Biofluid quantification of TWEAK/Fn14 axis in combination with a selected biomarker panel improves assessment of prostate cancer aggressiveness

Prostate cancer (PCa) is the fourth most common cancer in both sexes collectively and the second most common cancer in men [1]. PCa is a curable disease if detected at an early stage and there are several viable therapeutic options available; however, many patients fail to respond to first-line treatments. Conventional clinical prognosticators of PCa severity and progression include digital rectal examination, serum prostate-specific antigen (PSA) measurement, and transrectal ultrasound-guided histopathological staging [2]. PSA is encoded in humans by the kallikrein-3 (KLK3) gene [3] and its secreted protein is a pre-propeptide that can be activated by human glandular kallikrein-2 protease, encoded by KLK2 [4]. While the application of PSA serum testing as a screening tool for PCa has reduced mortality rates and the prevalence of advanced-stage disease at diagnosis, there is continuing debate over its efficacy as a specific marker for diagnosis and prognosis [5]. Accordingly, robust biomarkers are needed for the early screening of patients at risk of PCa.

Liquid biopsy is a promising non-invasive modality for molecular profiling in cancer, enabling the assessment of circulating molecules in various biological fluids for biomarker discovery [6]. Moreover, when combined with multi-marker analysis, liquid biopsy is a potentially powerful platform for prognosis and early assessment of treatment failure.

TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand/receptor pair belonging to the TNF receptor superfamily. TWEAK is synthesized as a membrane-anchored protein (mTWEAK) that is released into circulation in a soluble form (sTWEAK) after processing by a furin protease. Both sTWEAK and mTWEAK function through engagement with Fn14 [7]. TWEAK has pleiotropic effects on different cell functions, mediating pro-inflammatory and pro-angiogenic activity, and stimulating invasion, migration and survival [8, 9]. Fn14 is expressed at relatively low levels in normal tissues, but is significantly upregulated locally following tissue injury or disease, where it mediates tissue remodeling [7]. Interestingly, Fn14 overexpression promotes androgen-independent PCa progression and correlates with poor treatment outcome [10]. Many TNF receptors undergo proteolytic cleavage to generate soluble forms that are shed into biofluids, and a recent study reported the presence of high levels of soluble Fn14 (sFn14) in urine samples of patients with kidney injury [11], pointing to sFn14 as a potential biomarker for progression of kidney disease.

In addition to binding Fn14, sTWEAK can also bind to the CD163 (cluster of differentiation 163) receptor, which acts as a scavenger receptor since its binding does not transduce intracellular signals but instead mediates the elimination of sTWEAK, contributing to its degradation [12]. CD163 is expressed exclusively on monocytes/macrophages and is tightly regulated by inflammatory responses and anti-inflammatory signals [13]. Similar to Fn14, CD163 not only exists as a membrane-bound form, but is also released into circulation as a soluble form (sCD163), and is a sensitive indicator of macrophage activation [14]. Interestingly, the presence of CD163⁺ macrophages is strongly associated with less favorable clinical and pathological features in breast cancer [15].

Several chemokines are known to play key roles in the development and progression of PCa [16]. For example, the apoptosis-induced chemokine CXCL5 and its receptor CXCR2 have been reported to accelerate inflammation and growth of prostate tumor metastases in bone [17]. Moreover, CXCL5/CXCR2 expression was found to correlate with Gleason grading and clinical pathologic stages [18]. Another study examined the CCL7/CCR3 axis in a mouse model of PCa and obesity, specifically in the periprostatic adipose tissue surrounding the tumor, finding that this tissue secretes CCL7, which in turn stimulates the migration of CCR3-expressing tumor cells [19]. Notably, depletion of tumor CCR3 completely abolished the ability of obesity to promote tumor metastasis [15]. Crosstalk between CCL7 and CCR3 has also been found to promote colon cancer metastasis [20].

Because the aforementioned biomolecules, TWEAK, CXCL5 and CCL7 and their respective receptors (Fn14, CXCR2 and CCR3) as well as PSA-related genes (KLK3 and KLK2) are known to be linked to tumorigenesis and could therefore provide a growth advantage to tumors, we designed a study to quantify and examine these selected biomarkers in several biofluids from patients with PCa, to test whether they could predict tumor aggressiveness.

There is a desperate need to correctly identify patients with PCa and aggressive disease to guide treatment decisions and to avoid unnecessary prostate procedures. Biomarkers are used extensively in clinical practice to predict the presence of PCa and to distinguish aggressive from indolent forms [24]. While circulating serum total PSA is used extensively for general screening to diagnose the presence of PCa, new biomarkers are needed for the implementation of more personalized diagnosis and management of PCa.

To identify new biomarkers for PCa in liquid biopsies, in the present work we measured the circulating levels of sTWEAK and its receptors sFn14 and sCD163, in addition to two chemokines, CXCL5 and CCL7, all of which are closely related to prostate tumorigenesis [17, 19] but never analyzed in human PCa liquid biopsies. Additionally, we evaluated the expression of their related receptors (Fn14, CD163, CXCR2, and CCR3) in semen cell sediment as well as gene expression of the PSA-related genes KLK2 and KLK3, as possible biomarkers for PCa aggressiveness.

In the context of cancer, circulating levels of sTWEAK have previously been described as a biomarker in head and neck cancer, where low serum levels of sTWEAK were found to be related to reduced survival rate [25, 26], pointing to sTWEAK levels as a possible non-invasive biomarker in this patient group. To the best of our knowledge, no study has reported on the potential of sTWEAK as a PCa biomarker; however, it is important to consider that a pro-tumorigenic role of the TWEAK/Fn14 axis has previously been evaluated in vitro in several PCa cell types [8, 10, 27, 28].

Bivariate correlation analysis showed that semen levels of sTWEAK and sFn14 were positively related and also correlated with KLK2 mRNA expression in semen cell sediment. KLK2 is a member of the Kallikrein family of secreted serine proteases, which are associated with prostatic tissue [29]. KLK2 is highly expressed in prostate tumor cells and might be a prognostic marker for PCa risk [30]. Also, the expression of KLK2 is highly correlated with that of KLK3 [29], which allowed us to determine the prostate epithelial cell content in semen [3], suggesting that the positive correlation of sTWEAK and sFn14 in semen could be directly related to prostate cell epithelium.

Interestingly, a strong positive association was observed between sCD163 and sCXCL5 and sCCL7 in semen. All of these molecules are known to be expressed mainly in macrophages [31]. Additionally, sCD163 is specifically related to alternative “M2-type” macrophages, which are abundantly found in cancer [32], whereas CXCL5 protein expression levels are concordant with prostate tumor progression. Furthermore, both CD163 and CXCL5 are highly associated with inflammatory infiltrates, which are frequently detected in the lumen of malignant prostate glands [33].A relationship between the TWEAK/Fn14 axis and CD163 was also found in urine.

In serum, sTWEAK levels positively correlated with sCCL7 and sCXCL5, and it is known that high expression of CXCL5 is closely related to cancer progression [34‐36], which is in line with the association we observed in our cohort between PSA levels and sCXCL5 in urine. Of particular note is the association found between serum levels of sCD163 and glycemia-related variables (insulin, HOMA-IR index and fasting glycemia), corroborating previous findings for this biomarker in the context of diabetes and altered glucose metabolism [37, 38].

In serum, sTWEAK levels were unchanged between the high- and low-risk patient groups. Serum sTWEAK levels are reduced in obesity [39]. In our cohort, 51.6% of the patients were overweight and 26.7% were obese, but no association was observed between sTWEAK serum levels and BMI. However, we found that semen levels of sTWEAK were significantly altered according to Gleason histologic aggressiveness. The reduction in sTWEAK levels in semen in high-risk patients was accompanied by a tendency for an increase in Fn14 mRNA levels in semen cell sediment. This finding might indicate an active process of ligand–receptor interaction in this microenvironment, in which the ligand–receptor complex must be internalized, thereby reducing ligand availability and favoring the process of proliferation and migration, as described in PCa cells in vitro mediated by the TWEAK/Fn14 axis [10, 28].

Interestingly, the mRNA expression levels of CCR3 and CXCR2 in semen cell sediment showed a similar behavior to Fn14 mRNA in the ISUP high-risk patients, which might indicate a higher activity of the respective ligands CCL7 and CXCL5 at this site, although no significant changes were found for these soluble chemokines in our patients between high- and low-risk groups. We observed a decreased expression of KLK2 in semen cell sediment in high-risk patients, whereas PSA levels were higher. Although a positive relationship was expected, we do not have a clear explanation for this finding. Of note, it has been reported that KLK2 expression represents only 50% of total PSA [40].

Concerning the profiles of metabolic and biochemical parameters (lipidic, hepatic, renal and hormonal), we found differences only in the levels of serum glucose between low- and high-risk PCa patients in our cohort, with elevated levels of glucose in the latter group. This is consistent with a previous finding of an association between serum glucose levels and disease outcome in colorectal, breast, bladder, pancreatic and PCa patients [41]. By contrast, correlation patterns between anthropometric and metabolic variables across individuals with high-risk and low-risk were markedly different, as demonstrated by chord diagram analysis, pointing to an intrinsic metabolic behaviour in these groups. Serum cholesterol may be related to PCa biology [42, 43], in this sense, we observed an association between cholesterol levels and KLK3 and KLK2 gene expression, both related to PSA protein levels.

While PSA provides a sensitive marker for PCa diagnosis, it is not only confined to PCa, hence it cannot be used alone as a diagnostic PCa biomarker [44]. In our study, ROC curve analysis using PSA alone had no advantage in its predictive ability, either alone or combined with a classical clinical panel (age, T-classification, total PSA). These results prompted us to question whether we could establish another suitable non-invasive biomarker panel for PCa aggressiveness by inputting a combination of the new biomarkers studied. Indeed, ROC curve analysis demonstrated that this new non-invasive biomarker panel comprising sTWEAK in semen, glucose levels in serum and mRNA levels of Fn14, KLK2, CXCR2 and CCR3 in semen cell sediment had a better AUC than PSA and even the classical non-invasive clinical panel (age, T-classification, total PSA) (AUCs of 0.845, 0.716 and 0.721, respectively), and could classify patients according to PCa aggressiveness with higher specificity when compared with either total PSA classification or when using the non-invasive classical clinical panel (specificity of 76.2%, 48.4% and 48.3%, respectively). Moreover, the combination of the above biomarker panel to supplement total serum PSA improves the AUC stratification (91.3%) of tumor aggressiveness, as classified by ISUP high-risk or low-risk. This new panel (biomarker panel plus total serum PSA) could be used to correctly classify tumor aggressiveness in 90.9% patients with higher specificity (85.7%). Although our findings are encouraging, a larger cohort will be needed to support the biomarker panel for positive diagnosis.

A biofluid signature comprising sTWEAK in semen, serum fasting glycemia and the mRNA levels of Fn14, KLK2, CXCR2 and CCR3 in semen cell sediment, combined with total serum PSA levels, represents a non-invasive biomarker panel with high negative predictive value that can be used to classify PCa aggressiveness.