Targeting kallikrein-related peptidases in prostate cancer

Introduction: Novel therapeutic compounds are needed for prostate cancer (CaP), given the limitations of already used drugs and the disease’s mortality, often attributed to castrate resistance. Tissue kallikrein and kallikrein-related peptidases (KLKs) form a family of serine proteases aberrantly expressed and broadly implicated in human malignancies. In CaP, KLKs participate in the promotion of cell proliferation, extracellular matrix degradation, tumour cell invasion and metastasis. Areas covered: This review discusses the different ways of inhibiting, modulating and exploiting KLK activity and/or expression as emerging CaP therapeutics. KLKs are targeted by diverse naturally occurring substances, including proteinaceous inhibitors, low-molecular-weight peptides and Zn2+. Synthetic KLK inhibitors include protein/peptide-based inhibitors and small molecules. A re-engineered serpin-based KLK inhibitor is under evaluation in first-in-human trials as a CaP therapeutic, whereas additional potent and selective KLK inhibitors with relevance to CaP have been synthesized. KLK3-activated pro-drugs have entered Phase I and Phase II clinical trials as therapeutics for prostate tumours. The KLK3-based PROSTVAC vaccine is evaluated in Phase III clinical trials. Targeting KLK expression via RNA interference methods could represent another promising therapeutic approach for CaP. Expert opinion: Apart from their immense biomarker potential, KLKs also hold promise as the basis of novel CaP therapeutics

Loss of miR-378 in prostate cancer, a common regulator of KLK2 and KLK4, correlates with aggressive disease phenotype and predicts the short-term relapse of the patients

A large number of prostate cancer (PCa) patients receive treatment without significant benefits, strengthening the need for accurate prognosis, which can be supported by the study of miRNAs. In silico specificity analysis was performed for the identification of miRNAs able to regulate KLK2 and KLK4 expression. Total RNA was extracted from prostate tissues obtained from PCa and benign prostate hyperplasia patients. Thereafter, RNA was polyadenylated and reverse transcribed to cDNA, which was used for qPCR analysis. miR-378 was predicted to target both KLK2 and KLK4 and downregulated levels detected in PCa patients (p = 0.050). The reduction of miR-378 was correlated with higher Gleason score (p = 0.018), larger diameter tumors (p = 0.034), and elevated serum PSA (p = 0.006). Regarding prognosis, miR-378 was able to improve risk stratification according to Gleason score or tumor stage, while higher risk to recur highlighted for the patients expressing lower miR-378 levels. Finally, the loss of miR-378 was able to predict the short-term relapse of ‘high’- and ‘very high’- recurrence-risk patients, independent of Gleason score, tumor stage, PSA, and age as indicated by Kaplan-Meier survival curves (p = 0.030) and multivariate Cox regression analysis (p = 0.018). In conclusion, loss of miR-378 expression increases the risk for PCa progression and relapse, despite active treatment

Blood-based analysis of type-2 diabetes mellitus susceptibility genes identifies specific transcript variants with deregulated expression and association with disease risk

Despite significant progress by genome-wide association studies, the ability of genetic variants to conduce to the prediction or prognosis of type-2 diabetes (T2D) is weak. Expression analysis of the corresponding genes may suggest possible links between single-nucleotide polymorphisms and T2D phenotype and/or risk. Herein, we investigated the expression patterns of 24 T2D-susceptibility genes, and their individual transcript variants (tv), in peripheral blood of T2D patients and controls (CTs), applying RNA-seq and real-time qPCR methodologies, and explore possible associations with disease features. Our data revealed the deregulation of certain transcripts in T2D patients. Among them, the down-regulation of CAPN10 tv3 was confirmed as an independent predictor for T2D. In patients, increased expression of CDK5 tv2, CDKN2A tv3 or THADA tv5 correlated positively with serum insulin levels, of CDK5 tv1 positively with % HbA1c levels, while in controls, elevated levels of TSPAN8 were associated positively with the presence of T2D family history. Herein, a T2D-specific expression profile of specific transcripts of disease-susceptibility genes is for the first time described in human peripheral blood. Large-scale studies are needed to evaluate the potential of these molecules to serve as disease biomarkers

Study of L-DOPA decarboxylase and kallikreins gene expression regulation through microRNA molecules, in prostate and bladder cancers: evaluation of their differential diagnostic and prognostic value

One of the main features of prostate and bladder cancers is their clinical heterogeneity, in terms of disease progression rate, patients’ survival and treatment response. Therefore, the studying of the different molecular signatures of the disease and the identification of novel molecular biomarkers could offer, an alternative approach to improve disease prognosis and to support personalized treatment decisions, and is of first research and clinical priority.L-DOPA decarboxylase (DDC) was recently identified as a novel co-activator of androgen receptor (AR), enhancing the ligand-dependent AR transcriptional activity. Additionally, DDC represents also a marker of the neuroendocrine (NE) differentiation of the prostate. The NE prostate cells play essential role in prostate cancer progression. Tissue kallikrein (KLK1) and the kallikrein-related peptidases (KLK2-KLK15) constitute a family of 15 homologous secreted serine proteases. The value of KLKs as cancer biomarkers is highlighted by the previously established clinical utility of prostate specific antigen (PSA), as kallikrein-related peptidase 3 (KLK3) is largely known, for prostate cancer diagnosis and treatment monitoring. Finally, microRNAs (miRNAs) are a rapidly growing family of small (~22nt) non-coding RNAs, representing the most powerful gene expression regulators at the post-transcriptional level. Their function is crucial for cellular homeostasis, and the deregulation of miRNAs expression has become a hallmark of the majority of human malignancies.In the present Ph.D. Thesis is performed the expression analysis and the evaluation of the clinical significance of DDC, KLKs genes, and miRNAs molecules, which regulate their expression post-transcriptionally, in prostate and bladder cancers. To fulfill these objectives, a statistically significant number of 137 prostate and 279 blabber fresh frozen tissues were collected, processed and included in the study. Moreover, novel molecular methodologies were developed and optimized for the accurate and sensitive quantification of the tested genes and miRNAs in prostate and bladder tissue specimens. Additionally, a detailed database of patients’ history, clincopathological and follow-up data has been constructed for all patients. The analysis of the total number of tissue specimens and the extended statistical analysis of the results of the study highlighted the significant clinical value of the tested biomarkers for the differential diagnosis and the accurate prognosis of prostate and bladder cancers.Ένα ιδιαίτερο χαρακτηριστικό του καρκίνου του προστάτη και του καρκίνου της ουροδόχου κύστεως είναι η έντονη κλινική ετερογένειά τους, σχετικά με την πρόγνωση εξέλιξης της νόσου, την επιβίωση των ασθενών και την ανταπόκρισή τους στην θεραπεία. Για το λόγο αυτό, η μελέτη του μοριακού προφίλ των όγκων και η ταυτοποίηση νέων μοριακών δεικτών μπορεί να προσφέρει σημαντικά στην βελτίωση της πρόγνωσης και τον σχεδιασμό της εξατομικευμένης θεραπείας των ασθενών, εμφανίζοντας έντονο ερευνητικό και κλινικό ενδιαφέρον σήμερα.Η L-DOPA αποκαρβοξυλάση (DDC) έχει βρεθεί να αποτελεί ένα νεότερο συνενεργοποιητή του υποδοχέα των ανδρογόνων (AR) ενισχύοντας σημαντικά την μεταγραφική ενεργότητά του. Επίσης, η DDC αποτελεί δείκτη της νευροενδοκρινούς (NE) διαφοροποίησης του προστάτη, με τα ΝΕ κύτταρα του αδένα να διαδραματίζουν κεντρικό ρόλο για την εξέλιξη της νόσου. Οι καλλικρεΐνες (KLKs) αποτελούν μια οικογένεια 15 ομόλογων εκκρινόμενων πρωτεασών σερίνης, η ιδιαίτερη κλινική αξία της οποίας στις ανθρώπινες νεοπλασίες αντικατοπτρίζεται στον προσδιορισμό του PSA, όπως είναι ευρέως γνωστή η KLK3, του ορού του αίματος για την διάγνωση και την πρόγνωση του καρκίνου του προστάτη. Τέλος, τα microRNAs (miRNAs) αποτελούν μια διαρκώς αυξανόμενη οικογένεια μικρών (~22 nt) μη-κωδικών μορίων RNA, τα οποία αποτελούν τους σημαντικότερους ρυθμιστές της γονιδιακής έκφρασης σε μετα-μεταγραφικό επίπεδο. Η δράση τους είναι ιδιαίτερα σημαντική για την κυτταρική ομοιόσταση, ενώ η απορρύθμιση της έκφρασής τους παρατηρείται στην πλειοψηφία των κακοηθειών του ανθρώπου.Στην παρούσα διδακτορική διατριβή πραγματοποιείται η μελέτη της έκφρασης και αξιολόγηση της κλινικής αξίας των DDC, KLKs γονιδίων, καθώς και miRNAs, τα οποία ρυθμίζουν μετα-μεταγραφικά την έκφρασή τους, στον καρκίνο του προστάτη και της ουροδόχου κύστεως. Για την επίτευξη των στόχων της διατριβής πραγματοποιήθηκε η συλλογή και επεξεργασία ενός στατιστικά σημαντικού αριθμού 137 ιστοτεμαχίων προστάτη και 279 ιστοτεμαχίων ουροδόχου κύστεως, καθώς και η ανάπτυξη και βελτιστοποίηση νέων μοριακών μεθοδολογιών προσδιορισμού των επιπέδων έκφρασης των υπό μελέτη γονιδίων και miRNAs. Λεπτομερής βάση των κλινικοπαθολογικών χαρακτηριστικών και της πορείας τους (follow-up) έπειτα από την θεραπεία κατασκευάστηκε για το σύνολο των ασθενών. Η βιοστατιστική ανάλυση των αποτελεσμάτων ανέδειξε την ισχυρή κλινική αξία, ενός σημαντικού αριθμού των υπό μελέτη βιομορίων, για την διαφορική διάγνωση και πρόγνωση του καρκίνου του προστάτη και της ουροδόχου κύστεως