A role for estrogen signaling in urothelial carcinoma of the bladder (UCB) is suggested to be associated with more advanced disease with worse outcomes in women. Estrogen receptor β (ERβ) is the predominant receptor in bladder tissues. We aimed to ascertain whether ERβ correlates with clinicopathological predictors of aggressive bladder cancer and worse survival outcomes. ERβ was measured by immunohistochemistry in malignant and adjacent benign bladder tissues in patients (N=72) with UCB who underwent radical cystectomy. ERβ expression was tested for statistical association with clinicopathological variables and patient survival. ERβ expression was determined in bladder cancer cell lines, and the effects of the selective estrogen modulator tamoxifen and the ERβ agonist diarylpropionitrile on cell growth were determined. The ERβ level was significantly higher in malignant vs. benign urothelium (P<0.001) and was strongly associated with aggressive tumor histology characterized by lymphovascular (P=0.008) and perineural (P=0.006) invasion, and clinical histories of pelvic irradiation (P=0.005), hydronephrosis (P=0.022) and no intravesical chemotherapy (P=0.038). All patients with a high (>70%) percentage of ERβ positivity in tissue with >3-month follow-up developed recurrent disease (P=0.009). Higher ERβ level was predictive of worse recurrence-free and overall survival following cystectomy, after adjustment for tumor stage, and remained significantly associated with recurrence-free survival in the multivariable analysis including tumor stage, nodal stage and lymphovascular invasion. Activation of ERβ in bladder cancer cell lines led to significant increases in proliferation, while pharmacological inhibition with tamoxifen blocked cell growth. Our study supports a role for ERβ in aggressive UCB. Pharmacological targeting of ERβ warrants further investigation as a therapeutic strategy in UCB.
Urothelial carcinoma of the bladder (UCB) is the fourth and tenth most common solid malignancy among US men and women, respectively (
The estrogen receptor genes, ERα and ERβ, have differential tissue expression patterns and functions (
As ERβ is the predominant estrogen receptor isoform in normal and malignant bladder tissue, our study aimed to better delineate the role of ERβ in bladder carcinogenesis. We quantified ERβ expression in both malignant and benign tissues from UCB patients undergoing cystectomy and tested its association with tumor pathology, including for the first time histological features such as lymphovascular, perineural invasion and concomitant carcinoma
Institutional Review Board approval was acquired for this study. Tissue samples (N=129), including urothelial cell carcinomas (n=59) and benign urothelium (n=70), were obtained from May 2002 to December 2007 at the Department of Pathology and Laboratory Medicine, New York Presbyterian Hospital, Weill Cornell Medical College as previously described (
Immunohistochemical staining for human ERβ protein (14C8 antibody, 1:300 dilution; Novus Biologicals, Littleton, CO, USA) was performed using the Leica BOND-MAX Autostainer and the Leica Microsystems Refine Detection kit as previously described (
The HTB-1 (J82), HTB-5 (TCCSUP) and HT1376 UCB cell lines were derived from high-grade invasive urothelial carcinomas, and the HTB-3 (SCaBER) cell line was derived from an invasive squamous cell carcinoma of the bladder (ATCC, Rockville, MD, USA). RNA was extracted and cDNA was synthesized as previously described (
The effects of 17β-estradiol (E2)(Sigma-Aldrich, St. Louis, MO, USA) and selective ERβ agonist diarylpropionitrile (DPN) (Tocris, Minneapolis, MN, USA), with and without tamoxifen (Sigma Aldrich), were assessed on bladder cancer cell lines at a 10 nM concentration as previously reported (
Fisher’s exact test was used to test for statistical association between ERβ immunostaining scores and clinicopathological variables (
The majority of cases had only low (1+) intensity ERβ staining (
ERβ levels were also tested for association with clinical variables, including risk factors for UCB diagnosis or UCB-specific mortality (
Median and mean patient follow-up after radical cystectomy was 21 and 26 months, respectively. Recurrence occurred in 21/72 (29%) patients, 15/72 (21%) died from their disease, and an additional 10/72 (14%) patients died of other causes. All patients with a high (>70%) percentage of ERβ positivity in tissue at >3-month follow-up developed recurrent disease. Tumor stage, lymph node stage (P=−0.0001), LVI (P=0.03) and elevated ERβ positivity (P=0.009) were significantly inversely associated with recurrence-free, cancer-specific and overall survival in the Cox univariable analysis (
In Cox multivariable analysis including ERβ positivity and tumor stage, both variables remained significantly inversely associated with recurrence-free and overall survival; tumor stage (P=0.02) but not ERβ (P=0.07) remained significantly associated with cancer-specific survival, likely because of the limited number of cancer-specific death events. A second multivariable analysis of survival outcomes was performed with ERβ positivity, tumor stage, lymph node stage and LVI. In this second analysis, increased ERβ positivity still remained significantly inversely associated with recurrence-free survival (P=0.02) independent of these variables (
ERα and ERβ mRNA expression was evaluated in the HTB-1 (J82), HTB-3 (SCaBER), HTB-5 (TCC-SUP) and HT1376 (CRL-1472) bladder cancer cell lines. All four cell lines expressed transcripts for ERβ but not ERα as compared to the MCF-7 breast cancer cell line (
Identification of the molecular mechanisms governing bladder carcinogenesis is critical for the development of novel pharmacotherapies and biomarkers for this disease. Despite clinical observations of more advanced UCB and shorter survival among female patients, in addition to an established role for ERβ in bladder development and function (
We sought to clarify these findings by examining ERβ in a well-described UCB patient cohort (
To corroborate our patient findings, we determined the effect of ERβ activation on the
To explore ERβ as a therapeutic target, we measured the growth effects of the selective ER modulator (SERM), tamoxifen, in UCB cell lines, with or without simultaneous ERβ activation using DPN. Several prior studies of SERMs have described conflicting growth inhibitory effects in bladder cancer cell lines or murine xenografts (
In conclusion, we demonstrated ERβ upregulation in UCB compared to benign urothelium, consistent with an oncogenic function. Furthermore, we uncovered a stage-independent association between ERβ levels and: i) aggressive UCB histology and ii) clinical traits carrying a poor prognosis. We provide the first demonstration of ERβ as an independent predictor of poor cystectomy patient outcomes after adjustment for tumor stage, lymph node involvement and LVI. We demonstrated that ERβ activation increases UCB cell proliferation but can be effectively blocked by SERM pharmacotherapy. Although the relationship between ERβ and tumor stage and patient outcomes is disputed (
The authors gratefully acknowledge the institutional financial support of Weill Cornell Medical College, the University of Nottingham and an F31 Fellowship from NIH (NIDCR) (K.M.).
Immunohistochemical staining. Representative immunohistochemical staining for ERβ in (A) benign urothelium, (B) non-muscle invasive urothelial carcinoma (pTis, pTa, pT1), (C) muscle invasive carcinoma (pT2) and (D) extravesical (pT3) carcinoma. (All images were captured at ×400 total magnification; bar, 20 μm). (E and F) Distribution of staining positivity and intensity according to pathological stage. Separate scores were assigned to the stained tissue sections based on (E) the intensity of positively staining cells (0, none; 1, weak; 2, moderate; and 3, strong) and on (F) tissue positivity, i.e., the percentage of tissue staining positive (0–100%). Only nuclear staining was considered positive, and a minimum of 100 tumor nuclei were counted for each case. Please refer to
Kaplan-Meier survival estimates. Kaplan-Meier estimates stratified by percentage of ERβ positivity in tissue (square, 0–10%; open circle, 11–40%; black circle, 41–70%; star, 71–100%) for (A) recurrence-free survival (RFS) (P=0.030), (B) cancer-specific survival (CSS) (P=0.0018), and (C) overall survival (P=0.0061).
Expression of ERα and ERβ in human bladder cancer cell lines and effects of estrogenic agonists and antagonists on bladder cancer cell proliferation. mRNA expression of (A) ERα and (B) ERβ in bladder cancer cell lines normalized to the expression in MCF7 breast cancer cells. Comparison of the effects of β-estradiol and the ERβ-selective agonist (DPN), in the presence and absence of tamoxifen (TAM) on the proliferation of (C) HTB-1, (D) HTB-3, (E) HTB-5 and (F) HT1376 bladder cancer cells. Statistical significance is indicated relative to control treated cells, except where indicated by bars between treatment groups. (*P<0.05, **P<0.01, ***P<0.001; ns, not significant).
Characteristics of the cystectomy patients.
Characteristics | |
---|---|
Total patients, n (%) | 72 (100) |
Age (years) | |
Mean ± SD | 66.4±10.0 |
Median | 66.6 |
Range | 43.3–88.8 |
Gender, n (%) | |
Female | 21 (29) |
Male | 51 (71) |
Race, n (%) | |
Caucasian | 66 (92) |
Other | 6 (8) |
BMI, kg/m2 | |
Mean ± SD | 26.8±4.8 |
Median | 26.5 |
Smoking history, n (%) | 56 (74) |
Active smoker, n (%) | 15 (21) |
Prior pelvic irradiation, n (%) | 8 (11) |
Prior intravesical chemotherapy, n (%) | 21 (29) |
Prior neoadjuvant systemic chemotherapy, n (%) | 11 (15) |
Pre-operative hydronephrosis, n (%) | 20 (28) |
Pathological stage, n (%) | |
pT0 | 13 (18) |
pTa | 9 (13) |
pTis | 11 (15) |
pT1 | 7 (10) |
pT2 | 10 (15) |
pT3 | 11 (15) |
pT4 | 11 (15) |
Lymph node metastases, n (%) | 18 (25) |
N-stage | |
N1 | 10 (14) |
N2 | 5 (7) |
N3 | 3 (4) |
Lymphovascular invasion (LVI), n (%) | 15 (21) |
Perineural invasion, n (%) | 6 (8) |
Concomitant carcinoma |
34 (47) |
Association of ERβ with patient histopathological traits, disease recurrence and cancer-specific mortality in tumor specimens.
Tumor (n=59) | Benign urothelium (n=70) | |||
---|---|---|---|---|
|
| |||
Histopathological parameters | ERβ positivity % | ERβ intensity | ERβ positivity % | ERβ intensity |
Stage (overall) | 0.448 | 0.326 | 0.499 | 0.694 |
Non-muscle invasive vs. invasive | 0.197 | 0.290 | 0.540 | 0.288 |
NMI vs. MI | 0.218 | 0.149 | 0.425 | 0.493 |
BC vs. EVE | 0.249 | 0.457 | 0.105 | 0.043 |
Lymphovascular invasion | 0.389 | |||
Perineural invasion | 1 | 0.493 | 1 | |
Comcomitant carcinoma |
0.230 | 0.323 | 0.628 | 0.823 |
Positive bladder margin | 0.769 | 1 | 0.841 | 1 |
Fisher’s exact P-values are shown for each statistical association. Bold print indicates significance (P<0.05). Non-muscle invasive (Ta, T1 and CIS) and muscle invasive (T2, T3 and T4) bladder cancers. NMI, non-muscle invasive; MI, muscle invasive; BC, bladder confined; EVE, extravesical.
Association of ERβ with patient clinicopathological traits, disease recurrence and cancer-specific mortality in tumor specimens.
Tumor (n=59) | Non-tumor (n=70) | |||
---|---|---|---|---|
|
| |||
Clinical parameters | ERβ % positivity | ERβ intensity | ERβ % positivity | ERβ intensity |
Age (years) | 0.283 | 0.630 | 0.156 | 0.373 |
Gender | 0.791 | 1 | 0.952 | 0.199 |
Race | 0.183 | 0.801 | 0.398 | |
Smoking | ||||
Any history | 0.399 | 0.819 | 0.813 | 1 |
Active | 0.478 | 1 | 0.441 | 1 |
Body mass index | 0.198 | 0.928 | 0.139 | 0.677 |
Prior pelvic radiation | 0.310 | 0.418 | 0.771 | |
Hydronephrosis | 0.122 | 0.393 | 1 | |
Intravesical chemotherapy | 0.194 | 0.083 | 0.330 | |
Neoadjuvant chemotherapy | 0.292 | 0.462 | 0.701 | 0.385 |
Fisher’s exact P-values are shown for each statistical association. Bold print indicates significance (P<0.05).
Multivariable associations of survival outcomes.
Univariate analysis | Multivariable analysis 1 | Multivariable analysis 2 | |
---|---|---|---|
|
|
| |
(P-value) | pT stage (P-value) | pT stage, N stage and LVI (P-value) | |
Recurrence-free survival | |||
Percentage of ERβ positivity in tissue | 0.0090 | 0.029 | 0.017 |
pT stage | <0.0001 | <0.0001 | 0.0004 |
N stage | 0.0001 | - | 0.14 |
Lymphovascular invasion | 0.030 | - | 0.91 |
Cancer-specific survival | |||
Percentage of ERβ positivity in tissue | 0.0014 | 0.11 | 0.26 |
pT stage | <0.0001 | 0.016 | 0.071 |
N stage | 0.0012 | - | 0.83 |
Lymphovascular invasion | 0.0017 | - | 0.89 |
Overall survival | |||
Percentage of ERβ positivity in tissue | 0.0061 | 0.041 | 0.067 |
pT stage | <0.0001 | <0.0001 | 0.017 |
N stage | 0.0003 | - | 0.58 |
Lymphovascular invasion | <0.0001 | - | 0.035 |
Multivariable analysis 1 includes ERβ percentage and pT stage. Multivariable analysis 2 includes ERβ percentage, pT stage, N stage and lymphovascular invasion (LVI).