**1. Introduction**

Prostate cancer (PCa) is one of the most commonly diagnosed malignancies and the second leading cause of cancer deaths in the United States [1]. In Japan, the mortality rate of PCa is the sixth among those of all male malignancies, although the estimated incidence rates have slightly declined, possibly due to reduced prostate-specific antigen (PSA) screening, the same as in the United States [2]. In the early 1940s, Huggins and Hodges demonstrated growth and survival of PCa to depend on androgens [3]. Therefore, androgen deprivation therapy (ADT) has been a standard clinical procedure for the control of PCa growth, with patients mostly responsive to ADT at the beginning of the therapy, which is also called hormone-naïve prostate cancer (HNPC); however, most of those patients relapse thereafter, developing castration-resistant prostate cancer (CRPC). ADT is required to treat advanced PCa and biochemical recurrent cases after curative radical treatment; however, despite the reduction of serum testosterone (T) to castration levels and an observed tumor response in 80%–90% of the patients, residual concentrations of intratumoral 5 α-dihydrotestosterone (DHT) remain at 10%–40% of the pre-ADT levels in castration-resistant and hormone-naïve states [4,5]. The amount of residual androgens is substantial for triggering androgen receptor (AR) signaling, AR target gene expression, and cancer cell proliferation [6]. The de-novo pathway, which commences with cholesterol requiring multiple androgen synthetic enzymes, may be a result of the intratumoral androgen function; however, whether the complete repertoire of synthetic enzymes is required to generate androgens from cholesterol remains to be fully elucidated [7,8]. Circulating adrenal androgens, which are abundant in the form of dehydroepiandrosterone (DHEA) and with sulfated modification, dehydroepiandrosterone sulfate (DHEA-S), are other significant points of origin of the androgens. The acquired capacity of converting the adrenal androgens to more potent forms is a characteristic of CRPC. Therefore, abiraterone acetate, which is a potent CYP17A1 (17-hydroxylase/17, 20-lyase) inhibitor, is e ffective against CRPC, and has been recently approved for treating metastatic HNPC. Type 5 17 α-hydroxysteroid dehydrogenase, in another name, aldo-keto reductase family 1 member C3 (AKR1C3) is a crucial enzyme in the steroidogenesis pathway. It catalyzes Δ4-androsetene-3, 17-dione to T, DHT to 5 α-androstane-3 α, 17β-diol (3 α-diol), and 3 α-diol to androsterone; thus, it plays an important role in the formation of T and DHT [9]. Additionally, AKR1C3 can also reduce the weak estrogen, estrone, to the potent estrogen, 17β-estradiol, which might induce local estrogen production, contributing to PCa occurrence [10,11]. Estrogen and estrogen receptor (ER) (ER alpha: ER α and ER beta: ERβ) axes play an important role in both prostate carcinogenesis and progression to CRPC [12,13]. Although PCa co-expresses classical ERs, ER α and ERβ, and also non-genomic receptor, GRP30, complex interactions between ERs and AR, and those among various ligands in PCa cells need further investigation [12,13]. Moreover, AKR1C3 is known as prostaglandin (PG) F synthase that catalyzes the conversion of PGD2 to 11-βPGF2 α and PGF2 α prostanoids, hence contributing to proliferation and radio-resistance in PCa cells [14,15]. All these issues imply that AKR1C3 could have a potential role in PCa biology. Several studies have demonstrated that AKR1C3 expression levels are elevated in PCa cells than in benign cells; moreover, it is highly expressed in the CRPC cell lines and human CRPC tissues rather than in the hormone-naïve ones [9,16–22]. Nevertheless, most reports have focused on the PCa tissues derived from di fferent patients and compared the expression levels in normal/benign prostate hyperplasia tissues, localized cancer, and metastatic CRPC. This study aimed to compare the expression level of AKR1C3 between normal prostatic epithelium and cancer cells in the same patients. Moreover, we evaluated AKR1C3 expression and PSA progression-free survival after radical prostatectomy. We also investigated the expression level in hormone-naïve cancer and advanced CRPC in the same patients, to better understand the role of AKR1C3 in PCa progression.

### **2. Materials and Methods**

### *2.1. Human Prostate Tissue Samples*

All PCa patients included in this study were Japanese patients. Tissue-microarrays (TMAs) consisted of 175 radical prostatectomy (RP) specimens of patients with hormone-naïve PCa, who received RP between December 2004 and October 2012 at Kyoto University Hospital [23]. The TMA was developed with one core from each case. This study included 134 cases, which had both cancer and non-cancer tissues in each TMA core. We defined PSA failure as two consecutive measurements of PSA levels of ≥0.2 ng/mL, and the date of PSA failure as the time of the first measurement of PSA level of ≥0.2 ng/mL. When PSA levels after surgery did not decline below 0.2 ng/mL, we defined the date of PSA failure by the time of surgery. Consecutive prostate samples derived from 11 patients, both at hormone-naïve and castration-resistant states, were evaluated. HNPC specimens consisted of samples from needle biopsy or from transurethral resection of the prostate (TUR-P). CRPC specimens were collected from the TUR-P samples against urinary retention or gross hematuria, penectomy for pain control, and spinal laminectomy against spinal cord compression due to bone metastases.
