Exploring the role of NRF2 and circadian cycle as mediators of tumor resistance to chemotherapy

Abstract

Cancers figure among the leading causes of morbidity and mortality worldwide. The main limiting factor for cancer treatment failure is antitumoral therapy resistance. Several mechanisms command drug resistance and many of them can be tissue or drug specific. As the different pathways might proportionally contribute to tumor chemoresistance, it is fundamental to identify the key molecular regulator of those resistance factors, aiming to target it in order to achieve a better therapeutic efficacy. Glioma and melanoma are particularly aggressive and severe types of tumor. Temozolomide (TMZ) and cisplatin are among the most used chemicals to treat glioma and metastatic melanoma. However, chemotherapy has limited success with resistance development, and as consequence those types of tumor remain incurable. In this proposal, we intent to investigate the TMZ and cisplatin resistance mechanism mediators on both glioma and melanoma cell lines. We hypothesize that the antioxidant transcriptional factor NRF2 and circadian cycle may be central mediators for TMZ and cisplatin resistance. Thus, first we are going to generate cell lines knockout for NRF2 gene (NRF2-KO) and NRF2 constitutively activated (NRF2-Act) using CRISPR/Cas9 genome editing system. On those cell lines, we plan to evaluate, using both in vitro and in vivo models, important drug resistance mechanisms such as DNA repair capacity, glutathione intracellular level and apoptosis induction upon TMZ and cisplatin treatment. We also plan to investigate the role of circadian cycle on cell survival in cells treated with chemotherapy. For this part of the project, we will generate tumor cell lines expressing clock genes fused to reporter genes in order to easily monitor and control circadian cycle. These cell lines will be employed to analyze several TMZ and cisplatin resistance mechanisms and evaluate whether those factors are regulated by circadian cycle. Cutting edge molecular tools and unique in vivo models will be employed, as a consequence an intense international collaboration network with research groups from Massachusetts Institute of Technology, Cambridge, MA, USA, and Erasmus University, Rotterdam, The Netherlands, with DNA repair and circadian cycle experts, respectively. Ultimately, as consequence of the success of this proposal, we hope we will be able to design and implement more adequate and efficient protocols for clinical application to treat glioma and melanoma patients, aiming at improved chemotherapeutic efficacy and less adverse side effects. Thus, this proposal will contribute to improve our knowledge concerning drug resistance in glioma and melanoma cells what, in turn, will fundament future clinical trials to treat those patients. (AU)