Death receptor 5 expression is inversely correlated with prostate cancer progression

Prostate carcinoma (PCa) is one of the most common cancers in men. Prostate-specific antigen (PSA) has been widely used to predict the outcome of PCa and screening with PSA has resulted in a decline in mortality. However, PSA is not an optimal prognostic tool as its sensitivity may be too low to reduce morbidity and mortality. Consequently, there is a demand for additional robust biomarkers for prostate cancer. Death receptor 5 (DR5) has been implicated in the prognosis of several cancers and it has been previously shown that it is negatively regulated by Yin Yang 1 (YY1) in prostate cancer cell lines. The present study investigated the clinical significance of DR5 expression in a prostate cancer patient cohort and its correlation with YY1 expression. Immunohistochemical analysis of protein expression distribution was performed using tissue microarray constructs from 54 primary PCa and 39 prostatic intraepithelial neoplasia (PIN) specimens. DR5 expression was dramatically reduced as a function of higher tumor grade. By contrast, YY1 expression was elevated in PCa tumors as compared with that in PIN, and was increased with higher tumor grade. DR5 had an inverse correlation with YY1 expression. Bioinformatic analyses corroborated these data. The present findings suggested that DR5 and YY1 expression levels may serve as progression biomarkers for prostate cancer.

tivity may be too low to effectively predict mortality/survival rates (10). Conversely, increasing the sensitivity of PSA may result in false-positive diagnosis of the disease, since the majority of males with PCa will die of unrelated causes (11). Novel PCa biomarkers are required, which should have the ability to distinguish between a benign and a malignant disease (12,13).
Apoptosis through the activation of two predominant pathways may be induced by the binding of ligands to specific receptors on the cell surface, or by non-specific cellular stress (14). Both pathways converge at the level of the caspases, which mediate cell death via cleavage of various cellular substrates. Death receptors (DR) 4 and 5 bind to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which results in the recruitment of the Fas-associated death domain (FADD) to the intracellular death domain and subsequently induces the cleavage of procaspase-8 which initiates a cascade of events leading downstream to apoptosis (15)(16)(17)(18).
Reported studies have demonstrated that the surface expression of DR5 is a potentially useful prognostic marker in various cancers (19). The progression of melanoma has been previously associated with a decrease in DR5 and DR4 expression (20). Furthermore, DR5, along with other TRAIL receptors, was shown to be useful in determining the risk of breast cancer metastasis and patient survival (21). However, other studies have shown that DR5 was not a significant prognostic marker (22,23). These observations suggested that the prognostic value of DR5 may be cancer type-specific. In these cases, the role of DR5 repression in PCa was not examined.
A previous study by our group reported that the transcription factor Yin Yang 1 (YY1) may function in the pathogenesis of PCa. High expression levels of YY1 were associated with tumor progression, and patient survival was linked to lower levels of YY1 (24). In addition, it was previously demonstrated that YY1 may transcriptionally repress DR5 expression in prostate cancer cell lines (25)(26)(27).
The present study investigated the expression levels of DR5 in PCa and its clinical significance. Since it was reported that YY1 negatively regulates DR5 expression and that YY1 is overexpressed in PCa (24,25), it was hypothesized that DR5 expression may be inhibited in PCa and may be inversely correlated with YY1 expression. This hypothesis was tested by immunohistochemistry (IHC) on tissue microarray (TMA) constructs, prepared with tumor tissues derived from patients with PCa and PIN.

P ro s t a t e T M A c o n s t r u c t i o n .
F o r m a l i n -f i xe d , paraffin-embedded archival tumor specimens were prepared at the Immunology and Infection Research Unit, National Medical Center 'La Raza' and at the Oncology Disease Research Unit, Children Hospital of Mexico 'Federico Gomez', SSa. At least three core tissue biopsies (each 0.6 mm in diameter) were taken from morphologically representative regions of each prostate tumor and precisely arrayed as previously described (29)(30)(31). Tumor samples were accompanied by matching benign (morphologically normal or hyperplasic) and PIN lesions, where available. Tissues were arrayed into five TMA blocks. For staining, sections (4 µm) were transferred to glass slides using an adhesive slide system (PSA-CS 4; Instrumedics, Inc., St. Louis, MO, USA) to support cohesion of the array elements.
Immunohistochemical analysis. Slices cut at 4 µm for the TMA were placed on slides and either stained with Mayer's hematoxylin and eosin (H&E) for histopathological examination, or used for subsequent immunohistochemical analysis. The expression levels of DR5 and YY1 were determined using DR5 (Abcam, Cambridge, MA, USA) and YY1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies. Antigen retrieval was performed by immersing the slides in a solution of 0.01% sodium citrate pH 6.0 for 5 min in boiling water. Endogenous peroxidase activity was inhibited by immersing the slides in 3% H 2 O 2 -methanol and background-unspecific binding was decreased by incubating the slides in 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 60 min. The slides were incubated overnight at room temperature with predetermined optimal concentrations of anti-DR5 polyclonal antibody (1:500) and anti-YY1 polyclonal antibody (1:750). In order to decrease variability, all samples were processed at the same time in a single experiment, using a single batch of antibody diluted in PBS-BSA. Following washing, the slides were incubated with a biotinylated secondary antibody (Universal LSAB kit; Dako Corporation, Carpinteria, CA, USA) for 30 min at room temperature, followed by incubation with a streptavidin-horseradish peroxidase conjugate (Universal LSAB kit) for 30 min at room temperature and then with 3,3'-diaminobenzidine tetra-hydrochloride (liquid DAB, Dako Corporation). The reaction was stopped ny adding distilled water and the slides were counterstained with H&E. The tissue was washed in tap water for 5 min, dehydrated using an ethanol series (70, 90 and 100%) in xylene and mounted with E-2 mounting medium (Shandon lab, Pittsburgh, PA, USA). The slides were then analyzed by light microscopy (Olympus BX-40; Olympus Corporation, Tokyo, Japan).
Scoring of immunohistochemical staining. A semi-quantitative assessment of tissue antibody staining was conducted by an expert pathologist on prostate analysis, who was blinded to the pathological variables. The stained slides were verified by a second expert to ensure consistency in the scoring. Positive expression was scored based on either a positive staining on the membrane (DR5) or positive staining only in the nucleus (YY1). Data are presented as positively stained target cells per 100 cells (range 0-100% positive), per tissue region in the TMA (4 regions in each slide). In addition, the integrated optical density (IOD) in ~300 µm 2 regions per sample, selected randomly using the Image Pro-plus 6.4 software (Media Cybernetics, Bethesda, MD, USA), were analyzed.
The analysis of the expression levels on the TMAs was performed in a blind manner. The TMA spot was a second blinded quantitative assessment by the same pathologist. The target tissue for scoring was the glandular prostatic epithelium and the scoring of benign tissues did not include basal cells. The tissue spot histology and grading were confirmed on the counterstained study slides. Positive expression, indicated by a brown color, was quantified using the IODs. The density of the staining intensity in each region was analyzed using the Image Pro-plus 6.2 software (MediaCybernetics, Rockville, MD, USA) that was obtained with the diffusion of the light wavelength through the color density in the cells stained brown. The target tissue for scoring was performed in the malignant cells by consi dering the nuclear staining pattern for YY1 and the membrane cytoplasmic staining pattern for DR5. YY1 nuclear expression and DR5 cytoplasmic membrane expression were scored using two measures, negative and positive (weakly, moderately and strongly positive) intensities, in target cells. The data are presented as positively stained target cells per 100 cells (range 0-100% positive), per region on each spot (four regions in each spot), or as density, whereby four 100 µm 2 regions per spot were selected randomly to represent expression within each case. The mean pooled integrated intensity of the tumor or control spots was used.
Statistical analysis. The data were analyzed using Student's t-test for parametric data and the Mann-Whitney test for non-parametric data. To analyze normalized data, both the analysis of variance (ANOVA) and the Bonferroni tests were used. P<0.05 was considered to indicate a statistically significant difference.
The optimal cut-point for dichotomized PCa malignancy data was determined using the Gleason scoring system (32). The YY1-IOD-expression and DR5 expression were quantified by determining the sum of the IODs and the mean was calculated for each pathological group (malignant cells). For YY1 nuclear and DR-5 cytoplasmic membrane staining, the percentage of positive malignant cells (weak to strong brown staining) was obtained. Descriptive statistics were gathered for all the assembled data. For differences between the pathological groups that were used, Student's t test and the ANOVA Pearson's tests were used to analyze correlations of parametric data, respectively. Box plots to compare the various groups (central tendency, dispersion and symmetry of the data) were generated. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SSPS 11 statistical analysis program for Windows (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA).
Comparative metaprofiling of cDNA expression data. The Oncomine Premium database (Oncomine™ Compendia Bioscience, Ann Arbor, MI, USA) was used for analysis and visualization of the bioinformatics analyses (www.oncomine.com).
The differential expression analysis of YY1 and DR5 in existing prostate cancer microarray datasets was analyzed by setting a threshold value for gene rank at 10% and P<0.05.

Results
Evaluation of YY1 and DR5 protein expression in human prostate cancer tissues. Using immunohistochemical analysis, the YY1 and DR5 expression in PCa tissue microarray samples was examined. The expression of YY1 ( Fig. 1Aa  and b; Fig. 1Ba and b) and DR5 ( Fig. 1Ac and d; Fig. 1Bc and d) in human PCa tissues were examined in the PIN and malignant glandular epithelium (Figs. 1 and 2, respectively). YY1 was expressed predominantly in the nucleus and diffusely in the cytoplasm. DR5 was expressed on the membrane and in the cytoplasm. The expression of YY1 in the PIN samples was weak and predominantly localized to the cytoplasm. By contrast, the DR5 expression in the PIN samples was detected in the membrane and in the cytoplasm. In tumor samples, the YY1 expression was directly proportional to the malignant grade, whereby the highest expression was observed in HG PCa as compared with the expression in the LG PCa ( Fig. 2Aa and  b). By contrast, the DR5 expression was inversely proportional to the tumor grade (low in HG PCa and high in LG PCa) ( Fig. 2Ac and d).
Overexpression of YY1 and dowregulation of DR5 in PCa tissues. The expression of YY1 and DR5 was quantified by semiquantitative and quantitative assessments (Fig. 2B). There was a significant difference in the expression of YY1 between the LG PIN and HG PIN (Fig. 2Ba). In addition, there was a significant difference in the DR5 expression between the LG PIN and HG PIN samples (Fig. 2Bb) (P=0.001). YY1 was significantly elevated in PCa tissues as compared with the PIN tissues. YY1 expression was barely detectable in PIN tissues and was significantly increased (P<0.001, ANOVA) with an increase in tumor grade. This observation was similar in both the density quantification (Fig. 2Aa) and positive cell counts (nuclear) (Fig. 2Bc). An opposite effect was observed in the DR5 expression, whereby both the density of staining (Fig. 2Ab) and the positive cell counts (membrane) (Fig. 2Bd) were significantly decreased (P<0.001, ANOVA) as a function of the tumor grade. The highest levels of expression were observed in the PIN samples ( Fig. 2Ac and d).
Inverse correlation between DR5 and YY1 expressions in PCa tissues. A Pearson's analysis was performed, based on the YY1 and DR5 expression in all of the tumor samples. The expression levels of YY1 and DR5 were found to be inversely correlated in both the LG PCa (P<0.036, r Pearson=-0.406) and HG PCa (P<0.0001, r Pearson=�0.611) samples (Table I).
Bioinformatic analyses of YY1 and TNFRSF10B (DR5) gene expression in prostate adenocarcinoma. Analysis of the TNFRSF10B (DR5) expression levels in different prostate tumors was performed using a public data set of microarrays retrieved from the Oncomine database and gene expression Omnibus, derived from the published analysis reported by Vanaja et al (28) and Wallace et al (29). The microarray data included 69 prostate adenocarcinomas and were compared LG PCa and HG PCa. Both the PIN box plots are short since the YY1 IOD had a homogeneous distribution and minimal dispersion of cases. The YY1 IOD was low in both groups. The YY1 IOD expression in the PCa groups had a box plot greater than the PIN groups. The YY1 IOD was higher in both groups. The LG PCa box plot was less large, with a negative asymmetrical distribution, indicating that the majority of the measurements were lower than the HG PCa, but higher than the PIN groups. In addition, there was a low dispersion due to the homogeneity of the measurements. The HG PCa box plot had a positive asymmetrical distribution, meaning that the majority of the measurements were higher than those of the other groups. All the differences were statistically significant with P<0.0001. (b) DR5 IOD distribution in LG PIN, HG PIN, LG PCa and HG PCa. Both PIN box plots were shorter than those of the YY1 IOD, most markedly in the HG PIN group, where measures were greater. The dispersion in both groups was minimal and although there was asymmetry, this indicated that the measurements were homogeneous. The DR5 IOD expression in the PCa groups was very low, with a low dispersion, and a symmetrical and homogeneous distribution, which was due to low or absent expression. All differences were statistically significant with P<0.0001. * P= 0.002 and ** P= 0.01. YY1 Yin Yang 1; Dr5, death receptor 5; PIN, prostatic intraepithelial neoplasia; PCa, prostate carcinoma; TMA, tissue microarray constructions; IOD, integrated optical density; LG PCa, low grade prostate carcinoma; HG PCa, high grade prostate carcinoma; NL, normal prostate. with 20 samples from the prostate gland. In addition, YY1 and DR5 gene expression from the prostate adenocarcinoma samples were analyzed in the same data sets and compared with the prostate gland. The tumors showed low expression of DR5 as compared with the normal tissues (P=0.05 or 0.02, respectively). A higher expression of YY1 was observed in the same data sets as compared with the normal tissues (P<0.05) (Fig. 3). An inverse correlation of expression was observed between DR5 and YY1 in PCa. These findings were concordant with other database and gene expression analyses (30)(31)(32)(33).

Discussion
The present study has shown for the first time, to the best of our knowledge, the underexpression of DR5 in prostate cancer cell lines and patient-derived tumor tissues. The transcriptional regulation of DR5 was previously reported to be, at least in part, due to the repressor activity of YY1 (25). The present study therefore hypothesized that the transcriptional regulation of DR5 in human PCa would additionally be negatively regulated through the overexpression of YY1. The data of the present study

B A
confirmed this hypothesis and demonstrated an inverse correlation between the expression of DR5 and YY1. The experimental findings were corroborated by bioinformatic analyses. These data suggested that the expression levels of DR5 and YY1 in PCa may be novel prognostic factors in the progression of PCa.
The targeting of DR5 by TRAIL or anti-DR5 monoclonal antibodies has been the focus of various clinical trials in clinical cancers (34)(35)(36). The approach to inhibit the repression of DR5 through targeting YY1 will result in the upregulation of DR5 and its response to TRAIL-induced apoptosis. We have previously demonstrated that the inhibition of YY1 by YY1-specific siRNA in PCa cells resulted in sensitization of PCa cells to TRAIL apoptosis (37). It has been additionally shown that several anticancer agents overcome resistance to Apo2L/TRAIL through the upregulation of DR5 in malignant cells (25,38). In addition, the lower expression of DR5 has been noted in various types of cancer, including breast (39), lung (40), colorectal (41) and esophageal (42) cancers. Gene silencing of DR5 and DR4 was shown to abolish TRAIL-induced apoptosis (43).
Of note, the expression levels of DR5 have been described as a potentially useful prognostic marker in various cancers, including melanoma, where the decrease in DR5 expression was identified to be correlated with the progression as well as metastasis of the disease (20). Furthermore, DR5 expression was shown to be negatively correlated with the overall survival of breast cancer patients (22). DR5, however, was not found to be a significant prognostic marker in cervical or colon cancers (22,23). These observations suggested that the prognostic value of DR5 may be cancer type-specific. In PCa, the significance of DR5 expression had not been previously explored and the present findings suggested that it may be a significant novel prognostic marker in the progression of PCa.
The transcription factor YY1 is expressed in normal tissues and is upregulated in various types of cancer, including PCa, with positive and negative regulatory effects on gene expression. Elevated YY1 expression is correlated with the development of PIN and advanced prostate cancer (44,45). The results of the present study regarding the expression of YY1 and its correlation with the tumor grade are consistent with previous findings, which have linked higher YY1 expression to tumors, as compared with benign tissues.
In the present study, it was shown that YY1 was overexpressed in tumor samples as compared with PIN. The overexpression gradually increased with higher tumor grades ( Fig. 2A and B). By contrast, DR5 was considerably higher in PIN as compared to tumors. This downregulation of DR5 in the tumors decreased further with increasing tumor grade (Figs. 1  and 2). In addition, Pearson's analysis demonstrated an inverse correlation between the expression of YY1 and DR5 in PCa tissues (Fig. 3). This finding is consistent with the hypothesis that YY1 negatively regulates DR5 and this inverse correlation in PCa patient samples has an important implication in the development of this disease.
High DR5 expression has been linked to TRAIL-mediated apoptosis (15,(25)(26)(27). DR5 expression was shown to be low in the tumors as compared with PIN, which suggested that the tumor cells may be resistant to TRAIL-mediated apoptosis. This is relevant since the TRAIL-DR5-mediated apoptotic pathway is involved in the immune-mediated apoptosis signaling response by cytotoxic T lymphocytes and natural killer cells (46).
These results additionally suggested a mechanism whereby patient tumors with high YY1 expression and, therefore, low DR5 expression (repressed by YY1), would experience higher apoptosis resistance to both immune and chemotherapeutic drugs and, consequently, develop tumors of higher grades as a consequence of tumor unresponsiveness to therapy.
The present findings have only examined the expression of DR5 in PCa and several reports have demonstrated that DR5 is the preferential receptor-inducing signal for TRAIL apoptosis (25,27,39). The expression of DR4 and its role in TRAIL-induced apoptosis, however, cannot be ruled out. The expression of DR5 and DR4 is reduced in prostate tumors in comparison with that in benign tissues. Low expression of death receptors suggests resistance to apoptosis and, thus, increased tumorigenesis. However, YY1 has not been shown to regulate DR4 thus far to the best of our knowledge (47). Additional studies are therefore required to determine the role of DR4 in PCa and its clinical significance.
In conclusion, the present study demonstrated the clinical significance of DR5 downregulation in PCa and its inverse correlation with the expression of YY1. In addition, these data suggest a potential prognostic significance of both DR5 and YY1 in the progression of PCa.