Abstract B30: Src tyrosine kinase inhibitors activate protective, ULK1-dependent autophagy by downregulating oncomir-106-393-cluster expression in non-small cell lung cancer cells

Abstract

Abstract Autophagy has been proposed to play a role as cytoprotective mechanism for tumor cell survival under unfavorable conditions and upon anticancer treatment. In lung cancer autophagy is mostly studied as possible resistance mechanism to therapy. There is increasing evidence that protective autophagy occurs upon various types of anti-cancer treatments, including chemo- and radiation therapy. Src tyrosine kinase inhibitors (TKIs) have been shown to inhibit cell migration and invasion in non-small cell lung cancer (NSCLC) cell lines. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. In the current study we show a marked induction of autophagic activity upon incubation of NSCLC cell lines with Src TKIs as measured by an increased LC3-I to -II conversion, a significant increase in GFP-LC3 dot formation, and a decrease in p62/SQSTM1 protein expression. Increased autophagic activity was found in three NSCLC cell lines (A549, H460, H1299) with two different Src TKIs (saracatinib, dasatinib). Interestingly, the addition of pharmacological autophagy inhibitors such as chloroquine or bafilomycin resulted in cell death in combination with Src TKI treatment. Moreover, we found that Src TKI-induced autophagy is associated with ULK1 expression in all three cell lines investigated. This effect was Src-specific since knocking down endogenous Src using RNAi resulted in a similar induction of ULK1. Using shRNA targeting ULK1 we showed that Src inhibitor induced autophagy is ULK1-dependent. Furthermore, ULK1 is a novel target of microRNA (miR)-106a as shown by ULK1 3′-UTR luciferase experiments and ectopic expression of miR-106a and antimiR-106a in NSCLC cell lines resulting in decreased and increased ULK1 expression upon Src inhibition, respectively. Lastly, in human lung adenocarcinoma compared to matched normal lung tissue (n = 23) miR-106a levels were significantly increased (p<0.0001) whereas ULK1 mRNA expression levels were significantly lower (p<0.0002) in tumor tissue. In conclusion, Src inhibitor-induced protective autophagy might explain their low success in clinical trials. Autophagy induced by Src TKIs depends on ULK1 and combining Src with autophagy inhibitors results in massive cell death as compared to single treatments. Furthermore, downregulation of the ULK1 targeting miR-106a upon Src inhibition allows for the induction of protective autophagy. Combining Src and autophagy inhibitors or Src inhibitors and miR-106a expression may represent attractive treatment options for NSCLC.