Interactions between hypoxia, HIF, type 2 IP3 receptors and invasion of glioblastoma

Most cancers form solid tumours, within which cells inappropriately proliferate. Since the development of an effective blood supply lags behind growth of the tumour, the tumour cells are exposed to much lower oxygen concentrations than most cells. This hypoxia leads to expression of a protein (HIF) that coordinates adaptive responses to the lack of oxygen. HIF causes tumour cells to loosen their attachments to each other, and so become more likely to leave the primary tumour and migrate elsewhere. This is a major clinical problem because most cancer deaths are caused by tumours spreading to form new tumours elsewhere in the body (metastases).

Glioblastoma is the most common primary brain tumour. These tumours rarely spread beyond the brain, but they do invade surrounding brain tissue, preventing complete surgical removal and usually leading to death within months of diagnosis. As with other solid tumours, this invasion begins in a hypoxic environment, within which the tumour cells express HIF. Our analyses of glioblastoma cells suggest a pathway through which hypoxia and HIF may lead to disruption of adhesions between the cells and thereby increased invasion of surrounding brain tissue. Surprisingly this pathway involves down-regulation of a protein (IP3R2) that differs minimally from its close relatives that are much more abundant in glioblastoma cells. Since this protein is a channel through which calcium ions can leak into the cell to regulate cellular activities, we suggest that IP3R2 is unique among its relatives in selectively delivering its calcium signals to targets that regulate the adhesions between cells.

We will address the mechanisms linking hypoxia, through HIF, to down-regulation of the IP3R2 protein. We will then ask how that leads to disruption of adhesions between cells and to increased invasion. We suggest that understanding these early steps in invasion might identify new targets for more effective treatment of glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumour. Most patients die within months of diagnosis as the tumour diffusely invades surrounding brain tissue, preventing complete surgical removal of the tumour. Understanding the early steps in invasion might identify new targets for more effective treatment. As with most solid tumours, GBM tumours occupy a hypoxic environment that stabilizes hypoxia inducible factor (HIF), a transcription factor that orchestrates adaptive responses to hypoxia. Hence, GBM cell invasion starts in a hypoxic environment, where tumour cells must detach from their neighbours and begin their migration through brain parenchyma. Our analyses of human glioma cells has shown that hypoxia, through HIF, causes down-regulation of a minor subtype of an intracellular Ca2+ channel, the type 2 inositol 1,4,5-trisphosphate receptor (IP3R2). Hypoxia or siRNA-mediated down-regulation of IP3R2 causes loss of adhesions between glioma cells and an increase in invasion. The results are surprising because the effects are specific for IP3R2, which comprises <2% of the IP3Rs expressed in glioma cells. We will use patient-derived glioma neural stem cells with in vitro and ex-vivo assays (cell aggregation and adhesion, spheroid and organoid invasion), fluorescence microscopy (recording events at nascent adhesions), gene-editing of endogenous IP3R2, and biochemical manipulations (HIF, Ca2+ signalling, IP3R2 expression) to explore the relationship between hypoxia, HIF-1, down-regulation of IP3R2, and disruption of the intercellular junctions that prepare GBM cells for invasion. Our hypothesis is that the activity of IP3R2 is required to maintain intercellular junctions between GBM cells, and that hypoxia, through HIF, causes decreased transcription of the IP3R2 gene, leading to disruption of the junctions and increased invasion. We suggest that a better understanding of these early events in cell invasion might identify new targets for GBM treatment

Basic science
We address fundamental mechanisms in cell biology: how does a specific intracellular Ca2+ channel through spatially organized signalling pathways regulate adhesions between cells, and how does hypoxia impact this regulation. Our GBM model provides an extreme example of a single IP3R subtype uniquely regulating a biological activity, and thereby a paradigm to explore the importance of spatially organized Ca2+ signals. Our questions are framed to provide insight into the behaviours of glioblastoma cells that determine whether they will diffusively infiltrate brain parenchyma. We will work with patient-derived glioma neural stem cells (to maximise the relevance to GBM). Addressing fundamental questions, allied with their potential to identify new targets for intervention in the treatment of GBM, will ensure that our work delivers impact.

Health
GBM is a devastating disease from which most patients die within months as the primary tumour diffusively invades surrounding brain tissue. Our work will advance understanding of the biology of the early steps in this invasion. We will actively engage with clinicians and the pharmaceutical industry to ensure that our findings are presented at an early stage to communities with direct interests in clinical development, for example, by presentation to British Neuro Oncology Society and British Neurosurgical Research Group, of which Bulstrode is an active member.

Training
The Taylor lab provides an environment, supported by international/cross-disciplinary collaborations, where staff can develop projects that will initiate their independent careers. This is particularly relevant for Rossi and Bulstrode, who are establishing independent groups focused on GBM. Rossi has experience supervising project/PhD students, teaching practical classes and lecturing. These skills will be developed and, alongside engagement and mentoring by Taylor at every stage of the project, Rossi will be well-prepared to move to an independent post. The proposal will also provide training for a skilled TA. A major impact is Taylor's proven ability to train staff at every career stage to meet future needs of industry, the public sector and academia. Our public engagement activities further contribute to providing the UK with well-prepared scientists and a scientifically-informed population. Bulstrode has interests in clinical and biology of glioma, and seeks through this collaboration to extend his exposure to the underlying biochemistry and translational drug development. These aspects will be of value as he seeks to transition to independence with a Clinician Scientist Fellowship.

Public understanding and schools
Taylor's lab organises the Young Pharmas scheme, which seeks to inspire year-12 students, ensure that they appreciate the importance of creativity and critical evaluation in science, and the economic impact of pharmacology. Parents and teachers gain exposure to these activities through the final poster session/guest lecture evening. Our research into GBM will be assimilated into these activities. We provide at least one summer placement each year for an undergraduate to gain research experience before deciding on postgraduate options. Staff contribute to Cambridge Science Festival with a hands-on practical and visits to schools. These activities encourage informed interest in science from students who have not yet finalised career choices, and a more widespread appreciation of the importance of addressing fundamental questions in biology. We work with press offices to maximize the impact of our work by bringing it to diverse audiences. Bulstrode has previously been interviewed in relation to his Zika work on Radio 4's Today program, and profiled twice in the Careers section of Nature.