Heparan Sulfate Biosynthetic System Is Inhibited in Human Glioma Due to EXT1/2 and HS6ST1/2 Down-Regulation.

Heparan sulfate (HS) is an important component of the extracellular matrix and cell surface, which plays a key role in cell-cell and cell-matrix interactions. Functional activity of HS directly depends on its structure, which determined by a complex system of HS biosynthetic enzymes. During malignant transformation, the system can undergo significant changes, but for glioma, HS biosynthesis has not been studied in detail. In this study, we performed a comparative analysis of the HS biosynthetic system in human gliomas of different grades. RT-PCR analysis showed that the overall transcriptional activity of the main HS biosynthesis-involved genes (EXT1, EXT2, NDST1, NDST2, GLCE, HS2ST1, HS3ST1, HS3ST2, HS6ST1, HS6ST2, SULF1, SULF2, HPSE) was decreased by 1.5-2-fold in Grade II-III glioma (p < 0.01) and by 3-fold in Grade IV glioma (glioblastoma multiforme, GBM) (p < 0.05), as compared with the para-tumourous tissue. The inhibition was mainly due to the elongation (a decrease in EXT1/2 expression by 3-4-fold) and 6-O-sulfation steps (a decrease in 6OST1/2 expression by 2-5-fold) of the HS biosynthesis. Heparanase (HPSE) expression was identified in 50% of GBM tumours by immunostaining, and was characterised by a high intratumoural heterogeneity of the presence of the HPSE protein. The detected disorganisation of the HS biosynthetic system in gliomas might be a potential molecular mechanism for the changes of HS structure and content in tumour microenvironments, contributing to the invasion of glioma cells and the development of the disease.

Computer-aided engineering of stabilized fibroblast growth factor 21.

FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

Fibroblast Growth Factor 2 Protein Stability Provides Decreased Dependence on Heparin for Induction of FGFR Signaling and Alters ERK Signaling Dynamics.

Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

Conventional Anti-glioblastoma Chemotherapy Affects Proteoglycan Composition of Brain Extracellular Matrix in Rat Experimental Model in vivo.

Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of glioblastoma multiforme (GBM), often accompanied by dexamethasone (DXM) to prevent brain oedema and alleviate clinical side effects. Here, we aimed to investigate an ability of the drugs to affect normal brain tissue in terms of proteoglycan (PG) composition/content in experimental rat model in vivo. Age- and brain zone-specific transcriptional patterns of PGs were demonstrated for 8, 60, and 120 days old rats, and syndecan-1, glypican-1, decorin, biglycan, and lumican were identified as the most expressed PGs. DXM treatment affected both PG core proteins expression (mainly syndecan-1, glypican-1, decorin, biglycan, lumican, versican, brevican, and NG2) and heparan sulphate (HS)/chondroitin sulphate (CS) content in organotypic brain slice culture ex vivo and experimental animals in vivo in a dose-dependent manner. TMZ treatment did not result in the significant changes in PG core proteins expression both in normal rat brain hippocampus and cortex in vivo (although generics did), but demonstrated significant effects onto polysaccharide HS/CS content in the brain tissue. The effects were age- and brain zone-specific and similar with the age-related PGs expression changes in rat brain. Combination of TMZ with DXM resulted in the most profound deterioration in PGs composition and content in the brain tissue both at core protein and glycosaminoglycan levels. Taken together, the obtained results demonstrate that conventional anti-glioblastoma therapy affects proteoglycan structure and composition in normal brain tissue, potentially resulting in deterioration of brain extracellular matrix and formation of the favourable tumorigenic niche for the expansion of the residual glioma cells. During the TMZ chemotherapy, dose and regimen of DXM treatment matter, and repetitive low DXM doses seem to be more sparing treatment compared with high DXM dose(s), which should be avoided where possible, especially in combination with TMZ.