## 3 Results  
The Aberrant Expression of the Testosterone Metabolic Tetrad HSD17B2, HSD17B3, SHBG, and SRD5A1 Characterize Androgen-Addicted PCa and Is Associated with Disease Progression  
Computational analysis of the gene ontology (GO) biological process complete dataset from the GO Ontology database version 8 December 2020 ([[http://doi.org/10.5281/zenodo.4316524](http://doi.org/10.5281/zenodo.4316524)]([http://doi.org/10.5281/zenodo.4316524](http://doi.org/10.5281/zenodo.4316524)), accessed on 3 November 2020) using the [PERSON]'s exact over-representation test indicate that HSD17B2, HSD17B3, SHBG, and SRD5A1 are significantly enriched for androgen biosynthetic processes (fold enrichment > 100, \(p\) = 1.82 \(\times\) 10\({}^{-6}\)) and male genitalia development (fold enrichment > 100, \(p\) = 8.27 \(\times\) 10\({}^{-6}\)) (Figure 1A). Bioinformatics-aidedgene expression profiling showed that, compared to HSD17B2 (0.31-fold, \(p\) = 0.0092) and SHBG (0.93-fold, \(p\) = 0.78) mRNA expression levels, which are downregulated, HSD17B3 (2.96-fold, \(p\) = 1.11 \(\times\) 10\({}^{-16}\)) and SRD5A1 (1.3-fold, \(p\) = 4.02 \(\times\) 10\({}^{-5}\)) transcript levels are upregulated in patients with PCa from the TCGA PRAD cohort (n = 497) (Figure 1B). Furthermore, using samples from our PCa cohort (n = 56), compared to the moderate-strong immunoreactivity of HSD17B3 and SRD5A1 in high-grade PCa (Gleason score (GS) \(\geq\) 8), normal prostate tissue (GS \(\leq\) 5), and low-grade PCa (GS = 6-7) were characterized by null-mild protein expression, conversely, moderate-strong HSD17B2 and SHBG protein expression levels were observed in the normal prostate and low grade PCa tissues compared to null-mild protein expression in the high-grade samples (Figure 1C). Consistent with this, reanalysis of the TCGA PRAD cohort (n = 497) showed that tumor T-stage progression was associated with SRD5A1 and HSD17B3 gene amplification and gain, but shallow or deep deletion of SHBG and HSD17B2 genes (Figure 1D). The differential expression of HSD17B2, HSD17B3, SHBG, and SRD5A1 in patients with different PCa grades is suggestive of a dysregulated androgenic signal-driven oncogenicity in patients with PCa, where intracellular signaling skewed towards a dominant SRD5A1/HSD17B3 at the expense of SHBG/HSD17B2 signaling drives enhanced cancerization, and may inform therapeutic decision making and management strategy for patients with PCa. Variation in HSD17B2, HSD17B3, SHBG, and SRD5A1 Expression Co-Occur and Concertedly Bear Significant Prognostic Relevance in Patients with PCa  
Ruling out random individualistic expression pattern, our computational mutual exclusivity and co-occurrence probe of a pooled PCa cohort (n = 4369) showed that all four molecular components of the testosterone metabolic tetrad co-occur, and the co-occurrence is statistically significant (Figure 2A). Since molecular co-occurrence is often suggestive of functional inter-relatedness, we examined if and to what degree the tetrad is culpable in risk of death and recurrence in patients with PCa. We observed that, while HSD17B2, HSD17B3, and SHBG expression levels were equivocal for risk of death, high expression of SRD5A1 was strongly associated with high risk of death (Figure 2B,C). However, all four components of the tetrad were fully active in driving the propensity for relapse, with concomitant high expression of SRD5A1 and low HSD17B2, HSD17B3, and SHBG being associated with significantly enhanced risk of recurrence (Figure 2D,E). In corroboration, Kaplan-Meier curves were generated from survival analyses of HSD17B2/HSD17B3/SHBG/SRD5A1 co-expression in TCGA PRAD cohort (n = 497), showing that, compared to the low expression group, patients with SRD5A1-dominant high tetrad expression exhibited worse mid-term to long-term (\(\geq\)5 years) overall survival ((HR = 5.19 (95% CI: 1.09-24.63); \(p\) = 0.04) (Figure 2F), and a profoundly shorter time to recurrence ((HR = 3.6 (95% CI: 1.59-8.12); \(p\) = 0.002) (Figure 2G).