A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRAS G12V and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non-small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRAS G12V

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage.

microRNAs (miRNAs), short regulatory sequences at the posttranscriptional level, are important mediators of signaling pathways that act as backups of transcriptional control. To identify miRNAs implicated in epidermal growth factor receptor (EGFR) signaling, transformed bronchial epithelial BEAS-2B cells were retrovirally transduced with KRASG12V and alterations in miRNA expression were assessed by microarray analysis. Here we show that miR-29b is significantly induced by mutant KRAS in bronchial epithelial and non-small cell lung cancer (NSCLC) cell lines as well as in primary NSCLC tissue. In agreement with these results, inhibitors of EGFR and MEK resulted in reduced levels of miR-29b while inhibitors of PI3K had no effect. KRASG12V-transduced BEAS-2B cells were significantly more protected from extrinsic apoptosis than control transduced cells, but co-transduction of cells with KRASG12V and anti-miR-29b constructs sensitized cells to apoptosis indicating that miR-29b is a mediator of KRAS-induced resistance to apoptosis. Protection from extrinsic apoptosis was due to enhanced nuclear factor κB (NF-κB) activity. The ubiquitin-editing enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) is a negative regulator of NF-κB signaling. Enhanced NF-κB activity elicited by miR-29b was due to targeting TNFAIP3/A20. Overexpression of miR-29b-refractory TNFAIP3 restored NF-κB activity as well as extrinsic apoptosis, demonstrating that TNFAIP3 is a relevant target of miR-29b. Interestingly, miR-29b conferred sensitivity to cisplatin-induced intrinsic apoptosis by targeting Mcl-1. Thus, miR-29b tips the balance from extrinsic apoptosis towards intrinsic apoptosis. Our results indicate that miR-29b can act either as an oncogene or tumor suppressor gene depending on the external stimulus. Citation Format: Erik Vassella, Stephanie Langsch. miR-29b acts as an oncogene or tumor suppressor gene depending on the external stimulus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1107.

<p>Supplementary Fig. 1: Ectopic PIK3CAH1047R expression induces a modest activation of the EGFR downstream signaling pathway in BEAS-2B clones. Supplementary Fig. 2: the steady state level of miR-29b and the relative copy number of the miR-29b2 locus are significantly correlated in KRAS/EGFR wildtype tumors. Supplementary Fig. 3: miR-138 expression is not induced by mutant KRAS or mutant EGFR in adenocarcinoma tissues. Supplementary Fig. 4: Inhibitors of the EGFR signaling pathway downregulate expression of key downstream effectors. Supplementary Fig. 5: miR-29b confers resistance to TNFalpha+ActD-induced apoptosis. Supplementary Fig. 6: TNFAIP3 is a target of miR-29b. Supplementary Fig. 7: miR-29b sensitizes A549 cells to cisplatin-induced apoptosis.</p>