Free Prostate-specific Antigen Forms and Kallikrein-related Peptidase 2: Tools for Prostate Cancer Diagnostics

Prostate-specific antigen (PSA) is a marker that is commonly used in estimating prostate cancer risk. Prostate cancer is usually a slowly progressing disease, which might not cause any symptoms whatsoever. Nevertheless, some cases of cancer are aggressive and need to be treated before they become life-threatening. However, the blood PSA concentration may rise also in benign prostate diseases and using a single total PSA (tPSA) measurement to guide the decision on further examinations leads to many unnecessary biopsies, over-detection, and overtreatment of indolent cancers which would not require treatment. Therefore, there is a need for markers that would better separate cancer from benign disorders, and would also predict cancer aggressiveness. The aim of this study was to evaluate whether intact and nicked forms of free PSA (fPSA-I and fPSA-N) or human kallikrein-related peptidase 2 (hK2) could serve as new tools in estimating prostate cancer risk. First, the immunoassays for fPSA-I and free and total hK2 were optimized so that they would be less prone to assay interference caused by interfering factors present in some blood samples. The optimized assays were shown to work well and were used to study the marker concentrations in the clinical sample panels. The marker levels were measured from preoperative blood samples of prostate cancer patients scheduled for radical prostatectomy. The association of the markers with the cancer stage and grade was studied. It was found that among all tested markers and their combinations especially the ratio of fPSA-N to tPSA and ratio of free PSA (fPSA) to tPSA were associated with both cancer stage and grade. They might be useful in predicting the cancer aggressiveness, but further follow-up studies are necessary to fully evaluate the significance of the markers in this clinical setting. The markers tPSA, fPSA, fPSA-I and hK2 were combined in a statistical model which was previously shown to be able to reduce unnecessary biopsies when applied to large screening cohorts of men with elevated tPSA. The discriminative accuracy of this model was compared to models based on established clinical predictors in reference to biopsy outcome. The kallikrein model and the calculated fPSA-N concentrations (fPSA minus fPSA-I) correlated with the prostate volume and the model, when compared to the clinical models, predicted prostate cancer in biopsy equally well. Hence, the measurement of kallikreins in a blood sample could be used to replace the volume measurement which is time-consuming, needs instrumentation and skilled personnel and is an uncomfortable procedure. Overall, the model could simplify the estimation of prostate cancer risk. Finally, as the fPSA-N seems to be an interesting new marker, a direct immunoassay for measuring fPSA-N concentrations was developed. The analytical performance was acceptable, but the rather complicated assay protocol needs to be improved until it can be used for measuring large sample panels.

Identification and validation of novel prostate cancer biomarkers

Prostate cancer (PCa) has emerged as the most commonly diagnosed lethal cancer in European men. PCa is a heterogeneous cancer that in the majority of the cases is slow growing: consequently, these patients would not need any medical treatment. Currently, the measurement of prostate-specific antigen (PSA) from blood by immunoassay followed by digital rectal examination and a pathological examination of prostate tissue biopsies are the most widely used methods in the diagnosis of PCa. These methods suffer from a lack of sensitivity and specificity that may cause either missed cancers or overtreatment as a consequence of over-diagnosis. Therefore, more reliable biomarkers are needed for a better discrimination between indolent and potentially aggressive cancers. The aim of this thesis was the identification and validation of novel biomarkers for PCa. The mRNA expression level of 14 genes including AMACR, AR, PCA3, SPINK1, TMPRSS2-ERG, KLK3, ACSM1, CACNA1D, DLX1, LMNB1, PLA2G7, RHOU, SPON2, and TDRD1 was measured by a truly quantitative reverse transcription PCR in different prostate tissue samples from men with and without PCa. For the last eight genes the function of the genes in PCa progression was studied by a specific siRNA knockdown in PC-3 and VCaP cells. The results from radical prostatectomy and cystoprostatectomy samples showed statistically significant overexpression for all the target genes, except for KLK3 in men with PCa compared with men without PCa. Statistically significant difference was also observed in low versus high Gleason grade tumors (for PLA2G7), PSA relapse versus no relapse (for SPON2), and low versus high TNM stages (for CACNA1D and DLX1). Functional studies and siRNA silencing results revealed a cytotoxicity effect for the knock-down of DLX1, PLA2G7, and RHOU, and altered tumor cell invasion for PLA2G7, RHOU, ACSM1, and CACNA1D knock-down in 3D conditions. In addition, effects on tumor cell motility were observed after silencing PLA2G7 and RHOU in 2D monolayer cultures. Altogether, these findings indicate the possibility of utilizing these new markers as diagnostic and prognostic markers, and they may also represent therapeutic targets for PCa.

Role of fibroblast growth factors and their receptors in prostate cancer

Prostate cancer (PCa) is the most common non-cutaneous malignant disease among males in the developed countries. Radical prostatectomy (RP) is an effective therapy for most PCa patients with localized or locally invaded tumors but in some cases the cancer recurs after RP. PCa is a heterogeneous disease, which is regulated by many factors, such as androgen receptor (AR), estrogen receptors and  (ER and ER), fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, the role of ERβ, FGF8, FGF13 and FGFRL1 was investigated in PCa. Previous studies have suggested that ER is protective against PCa whereas FGF8 has been shown to induce PCa in transgenic mice. FGF13 and FGFRL1 are poorly understood members of the FGF and FGFR families, respectively. Transgenic mouse models were used to investigate the ability of inactivated ERβ to facilitate FGF8-induced prostate tumorigenesis. Human PCa tissue microarrays (TMAs) were used to study the expression pattern of FGF13 and FGFRL1 in PCa and the results were correlated to corresponding patient data. The targets and biological functions of FGF13 and FGFRL1 were characterized using experimental in vivo and in vitro models. The results show that deficiency of ERβ, which had been expected to have tumor suppressing capacity, seemed to influence epithelial differentiation but did not affect FGF8-induced prostate tumorigenesis. Analysis of the TMAs showed increased expression of FGF13 in PCa. The level of cytoplasmic FGF13 was associated with the PCa biochemical recurrence (BCR), demonstrated by increasing serum PSA value, and was able to act as an independent prognostic biomarker for PCa patients after RP. Expression of FGFRL1, the most recently identified FGFR, was also elevated in PCa. Cytoplasmic and nuclear FGFRL1 was associated with high Gleason score and Ki67 level whereas the opposite was true for the cell membrane FGFRL1. Silencing of FGFRL1 in PC-3M cells led to a strongly decreased growth rate of these cells as xenografts in nude mice and the experiments with PCa cell lines showed that FGFRL1 is able to modulate the FGF2- and FGF8-induced signaling pathways. The next generation sequencing (NGS) experiments with FGFRL1-silenced PC-3M cells revealed candidates for FGFRL1 target genes. In summary, these studies provide new data on the FGF/FGFR signaling pathways in normal and malignant prostate and suggest a potential role for FGF13 and FGFRL1 as novel prognostic markers for PCa patients. Keywords: FGF8, FGF13, FGFRL1, ERβ, prostate cancer, prognostic marker