University of Cambridge
Contributions of type 2 IP3 receptors to intercellular adhesions and invasion of glioblastoma cells
Abstract
dc:description.abstractGlioblastoma multiforme (GBM) is the most common and invasive brain tumour in adults. Treatment options are limited with a median survival of 15 months and only 12% of patients survive for more than 5 years. A challenge in GBM treatment is that cells infiltrate brain tissue, making complete surgical resection impossible and allowing recurrence after local therapy. Ca2+ is an ubiquitous intracellular messenger. Ca2+ signals are often initiated by inositol 1,4,5-trisphosphate receptors (IP3Rs), which mediate Ca2+ release from the endoplasmic reticulum. Mammals express three IP3R subtypes (IP3R1-3). I show that in glioblastoma cell lines, IP3R2 is the least abundant subtype (<5% of all IP3Rs), and that loss of IP3R2 selectively increases invasion and reduces intercellular adhesion. I used patient-derived GBM stem cells (GSC) and a human glioma cell line U87-MG to explore the mechanisms linking IP3R2 to cellular adhesion and invasion. I optimised two different intercellular adhesion assays and adapted one invasion assay to measure the effects of reducing IP3R2 expression using siRNA or shRNA. GSC cells with a reduction in IP3R2 levels significantly increased invasion by 43% and also resulted in a 13% increase in looser intercellular adhesions. To identify candidate proteins that could contribute to reduced intercellular adhesion after loss of IP3R2, I optimised two unbiased approaches, mass spectrometry (MS) and RNA-sequencing (RNA-seq). U87-MG cells treated with IP3R2 shRNA or control shRNA were cell-surface biotinylated, and the biotinylated proteins and their partners were captured using streptavidin before MS analysis. It was impossible to obtain replicates of the MS analyses, but the single analysis suggested that the adhesion molecules N-cadherin, Cadherin 11 and NCAM are upregulated due to a reduction in IP3R2. Hypoxia has been shown to reduce IP3R2 protein levels, reduce cell-to-cell adhesion, and increase invasion in U87-MG cells. Therefore, hypoxia was included in the RNA-seq experiment with the aim of finding a pathway that is both affected by hypoxia and siRNA against IP3R2. In total, 1541 significant differentially expressed genes were detected in samples with lower expression of IP3R2 compared to the control. For hypoxia treated cells, compared to control cells 6449 differentially expressed genes were detected. These results established that the loss of IP3R2 was associated with increases in mRNA for TGFBR2 and decreases for Connexin43 and CD63. I then assessed six candidates ZDHCC9, Connexin43, CD63, N-cadherin, Cadherin11 and TGFBR2 identified by the unbiased analyses in functional analyses of invasion and intercellular adhesion. Using siRNA against each of these candidates, I confirmed that of 3 of 6 proteins significantly alter invasion in GBM. Taken together, in GSC loss of the minor IP3R subtype, IP3R2, reduces cell-to-cell adhesion and increases invasion. Using an unbiased approach, 6 candidates were identified to be likely responsible for this phenotype. Using siRNA to reduce the expression of each of these candidates in GBM confirmed that 3 of 6 proteins significantly alter invasion in GBM.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Van Marrewijk, Laura Marina
- Advisors dc:contributor.advisor
-
- Taylor, Colin
- Rossi, ana
Subjects
dc:subject × 2Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.115775
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/379805