w09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway

Abstract

The EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

Targeting EGFR‐mediated autophagy as a potential strategy for cancer therapy

“…This implies that upregulation of autophagy by inhibition of EGFR could augment cell death under stress. However, our recent work demonstrated that active EGFR‐mediated RAS/RAF1/MAP2K/MAPK1/3 signaling pathway could contribute to increase autophagy‐dependent apoptosis in gastric cancer cells . Fulda and Kögel also reported that Obatoclax enhanced breast carcinoma cell death induced by lapatinib through suppressing the EGFR/AKT/mTOR signaling and enhanced autophagy …”

“…Moreover, there is extensive cross‐talk between autophagy and other forms of cell death, such as apoptosis and necrosis . Therefore, the effect of autophagy on cell death may also depend on its cross‐talk with apoptotic and/or necrotic pathways …”

“…The RAS/RAF/MEK/ERK cascade evidently regulates the activity of many proteins involved in apoptosis and autophagy . Depending on the duration, the magnitude and its subcellular localization, ERK activation controls various cell responses, such as proliferation, migration, differentiation and death .…”

“…In T‐cell acute lymphoblastic leukemia cells, resveratrol was further proven to induce autophagy through inhibiting AKT/mTOR pathway and activating the RAS/RAF/MEK/ERK pathway with apoptosis . We further identified a 2‐amino‐nicotinonitrile compound (w09), a novel autophagy inducer which through induction of the RAS/RAF/MEK/ERK pathway effectively induces an autophagy‐dependent apoptosis in EGFR‐dysregulated gastric cancer cells . Indeed, in autophagic cell death induced in human glioma cells by tetrahydrocannabinol (THC), an active component of cannabinoids, the AKT/mTOR signaling was inhibited and RAS/RAF/MEK/ERK pathway induces with apoptosis shown to be downstream autophagy .…”

“…Therefore a mechanism of autophagy induction should be that which ensures persistently high levels of autophagy in that specific tumor. High expression of proteins in the EGFR/RAS/RAF/MEK/ERK pathway in corresponding activating mutated tumors ensures this high level after exposure to the small molecule that targets this pathway . Development of autophagy inducers specific for the EGFR/RAS/RAF/MEK/ERK pathway mutations is specially critical as activating mutations in this pathway accounts for a large percentage of cancers.…”