Contribution by NRF2 upregulation to lung carcinogenesis, and the possible therapeutic value of NRF2 inhibition by GSK-3

In the UK about 41,000 individuals are diagnosed with lung cancer each year. It is the major cause of death from malignant disease worldwide. Therapeutic options for treating lung cancer are limited. It is now recognised that lung cancer patients ought to be treated in an individualized manner according to the genes that have been mutated and are responsible for development of their own tumour. In order to do this, biomarkers that reflect mutations in cancer-associated genes are required to guide clinicians in their choice of therapy. It has recently been discovered that mutations causing permanent activation of a master regulator of cellular antioxidant systems, called NRF2, are the 2nd and 4th most frequent events in the two major forms of lung cancer, but as NRF2 regulates many processes besides antioxidant systems it is uncertain why its upregulation might benefit the tumour (to the detriment of the patient). In this project we will identify mechanisms by which NRF2 upregulation aids development of lung cancer. We will also identify biomarkers associated with NRF2 that can be used by clinicians to identify tumours in which its upregulation contributes to the disease.

An important related question is that we do not know whether inhibiting NRF2 hinders development of lung cancer, and addressing this issue is frustrated by the lack of drugs that inhibit NRF2. We recently found NRF2 can be suppressed in lung cancer cells by switching on a kinase, called GSK-3, which triggers destruction of NRF2. In our experiments, we activated GSK-3 in cell lines using a drug called MK2206. We now want to use mice to test whether MK2206 can be employed to inhibit NRF2 within lung tumours in vivo.

The overarching objective of this grant is to assess whether the permanent activation of NRF2 can be exploited clinically in lung cancer.

The first aim is to establish how NRF2 upregulation aids lung cancer by examining whether it supports it by: 1) increasing antioxidants and preventing cell death caused by high levels of oxidants produced as byproducts of tumour-specific activities; 2) increasing the ability of chemicals in tobacco smoke to cause mutations by converting them to DNA-damaging derivatives; 3) increasing synthesis of cellular macromolecules by providing more NADPH; 4) increasing cellular activity by providing more energy in the form of ATP.

The second aim is to identify specific genes, small molecules and proteins within lung tumours that are altered as a consequence of NRF2 upregulation to explain how it aids lung tumour development, by using state-of-the art technologies to examine lung tumours created genetically in mice with incrementally varied levels of NRF2 [from none to large increases]. We will then utilize changes in these molecules as biomarkers of NRF2-directed processes that facilitate cancer.

The third aim is to establish the clinical prognostic significance of the increase in NRF2 and the biomarker proteins it controls using antibodies to probe a well-characterized panel of human lung tumours.

The fourth aim is to test in a mouse lung cancer model whether suppression of NRF2 by MK2206 decreases growth of lung tumours.

This grant will increase our understanding of how NRF2 contributes to the development of lung cancer. It is of direct relevance to patients with lung cancer as identification of tumours in which NRF2 is upregulated will help predict the outcome of disease. Moreover, the ability to identify lung tumours with upregulated NRF2 and overexpression of its target genes will provide biomarkers for the personalized treatment of cancer that will be of great value to clinicians treating lung cancer patients. The grant will also allow us to test if pharmacological suppression of NRF2 using MK2206 inhibits growth of tumours lacking KEAP1. Importantly, MK2206 is already in clinical trials for lung cancer, providing the exciting prospect that our findings might be translated rapidly into the clinic.

Lung cancer is the most common cause of death from malignant disease worldwide. Mutations that cause constitutive upregulation of the antioxidant transcription factor NRF2 are extremely common in lung cancer, but it is not known why this is the case. Thus the objectives of this grant are to determine why NRF2 is upregulated in a large proportion of lung tumours, and to examine whether suppression of NRF2 by activating its negative regulator GSK-3 inhibits lung tumourigenesis.

We will use phosphospecific antibodies to define the molecular mechanisms by which activation of GSK-3 suppresses NRF2, and will test in a human lung cancer cell line whether upregulation of NRF2 supports tumourigenesis by inhibiting apoptosis, increasing mutagenesis (arising from activating chemical carcinogens in cigarette smoke), and/or increasing production of NADPH and ATP for synthesis of macromolecules. Next we will use a lung cancer experimental model on 5 different genetic backgrounds in order to provide widely divergent and incremental increases in NRF2 activity (from null to high) in order to determine the impact of variation in NRF2 activity on lung carcinogenesis, and to identify biomarkers associated with its ability to support cancer. We will then translate these findings into man by examining the prognostic significance of NRF2 upregulation and associated biomarkers in large, clinically annotated, training and separate validation cohorts of 250 lung cancer cases collected between 2000-2006 with known outcomes. Lastly, we will examine whether suppression of NRF2, by activating GSK-3 with MK2206, decreases lung tumour burden in mice with high NRF2 levels.

This project will provide new mechanistic insight into how NRF2 supports lung cancer. Further, the results will enable NRF2-based stratification of patients with lung cancer for therapy. It will also allow us to test whether the drug MK2206, which activates GSK-3, is best targeted at patients with upregulation of NRF2.

Lung cancer patients and the NHS, as well as Biotechnology diagnostics companies, and the Pharmaceutical industry will all benefit from our proposed research.

Lung cancer patients represent a very sizable group that will benefit from our research, as evidenced by the fact that approximately 41000 new cases of lung cancer are diagnosed in the UK each year. There are currently few therapeutic options for patients with lung cancer. However, as between 25% and 35% of lung tumours possess permanently upregulated NRF2, it may be possible to sensitize a significant proportion of lung tumours to anticancer drugs by inhibiting NRF2. Before such NRF2-based personalized treatment of lung cancer can be considered, biomarkers that reflect upregulation of NRF2 in the tumour are required. Within the duration of the grant, the proposed research is of potential clinical importance for those in the NHS responsible for managing patients with lung cancer because it will provide a means by which tumours with upregulated NRF2 can be identified; this represents a first step that allows the prognostic significance of its upregulation to be assessed, as well as stratification of patients for therapy. If NRF2 upregulation is found to be of prognostic value, it is envisaged that within 2-5 years the NRF2-associated biomarkers could be exploited to establish if high NRF2 activity segregates with responsiveness to MK2206 because this drug is already in clinical trials to treat non-small cell lung cancer. Again in the near future (2-5 years), our research will be of benefit to those working in the NHS because it should allow lung cancer patients to be stratified into a group with tumours that overexpress NRF2, which appears to occur more frequently than the currently used biomarkers used to affect clinical practice in lung cancer, and should therefore benefit from adjuvant therapy that includes a GSK-3 activator to suppress NRF2 (e.g. MK2206) and a cytotoxic anticancer drug against which NRF2 confers resistance.

The commercial private sector, Biotechnology diagnostics and the Pharmaceutical industry, will benefit from our research in several ways. Firstly, our identification of NRF2-associated biomarkers that synergize with oncogenes such as KRAS to promote lung carcinogenesis will allow antibodies to be generated that could be used for diagnostic purposes and the selection of appropriate therapy. We therefore propose that the antibodies we generate to identify NRF2-associated biomarkers should be made available for academic research through collaboration with MRCT (MRC Technology). Secondly, should NRF2-associated proteins be shown to aid promotion of tumourigenesis, these would then represent possible drug targets; an example would be inhibition of members of the aldo-keto reductase 1C family to block their putative ability to initiate cancer by creating redox-active quinones that can damage DNA. Thirdly, a number of Pharmaceutical companies, including AstraZeneca, have programmes aimed at developing NRF2 inhibitors, and they will be interested in the biochemical and clinical data produced by this project.

Staff employed on this project grant will gain skills in general molecular cell biology techniques, GSK-3 related pharmacology, mouse cancer models, redox signaling, oncogene actions, gene expression profiling, metabolomics, proteomics, biomarker discovery, construction of human tissue microarrays, immunofluorescence, AQUATM software, and bioinformatics (TMANavigator.org), all of which are likely to be highly valued by the pharmaceutical industry and would therefore increase their employment prospects. The BBSRC CASE student currently in the lab of JDH, who is sponsored by AstraZeneca on an NRF2-related project, would also benefit from the research project through association with the staff to be employed.