Although chemical castration by ADT has been the standard frontline therapy for advanced-stage PCa, disease progression is predicted to occur when cancer develops into CRPC. To better understand the changes involved in the transition to the progressive state, thorough study of the mechanisms of ADT resistance is important. The upregulation of components of the AR signaling pathway in intraprostatic tissue is one of the resistance mechanisms in CRPC. This study found upregulation of AR and steroidogenic enzymes in ADT-treated PCa patients. Furthermore, this study, in accordance with other studies, found that CRPC can develop in less than 12 months after the commencement of ADT  However, until now, no study has evaluated when the mechanism that triggers resistance to ADT becomes active in the prostate during ADT.
This study delves into the early response to ADT by evaluating intraprostatic AR and steroidogenic enzyme changes using prostate tissue from patients who still experienced urinary retention during ADT. It revealed a notably unique finding in the subgroup of patients who had ADT for only 12 months or less. Two patients had high intratumoral AR gene and protein expression after 3 months of ADT. It can be speculated that the resistance mechanism to ADT  through upregulation of AR might start as early as 3 months after the beginning of ADT. To the best of our knowledge, this is the first study to show early AR upregulation in human PCa tissue during ADT. This early resistance mechanism should be a warning to clinicians that this process should be monitored when starting ADT .
Another interesting result is that AR was the only gene that was upregulated at the early stage (3 months). PCa cells might start to overcome low serum androgen levels due to ADT by increasing AR expression first [10,11,12,13]. This suggests that the early mechanism to overcome low serum androgen levels is increased AR expression [10,11,12,13]. Many in vitro studies have shown upregulation of AR expression, demonstrating the adaptations of prostatic cells that increase sensitivity to low androgen levels after treatment with ADT [21,22,23]. However, these phenomena can be seen only in patients with orchiectomy who receive ADT. This might show that an abrupt decrease in serum testosterone levels induces the upregulation of AR. Furthermore, there are many known mechanisms of AR changes, including gene amplification and mutation, which have also been reported in patients with ADT > 12 months [8,9,10]. However, this study examined only protein expression and did not further evaluate the other AR changes, namely, AR amplification and AR mutation.
PCa cell growth is promoted by androgens, especially DHT . This study found that 5α-reductase isoenzymes, which regulate the conversion of T to DHT [11, 12], were increased in ADT-treated PCa patients. Similar to other studies, SRD5A1 [14, 24] and SRD5A3 [14, 25] were upregulated in ADT-PCa patients compared with ADT-naïve PCa patients, and SRD5A2 was downregulated [14, 24, 26]. However, until now, there has been very limited information on whether the isozyme is involved in the process of androgen biosynthesis. Interestingly, this study found that SRD5A was the only steroidogenic enzyme that was upregulated in the ADT ≤12 months group. This SRD5A upregulation was also related to AR upregulation. This suggests that PCa cells upregulate the expression of SRD5A, which is the primary enzyme responsible for the conversion of T to DHT expression, after or at the same time as AR expression is upregulated . Thus, developing a new strategy or compound that targets SRD5A can reduce the risk of early resistance.
Among the PCa-ADT patients with ADT durations of less than 12 months, three patients showed upregulation of genes with increased protein expression. Three of four patients showed upregulation of AR, with one patient showing upregulation AR and SRD5A1, 2, and 3; one showing upregulation of AR, SRD5A1 and SRD5A2; and one showing only AR upregulation. These findings are novel, as no one has ever investigated below the cut-off of 12 months. This study suggests the possible upregulation of AR and steroidogenesis enzymes (namely, SRD5A1, SRD5A2, and SRD5A3) as a compensatory mechanism for the low testosterone level due to ADT . Our study showed that patients whose SRD5A1, 2, or 3 level becomes upregulated have increased expression of the proteins later (at 7–9 months of ADT) than those with upregulated AR (at 3–7 months of ADT). This suggests that the increase in AR is the first compensatory mechanism, followed by the increases SRD5A1, 2, and 3. However, further studies with more patients are needed to validate this compensatory response.
Many studies have shown that there is a shift to adrenal androgen usage for maintaining DHT levels via upregulation of AKR1C3 expression [10,11,12,13,14]. This study showed that AKR1C3 can only be found in patients treated with ADT for more than 12 months, which is in accordance with other studies . The next question is why AKR1C3 is not upregulated in the early state. Based on the steroidogenic pathway, AKR1C3 is an upstream enzyme that converts adrenal androgen to downstream androgens, which are needed as a source of DHT [12,13,14]. To support the previous statement, we hypothesize that in PCa cells, AKR1C3 expression is increased after AR and SRD5A upregulation. However, it has been shown that there are many variations in AKR1C3, SRD5A and AR expression among patients in the ADT > 12 months group. This might be due to the dynamic process of steroidogenesis. The AR or steroidogenesis enzymes are regulated based on ‘real-time’ conditions as needed by PCa cells.
The main limitation of our study is the small sample size. However, this is the first study that tried to evaluate AR signaling pathway changes during ADT in human prostate tissues. This study also revealed an important finding in which PCa cells may adapt to low androgen levels caused by ADT before PSA levels rise. This finding is not the first significant one with interesting information that is limited by a low sample size. One study performed by Alsinnawi M et al. contributed significant prognostic information in which high expression of the SLCO gene may result in worse disease-free survival (DFS), with only 11 samples included in the study . Although the sample size was small, the results of the mentioned study were in concordance with the results of Terakawa T et al.’s team, who examine similar outcomes but with more patients included (n = 494) . Similar studies had similarly small sample sizes yet showed significance in practice. With only a few samples, Tiwari et al. showed that AR and its transcriptional corepressor REST modulate SPINK1 expression and that SPINK1 plays a plausible role in the progression of neuroendocrine prostate cancer . Another study by Cheung et al., using only 11 samples per group, found that Actin alpha cardiac muscle 1 (ACTC1) gene expression plays a role in compensating ADT administration for PCa as a response to ADT-induced muscle loss .
In addition to the sample size, other limitations of our study include the limited number samples available due to some nonutilizable old specimens unsuitable for RNA extraction and protein expression evaluation. Another limitation of this study is its use of the median expression level of each gene in BPH tissues as the cut-off for defining upregulation in other samples. This was the only available method, as there is currently no official validated cut-off to define upregulation of AR or steroidogenic genes in immunohistochemistry staining.
In conclusion, AR and steroidogenic enzymes are upregulated in PCa patients who are treated with ADT. Early AR and SRD5A upregulation can be found at 3 months in ADT patients. This indicates that the early evaluation of AR and SRD5A expression in intraprostatic tissue should be done. Further strategic treatment should target AR and the SRD5A enzyme to overcome early resistance to ADT.