MICA: A molecular dissection of the interplay between diabetes and cancer: an integrated, multidisciplinary approach. II.

This research programme focuses on the detailed actions of human insulin and Insulin-like Growth Factors-1 and 2 (IGF1/2); these are closely related protein hormones. Through evolution they acquired separate biological functions, with insulin becoming a key regulator of metabolism, while IGF1/2 are major growth factors behind cell growth and differentiation. The levels of activity of these hormones determine how long and how healthy we live in the face of lifestyle, diet and disease. When released into the blood the hormones bind, tightly and specifically, to their receptors: Insulin- (IR) and IGF-1R, respectively, large complex protein molecules on the cell surface. Receptor binding, through which the hormone activity is delivered into cells, involves structural changes in both the hormone and the receptors. Here we aim to understand the key events in the translation of hormone signal from the outside to the inside of the cell.

Despite their fundamental medical importance, such as insulin signaling malfunctions in Type 1 (T1D) and Type 2 Diabetes (T2D), IGF1/2 are major drivers of cancer, it is still not understood how these hormones achieve their specific signals and induce different biological effects via their receptors. The complexity of insulin & IG1/2 molecular actions are further convoluted by the existence of two, very similar forms of the IR, and the ability of these hormones to bind in some way to all receptors. There are two forms of the IR; the IR-B form controls metabolic actions of insulin, while IR-A binds also IGF1/IGF2, and can stimulate cell growth and proliferation.

This very complex, and intertwined molecular activity of insulin and IGF1/2 is the basis of their huge societal and human health impact. ~£25mln/day of the NHS budget is spent on T2D, largely to treat associated complications such as cardiovascular and kidney disorders, cancer, and neurodegeneration. Hence there is an urgent need to understand insulin and IGF1/IGF2 specificity at the hormonal and receptor level. This could be then exploited in the design and delivery of new, safer, forms of insulin (analogues), and new IGF1/2 analogues with anti-cancer and beneficial (e.g. anti-neurodegenerative) selective properties, without side-effects seen in diabetes. This research programme is responding to this challenge by offering a consolidated, multidisciplinary (structural, chemical and cell biology) approach to these problems, addressing all the key aspect of insulin & IGF1/2 biology: hormones, receptors and cells.
Fundamental research is the foundation cornerstone of this programme. However, the advanced expertise of this group in applied biomedical sciences will enable thorough clinical translation for the benefit of patients with different conditions.

This programme will deliver:

- on the receptor level: (i) the description of insulin binding to its receptors IR-A and IR-B, (ii) delineation of the structural signatures in the IR-A and IR-B extracellular, hormone-binding parts, (iii) how the hormone-triggered signal is transduced to the inside of the cell in IR and IGF-1R receptors, (iv) what are the structural determinants of insulin and IGF1/2 specific actions through their receptors

- on the hormone level: (i) description of the metabolic and mitogenic elements of human insulin, (ii) description of IGF1/IGF2 hormonal determinants behind their specificities, (iii) development of highly-metabolic, safe insulin analogues, (iv) development of IGF-1R specific IGF1 and IGF2 analogues, including IGF-1R antagonist with anti-cancer applicability

-on the cell-level: (i) description of the contribution of IR-A, IR-B and IGF-1R to hormone-activated glucose uptake into human muscle and fat tissue, (ii) development of advanced human cell-systems with specific receptors to optimise development of insulin analogues, and for study of T2D, (iii) validation of the available human tissue models used in glucose transport studies.

The experimental paths are very different, due to diverse methodologies of the structural, chemical and cell biology.

Receptors. The structural studies in York (IR-A,IR-B,IGF-1R) are re-focused due to gains achieved in the current MRC grant. Receptors and their constructs will be expressed only in the baculovirus system (yielding currently more reproducible/better crystals). Complexes with insulin/IGF1/2 will be expanded for highly specific analogues to decipher their structural signatures. Hormone receptor affinities will be evaluated by ITC & Novo Nordisk proprietary assays. TK protein expression/structural work will be carried in Cork, while TK crystal studies will be done with York. Frequent BAG beam access to DLS (Didcot), and remote data collection are well established. The exploitation of the larger constructs (i.e. IR ectodomains) by cryoEM is initiated, and it will be continued. However, when suitable grids:constructs are established, the cryoEM work will form a separate grant application.

Hormones. Analogues of insulin, IGF1/2 will be semi-/fully-synthesised or expressed in E.coli; IOCB has key expertise here. The established wide range of analogue assays (receptor affinities and autophosphorylation, cell viability etc.), will be expanded for IGF-2R receptor essays; unique radio-active (125I, Eu) IGF2 tracers will be produced. All analogues with novel properties will be used in structural studies with the receptors, and optimised further in novel human muscle/fat-tissue cell-based platforms developed here.

Cells. CRISPR-Cas9 will be used to knockout all IR isoforms individually (and IGF-1R), creating a novel insulin unresponsive cell line. This line will be transfected to express specific receptor(s) profile, then the insulin/analogues responsiveness of these cell lines will be characterised with respect to i) glucose transport, ii) the proportion of HA-GLUT4-GFP expressed/present at the cell surface, iii) IR downstream effectors activation.

The multi-facetted approach of our proposal maximizes the cohort of researchers on whose work our studies will impact. Our work will be of especial interest to all members of the research community who work on various aspects of insulin action, metabolism and diabetes, as well as those working on cell-signaling pathways. Our research has potential to greatly impact the underexplored research area seeking to define the molecular cross talk between signals emanating from distinct, but closely related cell surface receptors, and how perturbations in such processes underlie patho-physiologies including diabetes and numerous types of cancers.

As a collective, we are well-connected both nationally and internationally to disseminate our findings informally as well as making them publicly available through presentation at scientific meetings and publication in open-access journals. Through the very nature of our programme we have distinct research profiles; an asset that, as well as ensuring our work addresses questions that are important in multiple fields, will facilitate impact of this work.

The cell biology platforms we aim to generate will facilitate a paradigmatic shift in approach to allow characterization of the consequences on cellular physiology of hormone analogues that have hitherto been studies using biophysical and biochemical techniques. By making these platforms available to other researchers our work will increase the impact of the whole field (hormone/receptor structure/function)

The focus of our work on growth factor-signaling means that the work will also have impact on the pharmaceutical industry which has a substantial interest in Type-2 diabetes, insulin-resistance and cancers. Our work will pave the way for development/design of diagnostic and therapeutic strategies for these disease states. Given the substantial problem that these conditions represent in the U.K (and globally) not only in terms of morbidity and mortality, but also from an economic perspective, it is hard to overstate the impact this could have.

Presentation of our work at national and international meetings attended by academics and representatives of major pharmaceutical companies will maximise its impact in these areas, as will the other forms of engagement outlined in our 'Pathways to Impact'. It should also be noted that the Universities of all partners have Communications Departments dedicated to highlighting research outputs, and Commercial Exploitation Units to assist with commercialisation of any research output as need arises.

Finally, all PIs have a track record in engaging the general public, for example by presentation at diabetes support groups, and work on National Diabetes groups. Such meetings are often attended by policy makers, providing opportunity for our research to influence directly the nation's health, wealth and culture. While such developments clearly represent a long-term investment, they are nonetheless important.