## 3 Results
Inhibition of SRD5A1 with Outasteride Synergistically Enhances the Anticancer Potential of Low Dose Docetaxel, Reduced Metastatic Burden, and Confer Survival Advantage, In Vivo  
To provide more insight into the mechanistic underlining and pathobiological implication of the differential but concerted alteration of the HSD17B2, HSD17B3, SHBG, and SRD5A1 testosterone tetrad in disease course and therapy response of patients with PCa, we further probed the discovery cohort of the GSE70768 dataset (n = 199), consisting of complete, quality-controlled HT12v4 data for 13 castration-resistant prostate cancer (CRPC), 113 tumor, and 73 matched benign samples. Consistent with earlier results, we observed that, compared with _HSD17B2_ (t = 5.17, \(p\) = 0.00025) and _SHBG_ (t = 0.54, \(p\) = 0.75) gene expression levels which were inversely correlated with the Gleason score, we found that _SRD5A1_ was positively correlated with increased Gleason score, and this was statistically significant (t = 2.61, \(p\) = 0.028), while the association between _HSD17B3_ expression and Gleason score was equivocal (Figure 7A). Using same PCa cohort data, we found that, while _SHBG_ expression was downregulated in patients with CRPC compared with their peers in the tumor or benign group, howebit statistically insignificant (t = 0.40, \(p\) = 0.67), relative to the benign group, _SRD5A1_ expression was significantly upregulated in the tumor and CRPC groups (t = 11.39, \(p\) = 0.000021) (Figure 7B). Because of this demonstrated implication of the testosterone tetrad in disease aggressiveness and poor prognosis, performing an in silico protein-compound association query using the DrugBank database ([[https://go.drugbank.com/](https://go.drugbank.com/)]([https://go.drugbank.com/](https://go.drugbank.com/)), accessed on 19 February 2021), we identified small molecule inhibitors of components of the testosterone tetrad, including the US FDA-approved SRD5A1 inhibitors, Mutasteride, and Finasteride (Figure 7C). Furthermore, we exposed androgen-independent metastatic prostate carcinoma PC-3 cells to 1-5 uM Docetaxel (DOC; US FDA-approved for hormone-refractory metastatic PCa) with or without 1-5 uM Mutasteride (DUT). Drug-response/effect analyses show that combining DOC with DUT significantly enhanced the pharmacologic activity of DOC (-3.3-fold, \(p\) < 0.001), and that this effect was largely synergistic in nature using the CompuSyn-generated isobologram and confirmed by a zero interaction potency (ZIP) synergy score of 13.24, where a score \(>\) 10 implies synergism, \(-\)10 to 10 represents additivity, and \(<\)\(-\)10 means antagonism, using the SynergyFinder web application version 2.0 ([[https://synergyfinder.fimm.fi/](https://synergyfinder.fimm.fi/)]([https://synergyfinder.fimm.fi/](https://synergyfinder.fimm.fi/)), accessed on 26 January 2021) (Figure 7D). Evaluating the replicability of these findings in vivo (Figure 7E), we demonstrated that, compared with the control mice bearing PC-3 WT cells, significant reduction in the tumor burden of the PC-3_SHBG_OE (1.81-fold, \(p\) < 0.001) or PC-3_sgSRD5A1 (1.87-fold, \(p\) < 0.001) mice was observed by day 43. Pharmacological-wise, by day 43, treatment with DUTDOC combo had elicited a 2.28-fold reduction in tumor burden (\(p\) < 0.0005) (Figure 7F). In addition, we demonstrated that, in comparison to the control mice, the PC-3_SHBG_OE and PC-3_sgSRD5A1 bearing mice exhibited a 40 or 60% survival advantage, respectively, while treatment with the DUTDOC combo conferred an 80% survival advantage (\(X^{2}\) = 60.20, \(p\) < 0.0001) (Figure 7G). Similarly, we found that, compared to the control mice, the PC-3_SHBG_OE, PC-3_sgSRD5A1, and DUTDOC combo mice enjoyed a 50, 80, or 90% metastasis-free survival advantage (\(X^{2}\) = 60.73, \(p\) < 0.0001) (Figure 7H), and this was consistent with the number of metastatic nodules found on excised lungs, livers, brains, and bones of the tumor-bearing mice (Figure 7I).