Androgen receptor mRNA expression is a predictor for recurrence-free survival in non-muscle invasive bladder cancer

Bladder cancer (BC) is a common malignancy, with an estimated 429,800 new cases and 165,100 deaths in 2012, worldwide [1]. Urothelial carcinoma is the most common type of BC, and it is stratified to non-muscular invasive bladder cancer (NMIBC) and muscular invasive bladder cancer (MIBC). Patients with MIBC have a high risk of disease progression and metastasis and usually need aggressive treatments, such as radical cystectomy or chemotherapy; whereas NMIBC has a better prognosis and can be treated curatively by transurethral resection of bladder tumor (TUR-BT). However, NMIBC has a high risk of recurrence and can progress to MIBC. To prevent recurrence and progression, intravesical instillation (IVI) of anthracycline or bacillus Calmette-Guérin (BCG) has been administered, but about 50% still recur. As the treatment has not changed for decades, new studies leading to new treatments are needed to prevent recurrence and progression.

Men tend to be diagnosed about four times more often than women [2], which implies that BC is an endocrine-related cancer. Androgen receptor (AR) signaling has been suggested to have an important role in BC occurrence and progression by previous studies [3‐18].

Patients’ characteristics are shown in Table 1. Patients’ median age at TUR-BT was 74.1 years (range: 40.8–88.8 years) and 48 (90.6%) patients were male. Of the 53 patients, 11 (20.8%) were recurrent BC and 9 patients (17.0%) were recurred within 1 year. Tumor grade was low in 39 patients (73.6%) and high in 14 patients (26.4%). Pathological T-stage was pTa in 42 patients (79.2%) and pT1 in 11 patients (20.8%). Tumors were single in 24 patients (45.3%) and multiple in 29 patients (54.7%). Tumor size was ≥3 cm in 7 patients (13.2%); concurrent carcinoma in situ (CIS) was found in 2 patients (3.8%). Immediate IVI using anthracycline was administered in 51 patients (96.2%) and additional adjuvant IVI using either BCG or anthracycline was administered in 12 patients (22.6%). Six patients (11.5%) underwent second TURs.

We found no significant association between AR mRNA expression level and other clinicopathological factors (Spearman’s rank-order correlation; Table 2).

The Kaplan–Meier curve for RFS is shown in Fig. 1. Within a median observation period of 10.2 (range: 0.4–29.5) months, recurrence occurred in 17 patients (32.1%).

We stratified the 53 patients into two groups: 43 in the high AR mRNA-expressing group and 10 in the low AR group. The high AR group showed a tendency for longer median RFS (not reached) than did the low-AR group (9.04 months, P = 0.112; Fig. 2).

Results of univariate and multivariate analyses to identify risk factors for RFS are shown in Table 3. In univariate analysis, female sex (hazard ratio [HR]: 5.009, 95% CI: 1.589–15.787, P = 0.006) and tumor size ≥3 cm (HR: 3.487, 95% CI: 1.282–9.480, P = 0.0141) were significantly related to shorter RFS. Multivariate analysis showed that female sex (HR: 7.360, 95% CI: 1.649–32.856, P = 0.009), tumor size ≥3 cm (HR: 23.697, 95% CI: 4.383–128.117, P < 0.001) and low AR mRNA expression (HR: 0.202, 95% CI: 0.048–0.841, P = 0.028) were independent predictors of shorter RFS.

Although the function of AR signaling in BC is unclear, it is known to play important roles in prostate cancer occurrence and progression, and reportedly affects kidney, lung, breast and liver cancers [20].

AR is a nuclear steroid hormone receptor, composed of several domains, N-terminal transactivation domain, DNA binding domain, a hinge region and a ligand-binding domain (LBD). In the cytoplasm, heat shock protein-90 (HSP90) binds with the LBD. As 5α-dihydrotestosterone binds to AR, AR releases HSP90 and translocates into the nucleus, binds to the androgen-response element, and promotes gene transcription [3‐6, 21].

AR signaling and the urinary tract may be critically associated. Studies in animal models have shown AR to be present in several tissues in lower urinary tract and may affect growth, differentiation and maintenance of urinary bladder tissue [6, 7, 22, 23].

Reportedly, AR signaling has a pivotal role in BC occurrence and progression. Testosterone increased the risk of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder tumors in female rats, whereas diethylstilbestrol, a synthetic form of female hormone estrogen, inhibited incidence in male rats [8]. Also, intact female rats administered with testosterone had higher incidences of both bladder calculi and tumors, but oophorectomized rats did not, which implies that testosterone in combination with estrogen may contribute to BC formation [9]. Moreover, flutamide, a nonsteroidal antiandrogen therapy, inhibited BBN-induced bladder tumor incidence in rats, whereas finasteride, a 5α-reductase inhibitor, did not; this suggests that testosterone itself has an effect in bladder carcinogenesis but its converting form, 5α-dihydrotestosterone, does not [10].

Studies with animal models have indicated that AR has an important role in BC. Miyamoto et al. found that both male and female AR-knockout mice (ARKO) did not develop BBN-induced tumors, which suggests that AR has a crucial activity in bladder carcinogenesis [11]. That study also found that 25% of ARKO mice administered with dihydrotestosterone (DHT) developed tumors, compared with 50% of castrated male wild-type rats and 92% of intact wild-type male rats. These findings suggest that absence or low levels of androgen could restore AR signaling, or that androgens could induce bladder carcinogenesis independently of AR [7, 24]. Another study showed that mice lacking AR only in the urothelial tissues had lower incidence of BBN-induced BC and a higher survival rate than wild-type mice [12].

Therefore, the association between BC and AR has been the focus of several recent studies. Expression of AR have indicated to have high risk in bladder cancer occasion, but as for recurrence, expression of AR have shown to contribute to longer RFS. An immunohistochemical study of 169 patients indicated that recurrence was less likely for patients with AR⁺ BC specimens [13]; another study showed that loss of AR was strongly associated with higher grade and more invasive tumors [14]. A study of the predictive value of AR mRNA expression in pT1 NMIBC showed that high AR mRNA expression was an independent predictor for longer RFS and cancer-specific survival [15], which was similar to our present findings that high AR mRNA level was an independent predictor for RFS in NMIBC (including pTa and pT1).

Reportedly, AR pathway inhibitors have been shown to be effective in preventing BC recurrence. In patients with double primary cancers of the prostate and the bladder, androgen-deprivation therapy (ADT) to treat their prostate cancers reduced the risk of recurrence of AR⁺ BC [16]. Enzalutamide, an AR-signaling inhibitor, is also reported to inhibit AR⁺ BC cell growth in vivo [17]. BCG IVI has been the most effective therapy for NMIBC; its mechanism may be due to interaction between BCG and the AR pathway. DHT down-regulates NF-κB-mediated IL-6 expression, whereas AR blockers inhibit the effect of DHT. This result suggests that AR pathway inhibitors could improve the efficacy of BCG treatment [18].

As described above, previous clinical studies demonstrated that higher AR expression in both mRNA levels and protein levels were correlated with favorable outcome in NMIBC [13‐15]. Moreover, our current study also demonstrated that higher AR mRNA expression levels were associated with longer recurrence-free survival in NMIBC. On the other hand, enhanced AR signaling pathway could play an important role in cancer initiation and progression in vitro and in vivo. There were discrepancies between our findings and previous reports from studies in vitro or in vivo [8, 10‐12]. Even though the reasons for discrepancies are unknown, several possible hypothesis have been suggested. It has to be considered that AR expression, and thus the role of AR, might change during the progression of bladder cancer, as indicated by the previously cited studies [14]. In addition, Sikic et al. reported that different AR subtype (AR 1 and AR2) has different role in bladder cancer carcionogenesis [14].

The mechanism of AR signaling in BC is still unclear, therefore, further studies of the AR pathway in NMIBC are needed.

Our study had several limitations. First, it was a retrospective study with relatively few patients and a short follow-up period. Second, CIS was not analyzed in this study. CIS is an essential risk factor of recurrence and progression in NMIBC, but our study cohort had only two BCs with coexisting CIS. Finally, for verify the role of AR pathway in NMIBC, not only AR mRNA expression levels, but also protein expression levels should be investigated in current study. Future clinical studies would need to estimate an appropriate sample size to include enough of these patients and investigate mRNA and protein expression levels simultaneously.