Investigating the role of vascular endothelial-cell senescence driving resistance to DNA-damaging therapies and metastasis formation in lung cancer

Lung cancer is the third most common cancer in the UK, and with a 5 year survival rate of only 19% is one of the leading causes of death worldwide (World Cancer Research Fund, 2020). Neo-adjuvant chemotherapy (with drugs that damage the DNA) can reduce the size of initial primary lung cancers prior to surgery. However, the cancer often comes back or spreads because of resistance to these types drugs. One way that resistance can occur includes a change in the cancer cells where they slow-down and become 'senescent'. But importantly, therapy induced senescence can occur not only in cancer cells but also in normal cells of our bodies that support the cancer, also known as the tumour microenvironment, or even in other non-cancerous organs.

In fact, senescence has been associated with many of the adverse secondary effects of chemotherapy in patients, with reports of cancer surviving patients developing age-related diseases such as vascular pathologies. Blood vessels are a major part of a tumour microenvironment, as they are the conduits by which blood and oxygen are delivered to the growing mass. More recently, endothelial cells (the cells that line blood vessels) have been shown to have an independent role, beyond their typical conduit role, as master regulators of a cocktail of molecules that are 'spat' out of the cell and can control how neighbouring cells behave or even travel through the circulation to other organs.
Focal adhesion Kinase (FAK) is a molecule that is found in cancer and several FAK-blocking drugs are presently in clinical trials for cancer treatment. However, FAK is expressed in many different cell types with sometimes opposing functions in them, therefore cell-type specific drugs need to be developed. In this context, we have shown that specifically targeting endothelial-cell FAK in tumour mouse models leads to increased sensitisation of tumour cells to DNA-damage therapies. Importantly, in a human clinical setting, we have shown that tumour-associated endothelial-FAK expression correlated with molecular subtype and prognosis in invasive breast cancer and that endothelial-cell FAK activation independently predicts improved survival in neoadjuvant-treated advanced breast cancer.

Based on our work so far we hypothesize that FAK is a regulator of therapy induced endothelial- cell senescence, which can be a mechanism by which cancer spreads. We will identify the molecules that regulate this with the overall goal of devising new ways to treat cancer spread and resistance to drugs better.

We hypothesise that targeting EC-FAK in combination with DNA-damaging therapy controls cancer metastases through senolytic effects. Here we will:
1. Investigate the role of EC-FAK in DNA-damaging therapy induced senescence and metastasis formation in vivo. Syngeneic lung cancer mouse cell lines transfected with a mNIS (sodium iodide symporter) will be injected into the left lung of immunocompetent EC-specific FAK KO and FAK-kinase-dead mice. Mice will receive chemotherapy prior to primary tumour radio-ablation, and monitored for metastasis development by SPECT/CT imaging. Mice will also be crossed with a p16-FDR line, to identify the functional consequence of ablating senescent cells in the regulation of chemoresistance and metastasis. Histologic analysis will determine cellular and molecular changes in primary cancers and metastases.
2. Identify the cellular and molecular mechanisms underlying EC-FAK regulation of metastasis after DNA-damaging therapy. ECs will be isolated and sorted by FACs from treatment naiive vs chemo-treated EC-FAK-knockout or EC-FAK kinase dead mouse pre-metastatic lungs for transcriptomic analysis. End-point metastatic lung foci will be analysed for gene expression. Bioinformatic analyses will compare these data with human lung cancer databases. The molecular mechanisms leading to senescence will be identified using immunofluorescence, western blotting, RIME and Chipseq.
3. Functional validation of the DNA-damage-induced EC-FAK driven mechanisms identified in (2) relevant to metastases formation. Genetic and pharmacological inhibition of the candidates identified above will be tested in physiological 3D microfluidic lung chip models with, and without, chemotherapy. Effects on lung cancer cell seeding and colonisation will be determined. It will also allow discovery and testing of novel alternative strategies, to FAK-inhibition, that target EC-senescence in combination with DNA-damaging therapies in pre-metastatic niche formation