Though castration by ADT has been the standard frontline therapy for advanced stage of PCa, disease progression is predicted to occur, into a state of castrate-resistant prostate cancer (CRPC). To better understand the changes towards the progressive state, elaborate study onto the mechanisms of resistance is important. The upregulation of AR signalling pathway 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 to other study, have found that CRPC can be developed in less than 12 months after commencement of ADT. (19) However, until now, there were no study evaluate how early the resistant mechanism in the prostate starts to overcome ADT.
This study delves into the early response to ADT, by evaluating intraprostatic AR and steroidogenic enzymes changes using prostate tissue from patients who still complained a urinary retention during ADT. It revealed a notable unique finding in the subgroup of patients who only had ADT under 12 months. There were two patients that had a high intratumoral AR gene and protein expression after 3 months of ADT. It can be speculated that the resistance mechanism to ADT (10) through upregulation of AR might start as early as 3 months. To best of our knowledge, this was the first study that showed an early AR upregulation in human PCa tissue during ADT. This early resistant mechanism should warn clinicians to detect this process when starting ADT. (20).
Other interesting result that is AR was the only gene which upregulated in early state (3 months). PCa cell might start to overcome low serum androgen level due to ADT by increasing AR expression first (10-13). It suggests that the early mechanism to overcome low serum androgen level is increasing an AR expression (10-13). Many in-vitro studies have shown upregulation of AR expression illustrating adaptations of the prostatic cells to increase sensitivity to low androgen level after treatment of ADT (21,22,23). However these phenomenon can only be seen in orchiectomy as an ADT patient. It might show that an abrupt decreasing serum testosterone level induces the upregulation of AR. Furthermore, here are many known mechanisms of AR changes, i.e. amplification, mutation, that have also been reported in ADT >12 months condition (8-10). However, this study only examined the protein expression and did not further evaluate the other AR changes i.e. AR amplification, AR mutation.
It has been known that PCa cell growth is promoted by androgen, especially DHT (12). This study found that the 5α-reductase isoenzymes, which regulate the conversion of T into 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 the ADT naïve PCa Patients, and SRD5A2 to be downregulated (14,24,26). However, up until now there has been very limited information whether the isozyme is involved in the process of androgen biosynthesis. Interestingly, this study found that SRD5A were the only steroidogenic enzyme which upregulated in ADT ≤12 months patients. This SRD5A upregulation were also related to AR upregulation. This speculates that after or simultaneously PCa cell upregulate AR expression, PCa cells upregulate SRD5A expression which is the closest enzyme is responsible the conversion of T to DHT (14). Thus, developing a new strategy or compound which targeted SRD5A can reduce the risk of early resistance.
Among the PCa-ADT patients with duration under 12 months, there were three patients showing upregulation of gene with increased protein expression. Three from four showed upregulation of AR, consisting of one patient with AR and SRD5A1, 2, and 3; one patient with upregulation of AR and SRD5A1 and 2; and one patient only had AR upregulation. These finding is novel to our research, as no one has ever investigated further below the cut-off of 12 months. This study would like to suggest that a possible upregulation of AR and the steroidogenesis enzyme (namely, SRD5A1, SRD5A2, and SRD5A3) as a compensation mechanism to the low testosterone level due to ADT (14). Our study showed, that patients that develop upregulated SRD5A1, 2, or 3 showed increased expression of protein later than those of AR, ranged from 7-9 months of ADT duration for SRDA1,2 and 3 compared with AR that occurred ranged from 3-7 months of ADT duration. This suggests that AR increased activity as a compensatory mechanism comes first, then followed by SRD5A1, 2, and 3. However, further studies with more patients is needed in order to conclude the compensatory response.
Many studies have shown that there is a shifting to an adrenal androgen usage for maintaining DHT level by upregulated of AKR1C3 expression (10-14). This study found that AKR1C3 can only be found in patients treated with ADT above 12 months duration, which is in accordance to other study (27). The further question is why the AKR1C3 is not upregulated in early state, based on steroidogenic pathway, AKR1C3 is an upstream enzyme that converts adrenal androgen to downstream androgen which needed as a source of DHT (12-14). To support the previous statement, we hypothesize that PCa cell will increase AKR1C3 expression after the AR and SRD5A upregulation. However, it has been shown that there are many variations of AKR1C3, SRD5A and AR expression in each ADT >12 months patients. It might due to the dynamic process in steroidogenesis. The AR or steroidogenesis enzymes were regulated based on ‘real time’ condition which is needed by PCa cells.
The main limitation of our study is the low sample size included. However, this is the first study which tried to evaluate the AR signalling pathway changes during ADT in human prostate tissues. This study also revealed an important finding in which PCa cell may adapt to low androgen level, caused by ADT, before rising its PSA level. This finding is not the first significant study with tantalizing information limited by low sample size. A study done by Alsinnawi M, et al has contributed a significant prognostic information where high expression of SLCO gene may result in worse Disease-free survival (DFS) with only 11 samples included in the study (28). Although small in number, the result of the mentioned study were in concordance with the result of Terakawa T, et al. team, examining the similar outcome, only with more number of patients included (n=494) (29). Similar studies had similarly low sample size yet give significance in practice. Tiwari et al showed AR and its transcriptional co-repressor REST modulate SPINK1 expression, and plausible role of SPINK1 in the progression of Neuroendocrine prostate cancer with only few samples (30). Another study by Cheung et al, using only 11 samples per group founded that the Actin alpha cardiac muscle 1 (ACTC1) gene expression would play a role in compensating ADT administration for PCa as a response to ADT-induced muscle loss (31).
Beside the lacking of sample numbers, other limitation of our study were limited samples available due to some non-utilizable old specimens unsuitable for RNA extraction and protein expression evaluation. Another limitation of this study, is using the median expression of each gene in BPH tissues as cut off in defining upregulation in other samples. This was the only available method, as currently no official cut off to define upregulation of AR and steroidogenic gene in immunohistochemistry staining that has been validated.
In conclusion, there are upregulation of AR and steroidogenic enzymes in PCa patients which were treated with ADT. The early AR and SRD5A upregulation can be found in 3 month in ADT patient. This indicates that the early evaluation of AR and SRD5A expression in intraprostatic tissue should be done. Further strategic treatment should be targeting AR and SRD5A enzyme to overcome early resistance to ADT.