**Downstream Neighbor of SON (DONSON) Expression Is Enhanced in Phenotypically Aggressive Prostate Cancers**

**Niklas Klümper 1,2,3, Marthe von Danwitz 1,2, Johannes Stein 1,2, Doris Schmidt 1,2, Anja Schmidt 1,2, Glen Kristiansen 2,4, Michael Muders 2,4, Michael Hölzel 2,3, Manuel Ritter 1,2, Abdullah Alajati 1,2,\*,**† **and Jörg Ellinger 1,2,\*,**†


Received: 3 November 2020; Accepted: 16 November 2020; Published: 19 November 2020

**Simple Summary:** Downstream neighbor of SON (DONSON) plays a crucial role in cell cycle progression and in maintaining genomic stability. We identified DONSON to be associated with an aggressive histopathological phenotype and unfavorable survival in prostate cancer (PCa) in different transcriptomic cohorts and on the protein level in our tissue microarray cohort. DONSON expression in the primary tumor was particularly strong in locally advanced, metastasized, and dedifferentiated carcinomas (TNM Stage, Gleason). Highly proliferating tumors exhibited a significant correlation to DONSON expression, and DONSON expression was notably upregulated in distant metastases and androgen-deprivation resistant metastases. In vitro, specific DONSON-knockdown significantly reduced the migration capacity in PC-3 and LNCaP, which further suggests a tumor-promoting role of DONSON in PCa. The results of our comprehensive expression analyses, as well as the functional data obtained after DONSON-depletion, lead us to the conclusion that DONSON is a promising prognostic biomarker with oncogenic properties in PCa.

**Abstract:** Downstream neighbor of Son (DONSON) plays a crucial role in cell cycle progression and in maintaining genomic stability, but its role in prostate cancer (PCa) development and progression is still underinvestigated. Methods: DONSON mRNA expression was analyzed with regard to clinical-pathological parameters and progression using The Cancer Genome Atlas (TCGA) and two publicly available Gene Expression Omnibus (GEO) datasets of PCa. Afterwards, DONSON protein expression was assessed via immunohistochemistry on a comprehensive tissue microarray (TMA). Subsequently, the influence of a DONSON-knockdown induced by the transfection of antisense-oligonucleotides on proliferative capacity and metastatic potential was investigated. DONSON was associated with an aggressive phenotype in the PCa TCGA cohort, two GEO PCa cohorts, and our PCa TMA cohort as DONSON expression was particularly strong in locally advanced, metastasized, and dedifferentiated carcinomas. Thus, DONSON expression was notably upregulated in distant and androgen-deprivation resistant metastases. In vitro, specific DONSON-knockdown significantly reduced the migration capacity in the PCa cell lines PC-3 and LNCaP, which further suggests a tumor-promoting role of DONSON in PCa. In conclusion, the results of our comprehensive expression analyses, as well as the functional data obtained after DONSON-depletion, lead us to the conclusion that DONSON is a promising prognostic biomarker with oncogenic properties in PCa.

**Keywords:** prostate carcinoma; DONSON; Downstream Neighbor of SON; biomarker; metastatic spread

#### **1. Introduction**

Prostate cancer (PCa) is the most common malignancy in men and contributes significantly to the overall mortality of malignant diseases [1]. Critical steps in PCa progression are the development of castration resistance and metastatic spread. The therapy of these advanced and castration-resistant PCa (CRPC) has improved considerably in recent years, but mortality remains high with limited therapy options in end-stage carcinomas [2,3]. A better understanding of the biology of this multi-facetted carcinoma can help to further improve the therapy of our PCa patients.

The Cancer Genome Atlas (TCGA) platform is a reliable source and an invaluable tool for cancer research [4]. A large cohort of primary PCa (pPCa) has already been comprehensively investigated by the TCGA Research Network, which has certainly contributed to a deeper understanding of this disease [5]. We hypothesized that genes that show a correlation to an unfavorable clinical course, and therefore to particularly aggressive tumors, represent interesting research targets. In an investigative approach, the PCa TCGA dataset was used to determine prognostically relevant genes [4,6], and in the present study, Downstream Neighbor of SON (DONSON) was identified as an interesting target gene for further analyses in PCa. Of note, in a comprehensive pan-cancer analysis of 30 distinct tumor entities using TCGA datasets, we recently found DONSON overexpression to be associated with unfavorable overall survival in diverse entities, suggesting tumor-independent oncogenic properties of this largely unknown gene [7]. Thus, DONSON was found to be a robust biomarker for risk stratification in clear cell renal cell carcinoma (ccRCC), and in vitro, DONSON was linked to a malignant phenotype in ccRCC cell culture models [7,8]. Mechanistically, it is known that DONSON represents a critical replication fork protein required for physiological DNA replication [9]. DONSON is pivotal for genome stability and integrity as severe replication-associated DNA damage was observed after depletion of DONSON [10]. Further, DONSON plays an important role in cell-cycle regulation and the DNA damage response pathway (DDR) signaling cascade [11]. Regulated cell division and the preservation of genomic integrity are essential to maintain cellular homeostasis, and disorders can lead to tumor formation [12].

Considering the apparently decisive role of DONSON on genome integrity and as DONSON seems to be associated with an aggressive PCa phenotype in the transcriptomic TCGA dataset, the question arises whether DONSON also plays an important role in the progression of PCa. However, a differentiated analysis of the role of this gene in PCa is still pending. Therefore, the aim of this study was to thoroughly analyze the expression pattern of DONSON in PCa cohorts and, subsequently, its functional role in vitro in established PCa cell culture models.

#### **2. Results**

#### *2.1. Downstream Neighbor of SON (DONSON) mRNA Expression is Associated with Aggressive PCa*

In order to analyze the relevance of the DONSON in PCa, we comprehensively associated clinical-pathological parameters and the patients' clinical course with the DONSON mRNA expression using the PCa TCGA dataset (*n* = 532). DONSON expression was significantly enhanced in the carcinoma samples compared to normal adjacent prostatic tissue (NAT) (Figure 1A). DONSON was associated with enhanced local tumor expansion (pT-stage, Figure 1B) and lymphonodal metastatic dissemination (pN-stage, Figure 1C). Furthermore, a strong association of the DONSON expression with the ISUP grading, derived from the PCa-specific grading parameter Gleason score [13], was evident (Figure 1D). After dichotomizing the PCa cohort using the median DONSON expression, there was a strongly reduced progression-free survival (PFS) for the DONSON overexpressing subgroup (Figure 1E). DONSON remained an independent predictor of unfavorable PFS in the PCA TCGA cohort after

adjustment for co-variables (TNM; age) using a Cox regression model (*p* = 0.001; HR = 1.87, 95% CI (1.31; 2.68); Table 1). Since PCa with a Gleason score of 7 is particularly difficult to stratify in terms of aggressiveness, we next investigated whether DONSON would have additive prognostic value in this subgroup. In this clinically highly relevant patient cohort, DONSON expression was again significantly associated with shortened PFS and remained an independent predictor of unfavorable clinical course in a multivariate Cox analysis (*p* = 0.01; HR = 3.82, 95% CI [1.44; 10.2]; Table 1) (Figure 1F). Of note, the proliferation marker Ki67 expression had no prognostic value in the Gleason 7 subgroup in univariate and multivariate Cox regression analyses, and DONSON remained an independent predictor of unfavorable PFS after co-adjusting for Ki67 additionally to TNM and age (*p* = 0.01; HR = 4.03, 95% CI [1.49; 10.9]). DONSON overexpression was also associated with worse overall survival (OS). However, the low number of events in the PCa TCGA cohort (*n* = 10) only permits a limited consideration of this important endpoint (Supplementary Figure S1A and Table S1).


**Table 1.** Multivariate Cox Regression Analyses in the evaluated prostate cancer (PCa) cohorts regarding progression-free survival (PFS).

PC—Prostate cancer, TCGA—The Cancer Genome Atlas, TMA—Tissue microarray, DONSON—Downstream Neighbor of SON.

Since the PCa TCGA dataset set only contains the expression profiles of primary carcinomas, we wanted to investigate further data sets to more precisely examine the role of DONSON during tumor progression. Of note, in a publicly available PCa progression cohort (GSE21032) [14], DONSON expression was strongly upregulated in the metastatic samples compared to pPCA, which might hint towards a role DONSON plays during the metastatic process (Figure 2A). Interestingly, comparing the sites of the metastatic samples, DONSON expression was significantly enhanced in locally extensive and distant metastatic samples (bone, brain, lung) compared to lymphonodal metastases (LNPC) (Figure 2B). In accordance with this, DONSON expression was strongly enhanced in *n* = 25 androgen-deprivation resistant metastatic samples (Met(CRPC)) compared to pPCa in a second PCa progression cohort (GSE6919, Figure 2C) [15–17]. It is known that fast-growing carcinomas indicate a particularly aggressive phenotype. The proliferation marker Ki-67 is therefore evaluated for assessing tumor aggressiveness, e.g., in breast carcinoma [18], and was also described as a risk stratifier in PCa patients [19]. Of note, we observed a significant positive correlation between DONSON and the proliferative activity of the carcinomas measured by Ki-67 in all of the three independent cohorts (Figure 2D–F).

**Figure 1.** DONSON is associated with clinical-pathological parameters of malignancy and progression-free survival (PFS) using the PCa TCGA dataset (**A**) DONSON expression is enhanced in primary PCa compared to normal adjacent prostatic glands (NAT). DONSON is associated with locally advanced tumor expansion (T Stage), positive lymphonodal metastatic status (N Stage) and the dedifferentiation ISUP score (**B**–**D**). (**E**,**F**) DONSON overexpressing PCa exhibit a shortened PFS when analyzing the whole (**E**) or only the clinically relevant (**F**) subgroup of Gleason 7 carcinomas of the PCa TCGA cohort.

**Figure 2.** (**A**–**C**), DONSON expression is significantly increased in metastatic samples compared to primary PCA, which was particularly evident in distant (**B**) and androgen-deprivation resistant metastases (Met [CRPC], (**C**). (**D**), Correlation heatmap depicting DONSON´s significant correlation to the proliferative activity of PCa in three cohorts. (**E**,**F**), Scatter plots with regression line included visualize the distribution of the TCGA and GSE21032 cohort with regard to the DONSON and Ki67 expression (parametric Pearson´s r is specified). \* *p* < 0.05, \*\*\* *p* < 0.001.
