Stress response through theCaMK2A/NRF2/GSTP1 axis strengthens cancer cell resilience and mediates stemness phenotype through ROS attenuation

Abstract

Lung cancer is the leading cause of cancer mortality. Adaptive responses to microenvironmental stresses are important survival mechanisms of cancer cells that lead to long term treatment failure, with accentuated resilience in cell subsets identified by cancer stem cell markers such as CD44 and ALDH. Reactive oxidative species (ROS) are harmful by-products of both cancer cell proliferation and treatment-induced cell death, the elimination of which is critical for maintaining cellular homeostasis and survival. We previously observed the calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) could enhance cancer stem cell (CSC) properties and attenuate ROS in lung cancer cells. To understand the molecular mechanisms, we focused on its relation to the master transcription factor for ROS regulation, NRF2, and the downstream pathways. To test if NRF2 could be a substrate activated by CaMK2A, total NRF2 proteins were measured in nuclear protein extracted from lung cancer cells. CaMK2A-overexpression (OE) increased, while shRNA-mediated CaMK2A-knockdown (shCaMK2A) reduced nuclear NRF2, indicating NRF2 is a mediator of CaMK2A. To identify downstream molecular pathways, screening for known NRF2 target genes was performed using qPCR. The results identified Glutathione S-transferase P1 (GSTP1), an anti-oxidant and detoxifying enzyme, was a target observed consistently in CaMK2A-OE and shCaMK2A cell lines. This finding was confirmed at the protein levels by western blot, supporting CaMK2A/NRF2/GSTP1 might be a candidate axis playing a supportive role for lung CSC. To obtain further support, we studied the expression levels of GSTP1 in 44 clinical lung cancers from our cohort and the TCGA database, respectively, which showed significant up-regulation in cancers compared to corresponding normal lung samples. Further investigations showed GSTP1 protein levels were higher in tumor-spheres than monolayers of five established lung cancer cell lines. Stable knockdown of GSTP1 led to significant reduction of tumor sphere formation, expression of CD44 and ALDH1, as well as mouse xenograft tumorigenicity. On the other hand, sensitivities to chemotherapy and mutant EGFR targeting drugs were increased. Thus, the in vitro and in vivo data supported that GSTP1 played a role in the maintenance of the stem cell-like phenotypic state in lung cancer. To assess if GSTP1 maintained the CSC phenotype through regulating oxidative stress, its effects on ROS levels was measured using cellROX deep red probes. In shGSTP1 cells, the ROS level was significantly raised while it was reciprocally reduced on GSTP1- OE. Furthermore, ROS challenge experiments using N-acetyl-L-cysteine (NAC) in shGSTP1 cell lines resulted in increased cell sphere formation. Together, the results support the CaMK2A/NRF2/GSTP1 axis increases cancer cell resilience through ROS elimination and enhancement of stem cell-like properties of lung carcinoma, indicating its potential role as a long-term treatment target.