Abstract 2263: Autophagy protects against Src tyrosine kinase inhibitor treatment in non-small cell lung cancer cells via a novel miR-106a/ULK1 pathway
Authors: Sacha I. Rothschild, Oliver Gautschi, Jasmin Batliner, Melanie Jost, Martin F. Fey, Mathias Gugger, Mario P. Tschan
Journal: Cancer Research (2012)

Abstract Autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Its role in tumorigenesis is still ambiguous. Low levels of basal autophagy are crucial in order to sustain cellular homeostasis thereby also contributing to tumor prevention, whereas autophagy induction upon a variety of anti-cancer treatments is often involved in resistance mechanism to therapy. Src tyrosine kinase inhibitors (TKIs) inhibit cell migration and invasion in non-small cell lung cancer (NSCLC) cells. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. We hypothesized that cytoprotective autophagy plays a role in resistance mechanisms against Src TKIs. We found a marked induction of autophagic activity in NSCLC cell lines (A549, H460, H1299) treated with two different Src TKIs (saracatinib, dasatinib) as measured by increased LC3-I to -II conversion, increased GFP-LC3 dot formation, and decreased p62/SQSTM1 protein expression. Most importantly, the addition of pharmacological autophagy inhibitors (chloroquine, bafilomycin) in combination with Src TKI inhibitors resulted in cell death as compared to only decreased migration by Src TKI treatment alone. Next, we identified ULK1 as a key player in Src TKI-induced autophagy using a set of lentiviral vectors expressing shRNAs targeting ATG genes. Src TKIs significantly induced ULK1 mRNA and protein expression and knocking down ULK1 significantly attenuated Src TKI-induced autophagy. Furthermore, to exclude Src inhibition-independent effects of saracatinib and dasatinib on ULK1 expression, we targeted Src by RNAi and found similar induction of ULK1 as seen with the Src TKI treatment. We further investigated if micro(mi)RNAs are involved in ULK1 regulation during Src inhibitor treatment. Using miRNA target identification software and 3′-UTR luciferase reporter assays we identified miR-106a as ULK1-targeting miRNA. Incubating NSCLC cells with Src inhibitors resulted in a dose-dependent decrease of miR-106a paralleled by an increase in ULK1 expression. Moreover, ectopic expression of miR-106a and anti-miR-106a in NSCLC cell lines caused decreased and increased ULK1 expression upon Src inhibition, respectively. Importantly, similar to the pharmacological autophagy inhibition and or knocking down ULK1, expression of miR-106a enhanced the cytotoxicity of Src TKIs. Lastly, we found significantly higher miR-106a levels in human lung adenocarcinoma compared to matched normal lung tissue (n=23), whereas ULK1 mRNA expression levels were significantly lower (p&amp;lt;0.0002) in tumor tissue than in normal lung tissue. In conclusion, Src inhibitors induce protective autophagy in NSCLC cells that is mediated via miR-106 and its target ULK1. Combining Src and autophagy inhibitors may represent attractive treatment option for certain NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2263. doi:1538-7445.AM2012-2263