Abstract 189: KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer

Abstract

Abstract EGFR tyrosine kinase or downstream components of this pathway, e.g. KRAS, are frequently mutated in NSCLC patients. These mutations enable a constitutive active downstream signaling pathway and result in lung cancer development by uncontrolled proliferation and survival. MicroRNAs (miRNAs) are noncoding short nucleic acids, and are implicated in NSCLC progression. We aim to identify miRNAs that are regulated by EGFR downstream key molecules and to investigate their role in malignant processes of NSCLC. We demonstrate that viral transduction of bronchial epithelial cells (BEAS-2B) with mutated KRAS induces an activated downstream signaling pathway. Subsequently, a miRNA array profiling of KRAS-transduced cells identified upregulated miR-29b expression. We confirmed the upregulation of miR-29b expression in individual clones of KRAS-transduced cells. Moreover, the upregulated miR-29b expression was affirmed in NSCLC tissue harboring KRAS mutations by comparing it to the expression in matched normal tissue. Furthermore, to elucidate the downstream signaling pathway involved in the underlying regulation of miR-29b expression, NSCLC cell lines were treated with EGFR and MEK inhibitors which lead to downregulated miR-29b expression. Next, to study the role of miR-29b in NSCLC apoptosis, we overexpressed miR-29b in adenocarcinoma cells (A549) and performed cell death assays. Interestingly, and in contrast to other cell systems, A549 cells transfected with precursor miR-29b demonstrated attenuated TNF-α plus actinomycin D-induced apoptosis compared to control-transfected cells. Conversely, the downregulation of endogenous miR-29b levels by overexpressing antagomiR-29b, lead to increased sensitivity to apoptosis compared to scramble-transduced A549 cells. Consistently, KRASG12V-transduced BEAS-2B cells showed reduced sensitivity to TNF-α plus actinomycin D-induced apoptosis, while the effect was reversed in KRAS/antagomiR-29b co-transduced cells, indicating that KRAS attenuates apoptosis by overexpressing miR-29b. Furthermore, miR-29b is predicted to target the pro-apoptotic BH3-only protein Bim and the tumor necrosis alpha-induced protein 3 (TNFAIP3/A20), an important negative regulator of the NFκB signaling pathway and thereby may confer resistance to apoptosis. We plan to verify the predicted regulation of these genes by performing luciferase reporter assays and Western blot analysis. In addition, NFκB activation will be assessed by verifying nuclear and cytoplasmic fractions of cells transfected with precursor miR-29b. Finally, we plan to stably overexpress TNFAIP3 in NSCLC cell lines and assess apoptosis by overexpressing miR-29b. In summary, we provide a novel regulation mechanism of miR-29b by KRAS signaling, leading to attenuated apoptosis in NSCLC. Citation Format: Stephanie Langsch, Stefan Haemmig, Ulrich Baumgartner, Mario P. Tschan, Erik Vassella. KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 189. doi:10.1158/1538-7445.AM2015-189