An old drug with new tricks; examining the anti-cancer potential of metformin in mouse and cell models of EML4- ALK+ non-small cell lung cancer

Lung cancer is an aggressive disease producing the highest cancer worldwide. Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. The discovery of oncogenic receptor tyrosine kinases (RTKs) has led to the development of targeted drugs, improving the survival of many patients. One such RTK occurs when a chromosomal rearrangement fuses the echinoderm microtubule-associated protein-like 4 (EML4) with the anaplastic lymphoma kinase receptor (ALK) producing the constitutively active EML4-ALK. Crizotinib is a first-generation tyrosine kinase inhibitor (TKI) which targets ALK fusions, preventing cancer cell growth, and increasing the progression free survival of patients. However, the effect of crizotinib and other TKIs is limited by the development of acquired resistance. To overcome this, various strategies are being explored, including repurposing existing drugs which may possess anti-cancer mechanisms. Metformin, a hypoglycaemic agent, has promising epidemiological evidence in reducing the risk of several cancers. This thesis aimed to examine if metformin is active against EML4-ALK+ lung cancer and if it provides synergistic benefit to crizotinib. The first section of this thesis examines if a combination of metformin and crizotinib produces toxicity, which was followed by an in vivo xenograft model study. The drugs in combination showed no liver or kidney toxicity. Metformin did not affect CYP3A activity or polypeptide levels, the main metabolising enzyme of crizotinib. Crizotinib, metformin and the combination slowed the tumour growth rate in an EML4-ALK+ lung cancer xenograft model. However, the combination was not significantly different to the drugs alone and therefore, the combination was not examined further. The second section of this thesis explored the potential anti-cancer effect of metformin by examining known targets using in vitro techniques. Metformin did not inhibit ALK phosphorylation in EML4-ALK+ cells, confirming that it has a different mechanism of action to that of crizotinib. Nor did metformin affect mTOR or AMPK, however, it did induce apoptosis and a G1 phase cell cycle arrest. Interestingly the mitochondrial membrane potential (Δψm) was reduced with metformin treatment, indicating that metformin is likely inducing mitochondrial dysfunction in cancer cells. An analogue of metformin, mito-metformin10, was shown to be far more cytotoxic and also reduced the Δψm at concentrations far less than that of metformin. To examine if crizotinib was limiting the entry of metformin into the cell, mass spectrometry was conducted. However, this experiment could not conclude whether the intracellular levels of metformin were reduced with crizotinib treatment, as the within experimental group variation was greater than that of the between group variation. This thesis demonstrated that metformin can produce anti-cancer effects in EML4-ALK+ lung cancer. Further experimentation should be conducted to confirm the new proposed hypothesis that metformin induces energetic stress in cancer cells due to an inhibition of mitochondrial complex I and reducing hepatogluconeogenesis, preventing glycolysis-induced ATP production. Nevertheless, this thesis supports the hypothesis that metformin may be a beneficial anti-cancer compound in EML4-ALK+ lung cancer