Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma

Abstract

Lung cancer is the most lethal of all cancers world-wide and the majority of patients require chemotherapy or targeted therapy at presentation. Survival is compromised by drug resistance through multiple mechanisms such as by-pass mutations, micro-environment activation of survival programs and induction of tumor initiating cells (TIC). Induction of TIC phenotypes could be accompanied by upregulation of embryonic or developmental pathways but molecular mechanisms are incompletely understood. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) is a multifunctional serine/threonine kinase involved in growth and stress signals integration. A tumor-supportive role has been reported in leukaemia and thyroid cancer but reports on its involvement in TIC regulation are limited. We investigated the role and molecular mechanism of CaMK2A in lung cancer TIC regulation using in vitro and in vivo models. The results showed CaMK2A was overexpressed in lung cancer cell lines and human lung adenocarcinomas compared to normal lung. In cancer cells with stable CaMK2A-knockdown, in vitro reduction in tumor spheres formation, anchorage independent growth and increased reactive oxygen species, as well as in vivo reduction of xenograft tumorigenicity were observed. The opposite effects were observed in cancer cells with stable CaMK2A over-expression. Further, specific pharmacological inhibition of CaMK2A by KN93 led to significantly reduced IC50 for gefitinib in lung cancer cells harboring activating EGFR mutation (HCC827) and in HCC827 exogenously induced for gefitinib resistance. In contrast, IC50 of cisplatin treatment was increased in cancer cells with genetically-induced CaMK2A over-expression. Moreover, CaMK2A knockdown led to reduced mRNA expressions of pluripotency genes (SOX2, NANOG, OCT4) and TIC markers (ALDH, CD166). These results suggested CaMK2A is involved in lung tumorigenicity through TIC regulation. To further investigate the molecular mechanism, we showed tumorigenicity was restored in xenografts with CaMK2A knockdown rescued with SOX2 over-expression. In CaMK2A knockdown cells, reduced SOX2 expression was associated with increased H3K27me3 repressive histone mark, which has not been reported in the literature. Taken together, we have shown CaMK2A plays a role in lung adenocarcinoma and TIC maintenance through histone modification and regulation of SOX2 expression. Targeting TIC through CaMK2A modulation might be useful for overcoming drug resistance and improving long term lung cancer survival.