Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell–microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.This study was supported by Fundació la Marató TV3, Project n° 111431

Molecular mechanisms of WNT signaling and receptor-transducer coupling [Elektronisk resurs]

Wingless and int-1 (WNT) signaling is a group of evolutionarily conserved signal transduction pathways that are vital for fundamental cellular processes. The signals are transduced via receptors at the cell membrane, Frizzleds (FZDs), which belong to class F of GPCRs. In this thesis, the focus is on WNT signaling, the binding of WNT-3A to FZDs, and their coupling to their primary intracellular transducer, Dishevelled (DVL), constitutively, i.e. in the absence of ligands. WNT-3A has been extensively studied for its role in beta-catenin dependent signaling. In this work, eGFP-WNT-3A was used in combination with HiBiT-tagged FZD1-10 to determine the binding kinetics in a live cell nanoBRET assay. The kinetic parameters were determined from both kinetic and saturation binding curves. Among all FZDs, except for FZD3 and FZD9 for which binding could not be detected, the determined affinities range from single to double digit nanomolar concentrations. Additionally, the utility of this assay for other experimental modes was shown, including competition binding with commercial preparations of WNT- 3A, WNT-5A, WNT-5B, WNT-10B, WNT-11 and WNT-16B. In addition to ligand induced signaling, constitutive activity, which is defined as coupling of the receptor to its transducer in the absence of stimulation by a ligand can have important functional consequences. To this end, G protein tri-cistronic activity sensors (G-CASE) were designed and in addition to measuring ligand induced G protein coupling, were validated specifically for determining constitutive coupling in GPCRs. FZDs couple constitutively to DVL. To this end, a FZD6-DVL3 complex was formed and stabilized for cryogenic electron microscopy (cryoEM) experiments, which enabled the determination of the structure at an overall resolution of 3.6 Å. The structure consists of FZD6 and residues 409-495 of DVL3 (DEP domain). The receptor exhibits a typical GPCR-like organization with seven transmembrane helices. The interaction of FZD6 with DEP is characterized by two sites: 1) a lipophilic cavity formed by residues L3935.69, V3965.72, V3995.75 and I4005.76 on FZD6 and residues L434 and I436 on DVL3 and 2) a polar site formed by residues R22612.49, K4126.28, R4166.32 and K4988.49 on FZD6 and K435 on DVL3, surrounding an unassigned density. Single, alanine point mutations in R22612.49, R4166.32, K4988.49, as well as the molecular switch W4937.55 in the vicinity, reduce the relative affinity of FZD6 to DVL3 (amino acids 243-496). The moiety in this unassigned density may play a central role in coordinating the polar residues surrounding this site. Overall, the work presented in this thesis adds to our knowledge on WNT signaling and receptor-transducer coupling methodologically with new approaches to study WNT-FZD kinetics and constitutive GPCR coupling, as well as mechanistically with novel information on the molecular interaction of FZD6-DVL3, with the first structure of a FZD-DVL complex reconstituted in a lipid environment. List of scientific papers I. Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBIT/BRET Assessments. Paweł Kozielewicz, Rawan Shekhani, Stefanie Moser, Carl-Fredrik Bowin, Janine Wesslowski, Gary Davidson, Gunnar Schulte. ACS Pharmacol Transl Sci. 2021 May 11;4(3):1235-1245. https://doi.org/10.1021/acsptsci.1c00084 II. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Hannes Schihada, Rawan Shekhani, Gunnar Schulte. Sci Signal. 2021 Sep 7;14(699):eabf1653. https://doi.org/10.1126/scisignal.abf1653 III. Structural insight into the functional interaction between Frizzled 6 and the transducer protein Dishevelled. Rawan Shekhani, Magdalena M Scharf, Jan Hendrik Voss, Jia Yu Ho, Fanny Peysson, Shi Min Tan, Allan H. Pang, Jitender Kumar, Vítězslav Bryja, Konstantinos Tripsianes, Sébastien Granier, Rémy Sounier, Yong Zi Tan, Gunnar Schulte. [Manuscript

Fibre Thermal Damage in Ring Spinning

Synthetic fibres are more susceptible to thermal damage and have to be processed at lower spindle speeds than cotton on ring spinning machines. Blends of polyester-cotton were spun using ring spinning machine at various spindle speeds. Analysis of the properties of the yarns and the fibres in the yarn revealed that spinning fibre blends with a high polyester content at higher spindle speeds resulted in weaker yarns. Thermal damage of the polyester fibre in the yarn could be seen from SEM photographs. Blending polyester with cotton significantly reduced the risk of thermal damage to the polyester fibre. Yarn hairiness decreased with the increase of spindle speed, but this reduction could not be attributed to fibre thermal damage alone.</jats:p

A systematic review of in vitro models of drug-induced kidney injury

Drug-induced nephrotoxicity is a major cause of kidney dysfunction with potentially fatal consequences and can hamper the research and development of new pharmaceuticals. This emphasises the need for new methods for earlier and more accurate diagnosis to avoid drug-induced kidney injury. Here, we present a systematic review of the available approaches to study drug-induced kidney injury, as one of the most common reasons for drug withdrawal, in vitro. The systematic review approach was selected to ensure that our findings are as objective and reproducible as possible. A novel study quality checklist, named validation score, was developed based on published regulatory guidance and industrial perspectives, and models returned by the search strategy were analysed as per their overall complexity and the kidney region studied. Our search strategy returned 1731 articles supplemented by 337 from secondary sources, of which 57 articles met the inclusion criteria for final analysis. Our results show that the proximal tubule dominates the field (84%), followed by the glomerulus and Bowman's capsule (7%). Of all drugs investigated, the focus was most on cisplatin (n = 29, 50.1% of final inclusions). We found that with increasing model complexity the validation score increased, reflecting the value of innovative in vitro models. Furthermore, although the highly diverse usage of cell lines and modelling approaches prevented a strong statistical verification through a meta-analysis, our findings show the downstream potential of such approaches in personalised medicine and for rare diseases where traditional trials are not feasible

Evaluation of Current Regulation and Guidelines of Pharmacogenomic Drug Labels: Opportunities for Improvements

Pharmacogenomic drug labels in the Summary of Product Characteristics (SmPC) provide an instrument for clinical implementation of pharmacogenomics. We compared pharmacogenomic guidance by Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), the US Food and Drug Administration (FDA), and by the European agencies the European Medicines Agency (EMA), College ter Beoordeling van Geneesmiddelen Medicines Evaluation Board (CBG-MEB), and Federal Institute for Drugs and Medical Devices (FIDMD), collectively assigned as EMA/FIDMD+MEB shortened as EMA/FM. Of 54 drugs with an actionable gene-drug interaction in the CPIC and DPWG guidelines, only 50% had actionable pharmacogenomic information in the SmPCs and the agencies were in agreement in only 18% of the cases. We further compared 450 additional drugs, lacking CPIC or DPWG guidance, and found 126 actionable gene-drug labels by the FDA and/or the EMA/FM. Based on these 126 drugs in addition to the 54 above, the consensus of actionable pharmacogenomic labeling between the FDA and the EMA/FM was only 54%. In conclusion, guidelines provided by CPIC/DPWG are only partly implemented into the SmPCs and the implementation of pharmacogenomic drug labels into the clinics would strongly gain from a higher extent of consensus between agencies.Personalised Therapeutic

A systematic review of in vitro models of drug-induced kidney injury

Drug-induced nephrotoxicity is a major cause of kidney dysfunction with potentially fatal consequences and can hamper the research and development of new pharmaceuticals. This emphasises the need for new methods for earlier and more accurate diagnosis to avoid drug-induced kidney injury. Here, we present a systematic review of the available approaches to study drug-induced kidney injury, as one of the most common reasons for drug withdrawal, in vitro. The systematic review approach was selected to ensure that our findings are as objective and reproducible as possible. A novel study quality checklist, named validation score, was developed based on published regulatory guidance and industrial perspectives, and models returned by the search strategy were analysed as per their overall complexity and the kidney region studied. Our search strategy returned 1731 articles supplemented by 337 from secondary sources, of which 57 articles met the inclusion criteria for final analysis. Our results show that the proximal tubule dominates the field (84%), followed by the glomerulus and Bowman's capsule (7%). Of all drugs investigated, the focus was most on cisplatin (n = 29, 50.1% of final inclusions). We found that with increasing model complexity the validation score increased, reflecting the value of innovative in vitro models. Furthermore, although the highly diverse usage of cell lines and modelling approaches prevented a strong statistical verification through a meta-analysis, our findings show the downstream potential of such approaches in personalised medicine and for rare diseases where traditional trials are not feasible