>Results</h3><p>Elevated BIRC6 protein expression was found in prostate cancer cell lines and clinical specimens as distinct from their benign counterparts. Increased BIRC6 expression was associated with Gleason 6–8 cancers and castration resistance. Reduction of BIRC6 expression in LNCaP cells led to a marked reduction in cell proliferation which was associated with an increase in apoptosis and a decrease in autophagosome formation. Doxorubicin-induced apoptosis was found to be coupled to a reduction in BIRC6 protein expression.</p> <h3>Conclusion</h3><p>The data suggest a role for BIRC6 in prostate cancer progression and treatment resistance, and indicate for the first time that the <em>BIRC6</em> gene and its product are potentially valuable targets for treatment of prostate cancers.</p> </div

Reduction of BIRC6 expression decreases prostate cancer cell viability and proliferation.

<p><b>A</b>, treatment of LNCaP cell cultures with siRNAs targeting BIRC6 leads to reduction of BIRC6 protein expression. BIRC6 protein expression in untreated LNCaP cells (Lane 1; at 78 h) and in LNCaP cells incubated with lipofectamine only (Lanes 2, 6, 10), siRNA-1 targeting BIRC6 (Lanes 3, 7, 11), siRNA-2 targeting BIRC6 (Lanes 4, 8, 12) or non-targeting siRNA (Lanes 5, 9, 13) for 30, 54 and 78 h after transfection. The results are representative of three independent experiments. <b>B</b>, treatment of LNCaP cell cultures with siRNA-2 targeting BIRC6 leads to reduced cell proliferation as shown by MTT assay. Cultures were treated for 30 h with lipofectamine only or with lipofectamine plus either non-targeting siRNA or siRNA-2 targeting BIRC6 and then incubated for 24, 48 and 72 h in fresh media. The relative cell numbers in the siRNA-2 cultures were considerably lower than those in the non-targeting siRNA treated cultures by 2.85%, 10.78% and 25.88% at 54, 78 and 102 h, respectively. Error bars depict standard deviations.</p

Decreased BIRC6 expression induces apoptosis and inhibits autophagosome formation.

<p><b>A</b>, apoptosis of LNCap cells transfected with Lipofectamine (Lipo), non-targeting control siRNA (NT) or <i>BIRC6</i> siRNA2 and subsequently incubated for 48 h, as assessed by flow cytometric analysis of Annexin V/PI stained cells. On the basis of Q2+Q3 values (apoptotic cell populations), <i>BIRC6</i> siRNA markedly increased apoptosis of LNCaP cells compared with Lipo (p = 0.0104) or NT siRNA treated cells (p = 0.0123). (Data are representative of 3 independent experiments); <b>B</b>, treatment of LNCaP cells with siRNA-2 targeting BIRC6 leads to activation of caspase-3 (as shown by appearance of cleaved caspase-3) and degradation of its substrate, PARP (as shown by loss of full length PARP and appearance of a cleaved PARP product). Untreated LNCaP cells (Lane 1); LNCaP cells incubated with lipofectamine only (Lane 2), siRNA-2 targeting BIRC6 (Lane 3) or non-targeting siRNA (Lane 4) for 96 h following transfection. <b>C</b>, treatment of LNCaP cells with siRNA-2 targeting BIRC6 leads to decreases in Beclin-1 expression and reduction of LC3B-II. Untreated LNCaP cells (Lane 1); LNCaP cells incubated with lipofectamine only (Lane 2), siRNA-2 targeting BIRC6 (Lane 3) or non-targeting siRNA (Lane 4) for 113 h following transfection. <b>D</b>, autophagosome formation in BIRC6-silenced cells was significantly lower compared to cells treated with non-targeting control siRNA. Cells transfected with non-targeting siRNA control (left) showed more LC3-GFP puncta than cells transfected with siRNA-2 targeting BIRC6 (right), which showed diffused expression of LC3-GFP. <b>E</b>, Reduced BIRC6-expressing LNCaP cells shows significantly fewer autophagic cells (33.6%) compared to control cells (51%). Autophagic cells were quantified by counting cells with more than 15 LC3-GFP puncta under a confocal microscope.</p

BIRC6 protein expression is elevated in prostate cancer cells in comparison with benign prostate cells.

<p>BIRC6 protein expression in malignant prostate cells (Lanes 1–6), positive control malignant cervical and ovarian cells (Lanes 7–8) and benign prostate cells (Lanes 9–10).</p

Doxorubicin-induced apoptosis in LNCaP cells is associated with a reduction in BIRC6 protein expression.

<p><b>A</b>, incubation (24 h) of LNCaP cells with doxorubicin (1 µg/mL) leads to reduction of BIRC6 protein expression and apoptosis (loss of full length PARP and appearance of cleaved PARP) as indicated by Western blot analysis. <b>B</b>, LNCaP cells incubated for 24 h (1) without doxorubicin and (2) with doxorubicin (1 µg/mL) at 100× magnification. <b>C</b>, Expression of BIRC6 and PARP cleavage were studied in LNCaP cells incubated with doxorubicin (500 ng/mL) for 4, 8, 12, 24 h. BIRC6 expression was decreased upon treatment in a time-dependent manner and the reduction precedes apoptosis induction as indicated by PARP cleavage at 24 h.</p

BIRC6 protein expression in TMA sections of prostate tissue samples.

<p><b>A</b>, arrows indicating weak cytoplasmic BIRC6 staining in benign prostatic luminal cells (score ‘1′). <b>B</b>, arrows indicating moderate cytoplasmic BIRC6 staining in Gleason grade 3 prostate cancer cells (score ‘2′). <b>C</b>, arrows indicating strong cytoplasmic BIRC6 staining in Gleason grade 4 prostate cancer cells (score ‘3′). <b>D</b>, arrows indicating weak cytoplasmic BIRC6 staining in Gleason grade 5 prostate cancer cells (score ‘1′). <b>E</b>, Black bars representing mean BIRC6 staining intensity in benign prostate epithelium (n = 35) and Gleason score 6 (n = 74), 7 (n = 23), 8 (n = 43) and 9–10 (n = 17) patient prostate cancer tissues. Grey bars representing frequency of BIRC6 expression (score ≥1). Elevated staining intensities were observed in Gleason score 6 (*<i>P</i> = 3.24×10−3), 7 (*<i>P</i> = 4.45×10−6) and 8 (*<i>P</i> = 1.63×10−3) prostate cancer tissues as compared to benign prostate epithelium. Error bars depict standard deviations. <b>F</b>, Black bars representing mean BIRC6 staining intensity in untreated high grade (Gleason score 8–10) prostate cancer tissues (n = 60) and castration-resistant prostate cancer (CRPC) tissues which had developed from Gleason score 8–10 cancers following neo-adjuvant hormone therapy (n = 10). Grey bars representing frequency of BIRC6 expression (score ≥1). Castration-resistant prostate cancer tissues showed a significantly higher mean BIRC6 staining intensity than untreated high grade prostate cancer tissues (*<i>P</i> = 1.38×10−3). Error bars depict standard deviations.</p

Pathological Parameters of Study Patients and PSA Levels.

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

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

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

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