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

在英国，每年约有4.1万人被诊断出患有肺癌。它是全世界恶性疾病致死的主要原因。治疗肺癌的治疗选择有限。现在人们认识到，肺癌患者应该根据突变的基因以个性化的方式进行治疗，这些基因对他们自己的肿瘤的发展负有责任。为了做到这一点，需要反映癌症相关基因突变的生物标记物来指导临床医生选择治疗。最近发现，导致细胞抗氧化系统的主要调节因子NRF2永久激活的突变，是两种主要形式肺癌中第二和第四常见的事件，但由于NRF2除了调节抗氧化系统外，还调节许多过程，因此尚不确定为什么它的上调可能有利于肿瘤(对患者不利)。在这个项目中，我们将确定NRF2上调促进肺癌发生的机制。我们还将识别与NRF2相关的生物标记物，临床医生可以用来识别其上调导致疾病的肿瘤。一个重要的相关问题是，我们不知道抑制NRF2是否会阻碍肺癌的发展，而解决这个问题是因为缺乏抑制NRF2的药物。我们最近发现，在肺癌细胞中，可以通过激活一种名为GSK-3的激酶来抑制NRF2，这种激酶可以触发NRF2的破坏。在我们的实验中，我们使用一种名为MK2206的药物激活了细胞系中的GSK-3。我们现在想用小鼠来测试MK2206是否可以用来在活体内抑制肺癌内的NRF2。这项拨款的首要目标是评估NRF2的永久激活是否可以用于肺癌的临床应用。第一个目标是确定NRF2上调是如何通过以下方式支持肺癌的：1)增加抗氧化剂，防止由肿瘤特异性活动产生的高水平氧化剂引起的细胞死亡；2)通过将烟草烟雾中的化学物质转化为DNA损伤的衍生物，增加它们引起突变的能力；3)通过提供更多的NADPH来增加细胞大分子的合成；4)通过以ATP的形式提供更多的能量来提高细胞活性。第二个目标是识别肺肿瘤中因NRF2上调而改变的特定基因、小分子和蛋白质，以解释它如何帮助肺癌的发展，方法是使用最先进的技术来检查NRF2水平逐渐变化的小鼠中遗传产生的肺癌[从无到大幅增加]。然后，我们将利用这些分子的变化作为NRF2指导的促进癌症过程的生物标记物。第三个目标是使用抗体来探测一组具有良好特征的人类肺癌，以建立NRF2及其控制的生物标记物蛋白增加的临床预后意义。第四个目标是在小鼠肺癌模型中测试MK2206抑制NRF2是否会减少肺癌的生长。这项拨款将增加我们对NRF2如何促进肺癌发展的理解。它与肺癌患者有直接关系，因为识别NRF2上调的肿瘤将有助于预测疾病的结果。此外，识别NRF2上调及其靶基因过度表达的肺肿瘤的能力将为癌症的个性化治疗提供生物标志物，这将对临床治疗肺癌患者具有重要价值。这笔赠款还将允许我们测试使用MK2206对NRF2进行药物抑制是否可以抑制缺乏Keap1的肿瘤的生长。重要的是，MK2206已经在肺癌的临床试验中，这为我们的发现可能迅速转化为临床提供了令人兴奋的前景。

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.