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Keywords:

  • castration-resistant prostate cancer;
  • hypogonadism;
  • intratumoural steroidogenesis;
  • testosterone replacement therapy

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

Intratumoural steroidogenesis may play a significant role in the progression of prostate cancer (PC) in the context of long-term ablation of circulating testosterone (T). To clarify the mechanism accounting for the progression of PC in a 74-year-old man who had undergone bilateral orchiectomy when he was 5 years old, we performed immunohistochemical studies of androgen receptor (AR) and steroidogenic enzymes in the prostate. We also measured steroid hormone levels in the serum and prostate, as well as mRNA levels of genes mediating androgen metabolism in the prostate. Positive nuclear staining of AR was detected in malignant epithelial cells. The levels of androstenedione (Adione), T, and 5-alpha dihydrotestosterone (DHT) in the serum of the patient were similar to those in PC patients receiving neoadjuvant androgen deprivation therapy (ADT), but were higher in the patient's prostate than in PC patients not receiving ADT. The gene expression of CYP17A1 and HSD3B1 was not detected, whereas that of STS, HSD3B2, AKR1C3, SRD5A1, and SRD5A2 was detected. Moreover, cytoplasmic staining of HSD3B2, AKR1C3, SRD5A1, and SRD5A2 was detected in malignant epithelial cells. Hence, in the present case (a man with primary hypogonadism), steroidogenesis in PC tissues from adrenal androgens, especially dehydroepiandrosterone sulphate, was the mechanism accounting for progression of PC. This mechanism might help elucidate the development of castration-resistant PC.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

Prostate cancer (PC) is the most common male malignancy in Western countries (Siegel et al., 2012). The steroid 5-alpha-reductase (SRD5A) converts circulating testosterone (T) into 5-alpha dihydrotestosterone (DHT), a high-affinity ligand for the androgen receptor (AR) in the prostate, and these active androgens and AR signalling play a central role in the development and progression of PC (Huggins & Hodges, 1941). Androgen deprivation therapy (ADT) with chemical or surgical castration is the standard treatment for advanced PC. Most patients with a more aggressive form of PC called castration-resistant PC (CRPC) go into relapse (Culig et al., 1998).

Previous studies have demonstrated that, in the absence of serum T with ADT, levels of both T and DHT in CRPC tissues are sufficient to activate AR (Mohler et al., 2004; Titus et al., 2005b), and that the expression of genes required for androgen synthesis is increased in CRPC tissues (Stanbrough et al., 2006). These findings suggest that androgen-AR signalling plays a significant role in the progression of CRPC. Therefore, measurements of intraprostatic androgen levels, as well as expression levels of the genes mediating androgen metabolism, are essential to elucidate the mechanism accounting for the development and progression of CRPC.

Primary hypogonadism is caused by testicular failure and is characterized by low levels of T in the serum (Bhasin et al., 2010). From the viewpoint of PC incidence in individuals with long-term ablation of circulating T, characterization of the endocrine environment of PC tissues in patients with primary hypogonadism might be useful for elucidation of the development of CRPC. However, this has never been investigated.

In the present report, we describe a case of PC in a patient with primary hypogonadism ascribable to bilateral orchiectomy in childhood. To elucidate the role of androgens in the development and progression of PC in this patient, we performed immunohistochemical studies of AR and steroidogenic enzymes in the prostate. Furthermore, we measured serum and intraprostatic steroid hormone levels, and mRNA levels of the genes required for androgen synthesis in the prostate.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

Case report

A 74-year-old man, who had had diabetes mellitus for 7 years visited a general hospital with a main complaint of general fatigue. His medical history revealed bilateral orchiectomy at age 5 years referable to testicular trauma in a traffic accident, and he had not been treated with testosterone replacement therapy (TRT) prior to the medical examination. Laboratory analyses revealed low total T levels (0.2 ng/mL) and high luteinizing hormone and follicle-stimulating hormone levels (14.5 and 58.5 mIU/mL respectively) in the serum. Prostate-specific antigen (PSA) levels in the serum were not examined. The patient was diagnosed with primary hypogonadism, and subsequently treated with testosterone enanthate (125 mg, once a month) by a physician at the general hospital. Six months after the initiation of TRT, his general fatigue disappeared and his haemoglobin A1c level improved from 8.5 to 6.0%. However, 2 years after the initiation of TRT, frequent urination and an elevated PSA level of 49.0 ng/mL in the serum were observed, and TRT was subsequently withdrawn. The patient's PSA level in the serum declined to 13.8 ng/mL 3 months after withdrawal of TRT, but it subsequently increased to 23.6 ng/mL 6 months thereafter. Four months after re-elevation of PSA levels, the physician referred him to our hospital. A physical examination revealed obesity, the absence of pubic hair, a small penis and an operative scar on the scrotum. In a digital rectal examination, a walnut-sized, stony, hard prostate with an irregular surface was palpated. Laboratory analysis revealed an elevated PSA level of 38.0 ng/mL in the serum. Transrectal ultrasound-guided transperineal prostate needle biopsy showed a poorly differentiated adenocarcinoma with a Gleason score of 4 + 5 = 9. Radiological imaging revealed gynecomastia and bone metastasis (Fig. 1), resulting in the diagnosis of stage D2 (T3aN0M1b) PC. We initiated treatment with bicalutamide (80 mg, once daily). Eight months after bicalutamide therapy, the patient's PSA level declined to 0.3 ng/mL, and he stopped taking medication ascribable to diarrhoea. One month after therapy cessation, the PSA level was 0.6 ng/mL, and he was subsequently treated with flutamide (135 mg, three times daily). One month after the initiation of flutamide therapy, the PSA level declined to 0.2 ng/mL. However, he died in a car accident soon after this measurement.

image

Figure 1. Radiological imaging. Bone scintigraphy showing abnormal radionuclide uptake in the right pubic bone (open circle).

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Immunohistochemical analysis of AR and steroidogenic enzymes

Immunohistochemical staining for AR, HSD3B2, AKR1C3, SRD5A1 and SRD5A2 was performed on 5-μm-thick paraffin-embedded prostatic tissue specimens. The methods for immunohistochemistry and the characteristics of the primary antibodies used in this study have previously been reported in detail (Nakamura et al., 2005, 2009). Mouse testis specimens, human adrenal gland and liver were used as positive controls for immunostaining. Negative control staining, in which primary antibody was replaced with phosphate-buffered saline, was also performed. No specific immunoreactivity was detected in these tissue sections (data not shown).

Hormone measurements in the serum and prostate

Serum samples were collected before prostate biopsy. The prostatic tissues were collected by biopsy of a hypoechoic area in the palpably indurated part of the prostate. After collection, the samples were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. The serum adrenocorticotropic hormone (ACTH) level was measured using the electrochemiluminescence immunity assay. Serum dehydroepiandrosterone sulphate (DHEA-S) and free T levels were measured using radioimmunoassay (BML, Tokyo, Japan). The serum unconjugated androsterone level was measured using radioimmunoassay (SRL, Tokyo, Japan). For comparison of androgen and oestrogen levels between the present case and other PC patients, serum samples and prostatic tissues were also obtained from eight PC patients at Kiryu Kosei General Hospital. Of these patients, three (median age 71 years) underwent radical prostatectomy for PC with neoadjuvant ADT, and five (median age of 68 years) underwent radical prostatectomy for PC without ADT. Neoadjuvant ADT was only performed with a luteinizing hormone-releasing hormone agonist, and the median duration of ADT was 5 months. Levels of DHEA, androstenedione (Adione), T, DHT, and estradiol in the serum and prostate and androstenediol (Adiol) levels in the prostate were measured using liquid chromatography-tandem mass spectrometry, according to our previously developed methods (Arai et al., 2010, 2011). The lower limits of quantification for DHEA, Adiol, Adione, T, DHT and estradiol were 2, 1, 1, 1, 1 and 0.15 pg/tube respectively. All samples were obtained in accordance with the requirements and approval of the Committee for Human Ethics and Experimentation at Gunma University and Kiryu Kosei General Hospital. Written informed consent was obtained from all patients and the experimental procedures were conducted in accordance with ethical standards of the Declaration of Helsinki.

Quantitative RT-PCR analysis of the genes required for androgen synthesis

Total RNA was extracted from frozen prostatic tissues using the RNeasy Plus Mini Kit (QIAGEN, Valencia, CA, USA), and was subsequently reverse-transcribed using random primers (Life Technologies, Carlsbad, CA, USA). Transcript levels were quantified using an ICycler IQ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. Amplification was performed using 2 μL cDNA and the following primers purchased from Life Technologies: CYP17A1 (Hs01124136_m1), STS (Hs00996676_m1), HSD3B1 (Hs00426435_m1), HSD3B2 (Hs00605123_m1), AKR1C3 (Hs00366267_m1), SRD5A1 (Hs00602694_mH) and SRD5A2 (Hs00165843_m1). Subsequently, PCR was performed as follows: 10 min at 95 °C followed by 45 cycles of 15 sec at 95 °C and 60 sec at 60 °C. Reactions were performed in triplicate for statistical evaluation. For standard curve generation and as an internal control, the 18S rRNA transcript level was measured according to a previous method (Koike et al., 2011). As tissue controls, adrenal glands, benign prostatic tissues and cancerous prostatic tissues (n = 3 each) were obtained from radical nephrectomy specimens from patients with kidney cancer, and prostate biopsy specimens were obtained from patients with benign prostatic hyperplasia (BPH; PSA ≤ 8) or PC (Gleason score ≥ 4 + 4 = 8).

Statistical analysis

Experimental quantitative RT-PCR data are expressed as means ± SD of three independent experiments (n = 3). All calculations were performed using IBM spss Statistics Version 20 (spss Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

Immunohistochemical analysis of AR and steroidogenic enzymes

Immunohistochemical studies showed positive staining of AR, HSD3B2, AKR1C3, SRD5A1 and SRD5A2 in the positive control mouse testes, human adrenal glands and liver [Fig. 2(F–J)]. In the tissue from the present case, positive nuclear staining of AR was detected in malignant epithelial cells (Fig. 2A), in which cytoplasmic staining of HSD3B2, AKR1C3, SRD5A1 and SRD5A2 was also detected [Fig. 2(B–E)].

image

Figure 2. Immunohistochemical localization of AR and steroidogenic enzymes. (A) AR, (B) HSD3B2, (C) AKR1C3, (D) SRD5A1 and (E) SRD5A2 in PC tissues with a Gleason score of 4 + 5 = 9 (×20 magnification). (F) AR in mouse testis (×20 magnification). (G) HSD3B2 and (H) AKR1C3 in human adrenal gland (×20 magnification). (I) SRD5A1 and (J) SRD5A2 in human liver (×20 magnification). Negative control staining without primary antibody yielded no specific immunoreactivity (data not shown). Scale bar: 50 μm.

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Serum and prostate hormone levels

The ACTH and DHEA-S levels in the serum of the present case were within the reference ranges for adult men provided by commercial laboratories, whereas free T and androsterone levels were below the lower limits of the reference ranges (Table 1). Adione, T and DHT levels in the serum of the present case were similar to those in PC patients receiving ADT, but intraprostatic Adione, T, and DHT levels were higher in the present case than those in PC patients receiving ADT (Table 2). Moreover, the intraprostatic T level in the present case was high compared with that in PC patients not receiving ADT (Table 2). Intraprostatic estradiol levels, as well as levels in the serum, were higher in the present case than in PC patients receiving ADT (Table 2).

Table 1. The serum hormone levels in the present case
Serum hormonesReference rangesaPresent case
  1. ACTH: adrenocorticotropic hormone, DHEA-S: dehydroepiandrosterone sulphate, free T: free testosterone.

  2. a

    The reference ranges were provided by commercial laboratories. The reference ranges of DHEA-S and free T were adjusted for age.

ACTH (pg/mL)10–6025.2
DHEA-S (ng/mL)50–2530794.0
Free T (pg/mL)4.5–13.8<0.5
Androsterone (ng/mL)0.18–0.910.08
Table 2. Patient characteristics and the hormone levels in the serum and prostate
Patient characteristics and hormonesPresent casePC with ADT (N = 3)PC without ADT (N = 5)p Valuea
  1. PC: Prostate cancer, ADT: Androgen deprivation therapy, PSA: Prostate-specific androgen, IQR: interquartile range, DHEA: dehydroepiandrosterone, Adione: androstenedione; T: testosterone, DHT: 5-alpha dihydrotestosterone.

  2. a

    The Mann–Whitney U-test was used for statistical comparison between PC patients receiving ADT and PC patients not receiving ADT.

PSA [ng/mL, median (IQR)] 3.87 (1.24–8.40)8.62 (6.28–13.73)0.101
No. Gleason score
6≥ 010.121
7 02
8≤ 32
No. pathological stage
T2N0M0 230.860
T3N0M0 12
Serum [median (IQR)]
DHEA (ng/mL)1.302.11 (1.79–2.44)1.71 (1.57–2.74)0.786
Adione (ng/mL)0.360.44 (0.27–0.82)0.40 (0.30–0.69)0.786
T (ng/mL)0.190.22 (0.09–0.37)4.35 (1.78–6.34)0.036
DHT (ng/mL)0.020.04 (0.02–0.05)0.29 (0.14–0.68)0.036
Estradiol (pg/mL)13.01.74 (1.40–2.78)20.12 (5.13–24.55)0.036
Prostate [median (IQR)]
DHEA (ng/g)7.3223.71 (19.89–36.47)34.89 (22.84–73.36)0.571
Adiol (ng/g)0.441.04 (0.78–2.41)2.40 (1.48–3.70)0.393
Adione (ng/g)0.360.06 (0.04–0.08)0.21 (0.06–0.76)0.250
T (ng/g)0.500.02 (0.01–0.03)0.09 (0.04–0.18)0.071
DHT (ng/g)2.770.75 (0.32–1.79)4.21 (4.00–4.69)0.036
Estradiol (pg/g)34.521.80 (8.70–22.45)23.10 (10.65–40.68)0.571

Expression of the genes required for androgen synthesis

In the present case, the expression of CYP17A1 and HSD3B1 was not detected in the prostate, whereas that of STS, HSD3B2, AKR1C3, SRD5A1 and SRD5A2 was detected (Fig. 3).

image

Figure 3. Quantification of transcript levels of (A) CYP17A1, (B) STS, (C) HSD3B1, (D) HSD3B2, (E) AKR1C3, (F) SRD5A1 and (G) SRD5A2 in the prostate and adrenal gland. n = 3 per group for BPH, PC, and adrenal gland samples. Quantitative RT-PCR was performed in triplicate to quantity mRNA levels, as described in 'Materials and methods'. For gene expression, the 18S rRNA transcript level served as an internal control. Data are expressed as fold change vs. internal controls and are presented on a logarithmic scale. Bars represent the mean ± SD.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

To the best of our knowledge, this is the first report of elevated intraprostatic steroid hormone levels as well as decreased serum levels in a man with primary hypogonadism suffering from PC. We demonstrated that AR protein was expressed in the PC tissues, and that intraprostatic Adione, T and DHT levels were high in the present case compared with those in PC patients receiving ADT. Moreover, the expression patterns of steroidogenic enzymes and genes mediating androgen metabolism in the prostate suggest that increased synthesis of T and DHT from DHEA-S in the PC tissues contributed to the progression of PC in the present case.

Primary hypogonadism is characterized by low T levels in the serum (Bhasin et al., 2010). TRT has the benefit of improving a variety of physical disorders and, although controversial, the potential disadvantage of inducing PC (Rhoden & Morgentaler, 2004). Indeed, a few reports have suggested a relationship between TRT and PC incidence in patients with Klinefelter syndrome (Hwang et al., 2003; Bydder et al., 2007). In the present case, elevated PSA levels in the serum were observed after 2 years of TRT. Although measurement of the patient's PSA level had not been performed prior to TRT, PC in the present case may have been induced by TRT.

The difference in intraprostatic endocrine environment between androgen-dependent PC and CRPC remains unclear. In healthy men, chemical castration for 1 month causes a 70–80% decline in the intraprostatic T level (Page et al., 2006). We previously demonstrated that the intraprostatic T level in androgen-dependent PC with ADT was reduced to 20–30% of that in androgen-dependent PC without ADT (Arai et al., 2011). Interestingly, the intraprostatic T level in CRPC is similar to that in the benign prostate (Titus et al., 2005b), and T levels in tissue are significantly higher in anorchid men with metastases of PC than in untreated eugonadal men with primary PC (Montgomery et al., 2008). These findings suggest that increased synthesis of T in PC tissues may induce resistance to long-term ablation of circulating T.

The adrenal glands are a major source of androgen precursors, which include DHEA-S, DHEA, and Adione (Adams, 1985). Importantly, the DHEA-S level in the serum is 100–500 times higher than that in the T level (Hsing et al., 2007). The adrenal androgens may be converted into T in PC tissues. STS, which converts DHEA-S into DHEA, and AKR1C3, which converts Adione into T, are expressed in the majority of PCs (Nakamura et al., 2005, 2006), while HSD3B, which converts DHEA into Adione, is expressed in the prostate (El-Alfy et al., 1999). Moreover, genes encoding enzymes that convert adrenal androgens into T in CRPC tissues show increased expression (Stanbrough et al., 2006). In the present case, AR protein was expressed in the PC tissues, and intraprostatic Adione and T levels were higher than those in PC patients receiving ADT. Moreover, although HSD3B1 gene expression was not detected, STS gene expression was detected in the prostate, and HSD3B2 and AKR1C3 proteins were expressed in the PC tissues. Notably, STS was the most highly expressed gene mediating androgen metabolism in the present case. These findings suggest that, in the present case, increased intraprostatic synthesis of T from DHEA-S may be the mechanism accounting for the development and progression of PC.

In contrast, the intraprostatic DHT level in CRPC is reduced to 10% of that in the benign prostate, which does not correspond to the intraprostatic T level in CRPC (Titus et al., 2005b). Although SRD5A2 is the predominant form in the benign prostate, the expression and activity of SRD5A2 are exceeded by those of SRD5A1 in CRPC tissues (Titus et al., 2005a; Stanbrough et al., 2006). The dominant pathway to DHT synthesis from adrenal precursors in CRPC follows an alternative route that bypasses T and requires 5-alpha reduction of Adione by SRD5A1 into 5-alpha androstanedione, which is then converted into DHT (Chang et al., 2011). Compared to SRD5A2, SRD5A1 requires a higher T concentration to achieve a 50% maximal rate of DHT production (Jin & Penning, 2001). Moreover, genes catabolizing DHT in CRPC tissues show increased expression (Stanbrough et al., 2006). Taken together, these findings suggest that although DHT is still predominantly synthesized by SRD5A1, decreased SRD5A2 activity and increased DHT catabolism may cause the reduction in the intraprostatic DHT level in CRPC. In the present case, the intraprostatic DHT level was reduced, but remained 66% of that in PC patients not receiving ADT. In addition, the expression of SRD5A2 was four times higher than that of SRD5A1. Although we did not investigate the expression of genes catabolizing DHT, these results may account for the high intraprostatic DHT level in the present case. Furthermore, intraprostatic DHT levels in CRPC may be maintained via the back-door pathway using 5-alpha-reduced steroid precursors such as androsterone (Locke et al., 2008). However, in the present case, the patient's androsterone level in the serum was below the lower limit of the reference ranges for adult men provided by commercial laboratories. These results suggest that the back-door pathway may not have been activated in the present case.

The limitations of our study include the issue of the heterogeneity of PC tissues. Recent studies have suggested that intratumoural androgen synthesis is heterogeneous in CRPC tumours (Mostaghel et al., 2010). Thus, our results for the expression of genes mediating androgen metabolism may only reflect a part of the intraprostatic endocrine environment in the present case. Moreover, our case is unique. Although it may inform the biology of CRPC, it represents the development of PC after many years of androgen deprivation and is not necessarily reflective of the most common clinical scenario of CRPC.

In summary, this is the first case report of elevated intraprostatic steroid hormone levels, as well as decreased levels in the serum, in a man with primary hypogonadism suffering from PC. It appears that steroidogenesis from adrenal androgens in the PC tissues was the mechanism accounting for the development and progression of PC in our patient. This mechanism might contribute to a better understanding of CRPC development.

Conflicts of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

This research did not receive any specific grant from any funding agency in the public, commercial, or non-profit sectors.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References

All authors were involved in writing the paper and approved the submitted versions.

Seiji Arai performed the research. Seiji Arai, Yasuhiro Shibata, Koshi Hashimoto, Kazuto Ito and Kazuhiro Suzuki analysed the data. Seiji Arai, Bunzo Kashiwagi, Takatoshi Uei, Yukio Tomaru and Yoshitaka Sekine contributed to the tissue collections. Seiji Arai, Yoshimichi Miyashiro and Seijiro Honma performed the hormone quantification. Yasuhiro Nakamura and Hironobu Sasano performed the immunohistochemical studies.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Funding
  9. Author contributions
  10. References
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