Project summary: Therapy resistance is one of the biggest problems currently facing clinical oncology. Emerging evidence indicates that the amino acid derivative L-carnitine and its transporter OCTN2 protects tumours from cell death through the synthesis of NADPH, the scavenging of free radicals, stabilisation of membranes, and promotion of antiapoptotic pathways. Here, we will develop novel molecular imaging agents for the assessment of carnitine utilisation in mouse models of lung cancer. The aims of the project are as follows:
-Assess carnitine uptake and metabolism in drug-sensitive and -resistant lung cancer. -Determine the affinity and specificity of fluorinated carnitine analogues for OCTN2 imaging using OCTN2 wild-type, knockout and overexpressing tumour cells (CRISPR-Cas9). Select a lead candidate for imaging (18F-carnitine). -Study 18F-carnitine pharmacokinetics, tumour uptake and radiotracer stability in mice. -Can 18F-carnitine measure tumour antioxidant capacity in cells following treatment with oxidising agents, antioxidants and chemotherapy? -Is 18F-carnitine a predictor of drug resistance in lung tumours?
Project description: Lung cancer is the most common cause of cancer death world-wide (~1.6 million deaths/year), with a ten-year survival rate of just 5%. This poor prognosis is the result of the ineffective treatment of patients with drug-resistant disease. Currently, there is no satisfactory way to identify patients that will not respond to standard-of-care treatments. Positron emission tomography (PET) imaging offers a potential solution to this clinical problem through the non-invasive assessment of molecular processes that underpin therapy-resistance. The identification of cancer patients that are refractory to treatment will allow the selection of second-line therapies that have the potential to improve patient response and survival.
The Molecular Imaging Group at King’s (Witney) is an interdisciplinary team of imaging scientists (comprising cancer biologists, synthetic and radiochemists, and clinical oncologists) focused on the development of novel PET radiotracers to image cancer drug resistance. Emerging evidence has suggested that the amino acid derivate L-carnitine (LC), which is transported into the cells by the Na+-dependent transporter OCTN2 (SLC22A5), is a strong antioxidant thought to be indispensable in therapy-resistant tumours. LC play an essential role in the transport of long-chain fatty acids across the inner mitochondrial or peroxisomal membrane for β-oxidation. Importantly, LC directly contributes to reactive oxygen species (ROS) scavenging, membrane stabilisation, antioxidant enhancement, and promotes anti-apoptotic pathways [1, 2]. Furthermore, High OCTN2 expression is associated with a poor overall patient survival .
For this doctoral thesis project, we will develop the first OCTN2/LC radiotracer to probe the molecular mechanisms of LC-induced therapy resistance. We hypothesise that therapy-resistant tumours will display elevated carnitine utilisation to neutralise drug-induced ROS and therefore survive treatment. The successful candidate will characterise a wide panel of human and mouse lung cancer lines with different driver mutations in terms of OCTN2 expression (western blotting), intracellular LC (NMR spectroscopy) and rate of fatty acid oxidation (metabolomics). Metabolic features associated with LC will be compared to differential sensitivity to cisplatin, the first-line treatment for lung cancer. Through gene editing (CRISPR/Cas9), tumour cell response to therapy will be assessed in OCTN2 knockout cells and following its overexpression.
To assess LC utilisation we will develop novel radiolabelled LC analogues. Cell uptake of lead candidate LC radiotracers will be measured in our panel of lung cancer cell lines. Radiotracer candidates with good target selectivity and high tumour uptake will be imaged in mice bearing orthotopic lung tumours. PET imaging studies will be performed dynamically to assess radiotracer pharmacokinetics. Following imaging, urine, plasma, liver and tumour tissue will be excised, homogenised and extracted in methanol to analyse radiotracer stability by radioHPLC . The specificity of tumour radiotracer retention will be evaluated in isogenic tumours with OCTN2 gene knockout. Finally, we will assess the ability of our lead candidate to predict therapy resistance in advanced mouse models of lung cancer. Together, this project will deliver a novel radiotracer for the prediction and monitoring of drug resistance in lung cancer.
Only home UK or EU/EEA candidates fulfilling the 3-year UK residency requirement are eligible for the EPSRC DTP studentships. EU/EEA applicants are only eligible for a full studentship if they have lived, worked or studied in the UK for 3 years prior to the funding commencing.
1. Li, J.L., Q.Y. Wang, H.Y. Luan, Z.C. Kang, and C.B. Wang, Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha. J Biomed Sci, 2012. 19: p. 32. 2. Pike, L.S., A.L. Smift, N.J. Croteau, D.A. Ferrick, and M. Wu, Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. Biochim Biophys Acta, 2011. 1807(6): p. 726-34. 3. Fink, M.A., H. Paland, S. Herzog, M. Grube, S. Vogelgesang, K. Weitmann, A. Bialke, W. Hoffmann, B.H. Rauch, H.W.S. Schroeder, and S. Bien-Moller, L-Carnitine-Mediated Tumor Cell Protection and Poor Patient Survival Associated with OCTN2 Overexpression in Glioblastoma Multiforme. Clin Cancer Res, 2019. 25(9): p. 2874-2886. 4. Pereira, R., T. Gendron, C. Sanghera, H.E. Greenwood, J. Newcombe, P.N. McCormick, K. Sander, M. Topf, E. Arstad, and T.H. Witney, Mapping Aldehyde Dehydrogenase 1A1 Activity using an [(18) F]Substrate-Based Approach. Chemistry, 2019. 25(9): p. 2345-2351.
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