Prognostic impact of kallikrein‐related peptidase transcript levels in prostate cancer

We aimed to study mRNA levels and prognostic impact of all 15 human kallikrein‐related peptidases (KLKs) and their targets, proteinase‐activated receptors (PARs), in surgically treated prostate cancer (PCa). Seventy‐nine patients with localized grade group 2‐4 PCas represented aggressive cases, based on metastatic progression during median follow‐up of 11 years. Eighty‐six patients with similar baseline characteristics, but no metastasis during follow‐up, were assigned as controls. Transcript counts were detected with nCounter technology. KLK12 protein expression was investigated with immunohistochemistry. The effects of KLK12 and KLK15 were studied in LNCaP cells using RNA interference. KLK3, ‐2, ‐4, ‐11, ‐15, ‐10 and ‐12 mRNA, in decreasing order, were expressed over limit of detection (LOD). The expression of KLK2, ‐3, ‐4 and ‐15 was decreased and KLK12 increased in aggressive cancers, compared to controls (P < .05). Low KLK2, ‐3 and ‐15 expression was associated with short metastasis‐free survival (P < .05) in Kaplan‐Meier analysis. PAR1 and ‐2 were expressed over LOD, and PAR1 expression was higher, and PAR2 lower, in aggressive cases than controls. Together, KLKs and PARs improved classification of metastatic and lethal disease over grade, pathological stage and prostate‐specific antigen combined, in random forest analyses. Strong KLK12 immunohistochemical staining was associated with short metastasis‐free and PCa‐specific survival in Kaplan‐Meier analysis (P < .05). Knock‐down of KLK15 reduced colony formation of LNCaP cells grown on Matrigel basement membrane preparation. These results support the involvement of several KLKs in PCa progression, highlighting, that they may serve as prognostic PCa biomarkers.


| INTRODUCTION
Prostate cancer (PCa) is the second most frequent cancer and the fifth leading cause of cancer deaths in men globally. 1 The prognosis of individual PCa patients is highly variable. 2 Histological classification, using Gleason grading and the Grade Group (GG) system, is one of the strongest predictors of PCa outcome. 3 Most new PCa cases cluster in 4 in which the highest or predominant Gleason grade pattern is 4. For this group, there are no good prognostic markers and, perhaps excluding GG4, tumor grading alone cannot adequately distinguish indolent cases from those who will develop aggressive PCa. 5 Thus, novel prognostic and predictive markers are needed.
Prostate-specific antigen (PSA, also known as kallikrein-related peptidase [KLK]-3) is a widely used PCa marker. 6 However, the accuracy of blood-based PSA tests to identify clinically significant PCa is poor. 7 The accuracy can be increased by analyzing, in addition to total PSA, different PSA forms, such as free PSA, PSA-density, and markers, such as KLK2. 8,9 PSA (referred hereinafter as KLK3) is a member of a human KLK family, consisting of 15 proteases. 10 Several KLKs are expressed in the prostate and are regulated by androgens, the most abundant being KLK3 and -2. 10,11 Although the blood concentrations of KLK2 and -3 are increased in PCa, often positively correlating with disease burden, the opposite has been found in PCa tissue, where low KLK2 and -3 expression is associated with dedifferentiation and poor prognosis. [12][13][14][15] In addition to KLK2 and -3, other KLKs are potential markers in various cancers, 16,17 including PCa where they are differentially expressed compared to benign prostate, and may have prognostic significance. 16,18 The physiological function of the major prostatic KLKs has been proposed to relate to promotion of sperm motility by dissolving seminal clot formed after ejaculation. 10 KLKs may drive PCa progression, although the opposite has also been suggested for some of them. 10,[19][20][21] Perhaps the most studied, in this respect, is KLK3, which has been proposed to have activities that both promote and suppress the PCa growth and progression. [20][21][22][23] KLKs are involved in proteolytic cascades, which may facilitate PCa growth and metastasis. 20,24 Several KLKs are able to activate growth-factors and protease-associated receptors (PARs), which are involved in tumor growth and metastatic dissemination. 10,[25][26][27][28][29] Especially, PAR1, also known as thrombin receptor, has been strongly implicated in promotion of cancer cell invasion. 30,31 Overexpression of PAR1 has also been shown to predict biochemical recurrence in PCa and is a marker of late-stage disease. 32,33 Among the four human PARs (PAR1-4, encoded by genes F2R, F2RL1, F2RL2 and F2RL3, respectively), especially the activation of PAR1 and -2 have been found to be regulated by several KLKs. 27,28 PARs can also be activated by other proteases, including thrombin and matrix metalloproteinases, present in the prostate tumor microenvironment. 28 Notably, PAR-activation by different proteases may lead to different responses. 28 Most of the studies addressing either the protein or mRNA expression of KLKs and PARs in PCa have included a relatively small number of nonaggressive to highly aggressive cases, 16,18 not reflecting the clinical need for prognostic biomarkers in  PCa. Previous studies have also addressed only a single or a few KLKs at a time. Thus, we aimed to study tissue mRNA levels of all human KLKs and PARs and their prognostic value in the clinically challenging GG2-4 PCa. KLK12 was also studied by immunohistochemistry and, together with KLK15, using RNA interference (RNAi).

| Patient summary and study design
The primary case-control study for mRNA analyses consisted of 165 GG2-4 patients treated by radical prostatectomy in Helsinki University Hospital in 1992-2015. The selection process of patients has been previously described. 34 Seventy-nine patients with metastatic progression or PCa-specific death, during the median followup of 11 years, were selected to present cases. Patients with similar clinical baseline characteristics, but no metastasis during the followup (n = 86), were assigned as controls, representing a clinically indolent phenotype of GG2-4 PCa. Additionally, 12 samples from distant PCa metastases, seven samples of castration-resistant PCa (CRPC) and six neuroendocrine PCa (NE) were included. Nineteen benign prostate samples from men without detected PCa, but with benign prostatic hyperplasia, were also studied. One of the metastases was from a para-aortic lymph node, from the level of renal vein and was extracted 10 years after RP. The other metastases were from bone (vertebra, humerus, femur, n = 5), bone marrow (iliac crest, n = 1), spinal cord (n = 1), liver (n = 1), appendix (n = 1), testis (n = 1) and epididymis (n = 1). All patients in the CRPC group, 10 in the metastasis group and three in the NE had received hormonal treatment before the samples were extracted. Patients in other groups were hormone-naïve at the time of sample extraction. During quality control, eight aggressive cases, four controls and three metastasis samples were excluded from the final analyzes. The histological slides of all the patients were rereviewed by an expert uropathologist (TM) and the index cancer lesions were annotated for RNA extraction and tissue microarray (TMA) construction using the formalinfixed paraffin-embedded (FFPE) blocks. Detailed demographics of analyzed samples are available in Table 1.   also detected in conditioned cell culture supernatants by immunoassays. 11 For this, 40 000 cells were grown on wells of 12-well plate for 7 days in 1.2 mL medium. KLK12 was also detected by immunocytochemical staining (ICC). For ICC the cells grown on glass surface were fixed for 5 min with acetone. After washing twice with PBS, essentially the same protocol, including the antibody, as used for IHC staining of KLK12 in tissue sections was followed, but without antigen retrieval and steps before that.

| Cell growth assays
To assess the effect of siRNAs on cell viability and growth on plastic were analyzed using Fiji ImageJ. 37 The background subtraction was performed using the Gaussian blur method and image thresholding using Otsu method. The Analyze particles-command was used to analyze the cell clusters in the segmented images. The minimum particle size was set to 200 pixels to remove the background debris. Cell clusters touching the border were excluded from the analysis. Median size of cell clusters from each siRNA treated sample was multiplied by the number of the cell clusters to obtain a colony formation score.

| Invasion and migration
The invasion assays were performed using Nunc Polycarbonate cell culture inserts (Thermo Scientific) and migration assays using Diego, CA) for 10 minutes at room temperature. One hundred microliters of the extracts were then transferred to a 96-well plate and the absorbance was measured at 560 nm using Multiskan EX plate reader.

| Statistical analyses
Statistical analyses were performed using nSolver Analysis Software  Among the significantly expressed KLKs, the expression of KLK4, -12 and -15 was increased, and that of KLK10 and -11 decreased, in cancer (cases and controls combined), compared to benign prostate (P < .05 for all) ( Figure 1A and Table 2). However, when aggressive cases were compared to controls ( Figure 1A and  Figure 1A, although no statistical tests were performed due to low number of samples in these groups. Low KLK2, -3 and -15, and high PAR1 and PAR4 expression was associated with poor metastasis-free survival (Table 2). Further, low KLK15 and high PAR1 and PAR3 were associated with poor PCa-specific survival ( Table 2). Survival plots depicting association of KLK15 with metastasis-free and PCa-specific survival are shown in Figure 1B Table S2.   Table S3.

| High KLK12 protein expression is associated with poor survival
We also studied KLK12 protein expression using IHC (Figure 3).
The intensity of KLK12 expression was stronger in canceradjacent benign spots (mean score = 2.46) compared to cancerous spots (mean = 0.96, P < .0001), and similar to that in benign spots from patients without detectable cancer (mean = 2.73, P = .21). In case and control samples, high KLK12 scores were associated with poor metastasis-free and PCa-specific survival

| Knockdown of KLK15 reduces the formation of LNCaP colonies on Matrigel
To investigate the functional effects of KLK12 and KLK15 expres-    (Table S4 and Figure S1).
The overexpression of KLK12 and KLK15 did not significantly change the cell proliferation on plastic surface (Table S4).
Also chemotactic migration and invasion were unaffected by overexpression.

| DISCUSSION
We the expression levels of KLK4, -12 and -15 were found to be higher, and those of KLK10 and -11 lower in cancerous than in benign prostate, agreeing with many, but not all studies. 16,38,39 Observed variation between studies may arise from differences in sample sets and methodology, including the probes used for mRNA detection. Notably, in our study, benign samples were from men who were not diagnosed with cancer. Most studies have used benign tissues adjacent to cancer as "benign" controls. Such tissues may differ from truly benign ones, perhaps due to paracrine regulation. 40,41 KLK splicing variants appear to be common in PCa. 42  associated with advanced cancer, should be identical across our study groups and, therefore, not compromise the major goal of our study, that is, elucidation whether changes in KLK mRNA levels have prognostic significance.
When mRNA expression levels of KLKs in aggressive cases were compared to those with indolently behaving control group, the expression levels of KLK2, -3, -4 and -15 were found to be decreased, and that of KLK12 increased. Low expression of KLK2, -3 and -15 was associated with poor metastasis-free survival and, in case of KLK15, also with poor PCa-specific survival. Association of low KLK2 and KLK3 protein levels and poor prognosis has been reported. [12][13][14]43 Others have also reported higher KLK15 expression in cancer than in benign prostate, but, contrary to our results, found that high protein or mRNA expression was associated with advanced PCa and shorter progression-free survival. [44][45][46] An integrated analysis of KLK expression data found in Oncomine and OncoLnc databases has suggested that dysregulation of especially KLK1-KLK5, KLK11 and KLK12 are associated with metastatic progression of PCa. 17 A study with prostate and breast cancer cell lines suggests that KLK12, like many other KLKs, is up-regulated by androgen and other steroid hormones. 10,47 However, apparently there are other mechanisms involved as well, as neuroendocrine tumors that had very low AR expression showed prominent expression of KLK12. Indeed, also hypoxia and HIF1α may regulate KLK12 expression. 48 As hypoxia is linked to prostate cancer progression, 49 this may explain why we found higher KLK12 expression in aggressive cases than in less aggressive ones. Importantly some patients with neuroendocrine tumors had received hormonal treatment before sample extraction.
PARs showed higher expression in cancerous than benign tissue.
While PAR1 and -4 expression was higher in aggressive cases compared to controls, the opposite was observed with PAR2. High PAR1 and -4 expression was associated with poor metastasis-free survival and high PAR1 was also associated with poor PCa-specific survival. In accordance with our results, a small IHC study identified PAR1 protein overexpression as a predictor of biochemical recurrence after radical prostatectomy. 32 Interestingly, this was observed in periglandular stromal cells, but not in epithelial cells, and PAR2 and PAR4 were not found to predict the outcome. 32 Another study found PAR1 to be overexpressed in metastatic PCa compared to localized disease, on both transcript and protein levels. 33 Notably, in the study by Kaushal and coworkers, the metastatic PCa more resembles our CRPC samples, than the aggressive cases in our case-control study, since the metastasis patients were not baseline-matched and were undergoing androgen ablation therapy. 33  have been reported to correlate with AR status. 10 We also found such correlation with expression levels of these KLKs and AR, while KLK15 correlated with AR in nonaggressive control group, but not in the aggressive PCas. The hormonal regulation of KLK15 is controversial, some studies suggesting regulation by steroid hormones, while the others have not found such an effect. 10,50,51 Since we did not find any difference in AR expression between the aggressive cancer and control groups it is unlikely that the observed differences in expression of androgen-regulated KLKs are explained by AR expression.
IHC staining of KLK12 revealed that while the benign glands showed significantly higher staining intensities than the cancerous tissue, high KLK12 protein expression was associated with poor metastasis-free and PCa-specific survival. Similar observation of differential KLK12 staining of the benign and malignant prostatic tissue has been previously reported. 35

AUTHOR CONTRIBUTIONS
The work reported in the paper has been performed by the authors, unless clearly specified in the text.