Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.

Urokinase plasminogen activator (uPA) system regulates extracellular matrix remodelling by activating ubiquitous protease plasmin in many important physiological processes. The system components include uPA, plasminogen activator inhibitors (PAIs) and uPA receptor (uPAR). Besides its role in physiological processes, uPA system is active in most tumour types where its aberrant regulation has been associated with the development of metastatic phenotype. In vitro and in vivo studies have shown that the over-expression of uPA, PAI-1 and uPAR not only enhances tumour cell invasion capacity and metastasis, but also corresponds to a higher risk of disease correlating with traditional clinicopathological features which makes them potential prognostic biomarkers and therapeutic targets in a wide range of human malignancies. This review focuses on uPA system's prognostic and predictive role in several types of human cancers, summarizing its activities in cancer development and highlighting the importance of addressing all unanswered questions before bridging the gap between laboratory findings to clinic use of uPA system's components as cancer biomarkers.

Glucose, the key source of metabolic energy, is imported into cells by two categories of transporters: 1) facilitative glucose transporters (GLUTs) and 2) secondary active sodium-glucose cotransporters (SGLTs). Cancer cells have an increased demand for glucose uptake and utilisation compared to normal cells. Previous studies have demonstrated the overexpression of GLUTs, mainly GLUT1, in many cancer types. As the current standard positron emission tomography (PET) tracer 2-deoxy-2-( ¹⁸ F)fluoro-D-glucose (2-FDG) for imaging tumour cells via GLUT1 lacks in sensitivity and specificity, it may soon be replaced by the newly designed, highly sensitive and specific SGLT tracer α-methyl-4-(F-18)fluoro-4-deoxy-Dglucopyranoside (Me-4FDG) in clinical detection and tumour staging. This tracer has recently demonstrated the functional activity of SGLT in pancreatic, prostate, and brain cancers. The mRNA and protein expression of SGLTs have also been reported in colon/colorectal, lung, ovarian, head, neck, and oral squamous carcinomas. So far, SGLTs have been poorly investigated in cancer, and their protein expression and localisation are often controversial due to a lack of specific SGLT antibodies. In this review, we describe current knowledge concerning SGLT1 and SGLT2 (over)expression in various cancer types. The findings of SGLTs in malignant cells may help in developing novel cancer therapies with SGLT2 or SGLT1/SGLT2 inhibitors already used in diabetes mellitus treatment.