*2.2. DONSON Protein Expression on a PCa Tissue Microarray (TMA)*

To test the prognostic potential of DONSON at the protein level, we stained and evaluated a large PCa TMA cohort immunohistochemically against DONSON. DONSON was expressed in the cytoplasm, which is in accordance with the staining pattern observed in the PCa and normal prostate gland specimens of The Human Protein Atlas cohort (HPA, www.proteinatlas.org) [20,21] (Figure 3A). Immunocytochemical DONSON staining in PC-3 cells with and without DONSON knockdown, induced via transfection of specific antisense oligonucleotides, was performed to confirm the cytoplasmic staining pattern and antibody specificity (Supplementary Figure S2). Interestingly, DONSON revealed a heterogeneous expression throughout the investigated cohort (DONSON expression negative/weak *n* = 48; DONSON expression moderate/strong *n* = 68). Of note, enhanced DONSON expression was associated with an advanced pT-stage (Figure 3B). In addition, the aggressive Gleason ≥ 8 PCa (ISUP IV+V) exhibited a significantly increased DONSON expression compared to Gleason ≤ 7 (ISUP I-III) (Figure 3C). No further significant associations between DONSON and clinical pathological parameters were evident, which may be due to the low sample size.

**Figure 3.** Immunohistochemical staining (IHC) against DONSON on a comprehensive PCa TMA with subsequent expression analysis (**A**), Representative images of the heterogeneous DONSON expression throughout the primary PCa cohort are depicted in three cases; 10× and 40× objective magnification. PCa 1 represents a well-differentiated DONSON-negative carcinoma. PCa 2 + 3 represent cases with particularly strong DONSON protein expression, wherein PCa 3 additionally exhibits an aggressive phenotype with fusing glands and components of a solid carcinoma. (**B**,**C**), DONSON expression is associated with advanced T Stage and Gleason score. (**D**), DONSON overexpression, defined as DONSON moderate/high (Score ≥ 2), predicts shortened PFS compared to the negative/low expression subgroup. (**E**), A strong statistical tendency for an increased nuclear AR expression was evident in the DONSON overexpressing subgroup; overexpression = OE, underexpression = UE.

In line with its potential as a risk stratifier in the PCa TCGA cohort, DONSON overexpression also showed a significant association with progression-free survival (PFS) at the protein level in the investigated cohort (Figure 3D). Further, a strong statistical trend was seen for DONSON to be an independent predictor of unfavorable PFS (*p* = 0.13; HR 1.48, 95% CI (0.89; 2.47); Table 1) measured by multivariate Cox regression co-adjusting the TNM stage and age.

The androgen receptor (AR) signaling pathway plays a crucial role in the progression of PCa, and nuclear expression of AR predicts an unfavorable clinical outcome and shorter time to the

development of castration resistance [22]. Interestingly, in the examined PCa cohort, a strong trend for increased AR expression (studied earlier in [23]) in the DONSON overexpressing subgroup was evident (Figure 3E). In accordance with this, in both PCa progression cohorts a significant correlation of AR and DONSON mRNA expression was observed (GSE21032: Pearson's r = 0.204, *p*-value = 0.012; GSE6919: Pearson's r = 0.549, *p*-value < 0.0001).
