Histopathological and prognostic significance of the expression of sex hormone receptors in bladder cancer: A meta-analysis of immunohistochemical studies

<div><p>Objective</p><p>Emerging preclinical evidence suggests the involvement of sex hormones and their receptor signals in the development and progression of bladder cancer. Meanwhile, previous studies have demonstrated conflicting results on the relationship between the status of sex hormone receptors in urothelial tumors and histopathological characteristics of the tumors or patient outcomes. We therefore conducted this meta-analysis to assess the clinicopathological impact of the expression of androgen receptor (AR) and estrogen receptors (ERs) in bladder cancer.</p><p>Methods</p><p>A comprehensive literature search in databases (<i>i</i>.<i>e</i>. PubMed, Web of Science, Cochrane) was performed for all immunohistochemical studies stained for AR, ERα, and/or ERβ in surgically resected bladder cancer specimens and analyzed for patient outcomes. We selected eligible studies in accordance with the PRISMA guidelines and analyzed data using R software.</p><p>Results</p><p>A total of 2,049 patients from 13 retrospective studies were included in this meta-analysis. The difference in ERα expression between non-tumors and tumors was significant [odds ratio (OR) = 0.412; <i>P</i><0.001], while those of AR (OR = 3.256; <i>P</i> = 0.336) or ERβ (OR = 0.580; <i>P</i> = 0.674) were not statistically significant. AR positivity in tumors was strongly correlated with gender (male <i>vs</i>. female: OR = 0.658; <i>P</i> = 0.027) or tumor grade (low-grade <i>vs</i>. high-grade: OR = 0.575; <i>P</i><0.001). ERβ positive rates were significantly higher in high-grade (OR = 2.169; <i>P</i><0.001) and muscle-invasive (OR = 3.104; <i>P</i><0.001) tumors than in low-grade and non-muscle-invasive tumors, respectively. Survival analysis in patients with non-muscle-invasive bladder cancer revealed associations between AR expression and better recurrence-free survival [hazard ration (HR) = 0.593; <i>P</i> = 0.006) as well as between ERβ expression and worse recurrence-free (HR = 1.573; <i>P</i> = 0.013) or progression-free (HR = 4.148; <i>P</i> = 0.089) survivals.</p><p>Conclusions</p><p>These data suggest down-regulation of ERα expression in bladder tumors, compared with non-neoplastic urothelial tissues. AR or ERβ expression was down- or up-regulated, respectively, in high-grade and/or muscle-invasive bladder cancers. Moreover, immunohistochemistry of AR/ERβ in surgical specimens may serve as prognosticators in patients with non-muscle-invasive bladder tumor.</p></div

Flowchart of literature search and selection process.

<p>(a) AR and (b) ER.</p

Eligible immunohistochemical studies assessing the expression of sex hormone receptors in bladder tumors.

<p>Eligible immunohistochemical studies assessing the expression of sex hormone receptors in bladder tumors.</p

Meta-analysis between sex steroid hormone receptor expression and clinicopathological features of bladder cancers.

<p>Meta-analysis between sex steroid hormone receptor expression and clinicopathological features of bladder cancers.</p

Infection of RANKL-primed RAW-D macrophages with <i>P. gingivalis</i> induces osteoclastogenesis.

<p>(A) (B) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces the formation of TRAP-positive MNCs. (C) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces mRNA expression of the osteoclast-specific gene, cathepsin K. Total RNA was isolated, and cathepsin K expression was assessed by real-time PCR. Expression levels were normalized to GAPDH. Effect of pretreatment (D), or OPG (E) on osteoclastogenesis induced by infection with <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) or TNF-α (10 ng/ml) for 22 h, then infected with <i>P. gingivalis</i>, and cultured for 24–48 h. After 24 h, RNA was extracted and analyzed for gene expression. After 48 h, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared to uninfected control (B, C) or untreated control (D).</p

Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis in the absence of TNF-α.

<p>(A) Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis. Representative photographs are shown. (B) Effect of neutralizing antibody against mouse TNF-α on osteoclastogenesis in RANKL-primed BMM induced by TNF-α or live <i>P. gingivalis</i>. BMM were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated by TNF-α or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. At the end of culture, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared with control IgG1.</p

Possible role of TNF-α in osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.

<p>Macrophages respond to infection with <i>P. gingivalis</i> by producing TNF-α, which stimulates osteoclastogenesis in osteoclast precursor cells in the absence of <i>P. gingivalis</i> (A). However, osteoclast precursor cells primed with RANKL do not produce TNF-α and respond differentially to various stimuli. (B) Cells that are not stimulated do not differentiate into osteoclasts. (C) Cells that are continuously re-stimulated with RANKL differentiate into osteoclasts in an NFATc1- and NF-κB-dependent manner in the presence of <i>P. gingivalis</i>. (D) Cells that are infected with <i>P. gingivalis</i> differentiate into osteoclasts in an NFATc1-dependent and NF-κB-independent manner. TNF-α does not stimulate osteoclastogenesis in osteoclast precursor cells in the presence of <i>P. gingivalis</i>, whereas RANKL stimulates osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.</p

<i>P. gingivalis</i> induces osteoclastogenesis in RANKL-primed RAW-D cells in the absence of TNF-α.

<p>Analysis of TNF-α mRNA expression (A) or production of TNF-α protein (B) by <i>P. gingivalis</i> infected RANKL-primed RAW-D cells and unprimed cells. (C) Effect of neutralizing antibody against mouse TNF-α on osteoclast formation in RANKL-primed RAW-D cells induced by TNF-α or live <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then retreated with TNF-α or live <i>P. gingivalis</i> in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. After 24 h, RNA was extracted, and TNF-α mRNA expression was assessed by real-time PCR. After 48 h, cell supernatants were collected and analyzed for TNF-α by ELISA. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed infected RAW-D or RANKL-primed uninfected control (A), unprimed control (B), or control IgG1 (C).</p

TLR4 is not involved in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection with <i>P. gingivalis</i>.

<p>Effect of <i>E.coli</i> LPS or Pam3CSK4 (A) or <i>P. gingivalis</i> LPS (B) on osteoclastogenesis in RANKL-primed RAW-D cells. (C) Effect of heat treatment of <i>P. gingivalis</i> on osteoclastogenesis in RANKL-primed RAW-D cells. (D) Effect of polymyxin B on osteoclast formation in RANKL-primed RAW-D cells induced by <i>E. coli</i> LPS, Pam3CSK4, live <i>P. gingivalis</i>, or <i>P. gingivalis</i> LPS. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then treated with <i>E. coli</i> LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), <i>P. gingivalis</i> LPS (10 µg/ml) or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence of various concentrations of polymyxin B. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01 compared to untreated controls (A, B, and C) or controls without polymyxin B (D).</p

Expression of osteoclast signaling proteins in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection.

<p>RAW-D cells were stimulated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 22 h. RANKL-primed RAW-D cells were then retreated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 24 h. Total RNA was prepared, cDNA was synthesized, and real-time PCR analysis was performed using NFATc1 (A), c-fos (B), or IFNβ (C) Taqman probes. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed control or RANKL-primed control.</p