Lung cancer resilience and stem cell phenotypes are enhanced through the CaMKIIα/NRF2/GSTP1 axis

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide. Cancer stem cells (CSC) constitute a small subset of cancer cells that enhance tumorigenesis, metastasis and drug resistance. Glutathione S-transferase pi (GSTP1) is an enzyme that catalyzes glutathione (GSH) conjugation, mediates cellular elimination of chemicals and toxic compounds, and has been reported to induce cancer drug resistance. However, its role in other cancer functions and CSC phenotypes has seldom been studied and not reported in lung cancers. It was observed GSTP1 mRNA was significantly up-regulated in lung cancers compared to normal lung in local patients, and correlated with poor outcome in patients of the TCGA database, indicating its potential carcinogenic role. Hence, this study aimed to investigate the role and regulation of GSTP1 in lung cancer. Results of genetic manipulation of GSTP1 expression showed GSTP1 played a supportive role in CSC phenotypes of lung cancer cells, including the ALDH+/CD44+-CSC proportions, in vitro self-renewal, in vivo tumorigenesis, cell motility and responses to both chemotherapy and targeted therapy. These CSC regulatory functions were attributed to suppression of reactive oxygen species (ROS), evidenced by the restoration of tumorsphere formation and reversal of multiple drug resistance by the introduction of the antioxidant NAC to GSTP1-knockdown cancer cells. Previous work in this laboratory has demonstrated the calcium signaling mediator CaMKIIα could maintain CSC phenotypes. Since both CaMKIIα and GSTP1 are involved in cellular stress reactions, the postulation that these two molecules might form a functional pathway was studied. Indeed, in cells with CaMKIIα overexpression, GSTP1 knockdown significantly reduced tumorspheres, the ALDH+/CD44+-CSC population and cisplatin resistance; reciprocally, concurrent CaMKIIα knockdown and GSTP1 overexpression significantly restored self-renewal and tumorigenesis both in vitro and in vivo, indicating CaMKIIα and GSTP1 were biochemically connected in serving their CSC supportive roles. To query how the two formed a functional axis, the candidate pathway CaMKIIα-NRF2-GSTP1 involving the ROS regulatory transcription factor NRF2 was tested. First, up-regulation of CaMKIIα could significantly promote NRF2 nuclear translocation. Next, NRF2 binding sequences were identified in the GSTP1 gene and their transcriptional capacities were demonstrated by dual luciferase assays. Last, direct interaction between CaMKIIα and NRF2 was demonstrated using the BiFC assay in which corresponding fluorescence was emitted upon the combination of the 2 differentially labeled proteins. Together, the experiments supported the presence of the CaMKIIα-NRF2-GSTP1 axis in supporting lung CSC. The above findings implicated the CaMKIIα-NRF2-GSTP1 cascade might provide a stress response pathway to maintain CSC plasticity. To substantiate this hypothesis, the effect of hypoxia on lung cancer cell self-renewal and tumorigenicity, and the involvement of CaMKIIα/GSTP1 was verified. Hypoxia stimulated calcium influx into cancer cells and activated the CaMKIIα/GSTP1 axis, which could be inhibited by adding the antioxidant NAC. The findings implicated the CaMKIIα-NRF2-GSTP1 pathway could support hypoxia-induced CSC phenotypes. Together, this study has unveiled the functions of GSTP1 in supporting cancer stem cell phenotypes and indicated GSTP1 might be a novel therapeutic target for lung cancer. (483 words)