Abstract
ABSTRACTMacroautophagy/autophagy inhibition is a novel anticancer therapeutic strategy, especially for tumors driven by mutant RAS. Here, we demonstrate that autophagy inhibition in RAS-mutated cells induces epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor invasion. This is at least partially achieved by triggering the NFKB/NF-κB pathway via SQSTM1/p62. Knockdown of ATG3 or ATG5 increases oncogenic RAS-induced expression of ZEB1 and SNAI2/Snail2, and activates NFKB activity. Depletion of SQSTM1 abolishes the activation of the NFKB pathway induced by autophagy inhibition in RAS-mutated cells. NFKB pathway inhibition by depletion of RELA/p65 blocks this EMT induction. Finally, accumulation of SQSTM1 protein correlates with loss of CDH1/E-cadherin expression in pancreatic adenocarcinoma. Together, we suggest that combining autophagy inhibition with NFKB inhibitors may therefore be necessary to treat RAS-mutated cancer.Abbreviations: 4-OHT: 4-hydroxytamoxifen; DIC: differential interference contrast; EMT: epithelial-mesenchymal transition; ESR: estrogen receptor; MAPK/ERK: mitogen-activated protein kinase; iBMK: immortalized baby mouse kidney epithelial cells; MET: mesenchymal-epithelial transition; PI3K: phosphoinositide 3-kinase; RNAi: RNA interference; TGFB/TGF-β: transforming growth factor beta; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6.
Highlights
Autophagy is an evolutionarily conserved biological process that degrades long-lived proteins and cytoplasmic organelles
These data suggest that epithelial-mesenchymal transition (EMT) is induced in autophagydeficient RAS-mutated tumors in vivo. These results demonstrate that autophagy inhibition is able to activate the EMT program, in RASmutated cancer cells, which is supported by the induction of EMT transcription factors and, a reduction in CDH1 expression
To investigate whether RAS mutational status influences the effect of autophagy in regulating EMT, we used RNA interference (RNAi) to deplete ATG5, an important component of the autophagic machinery, in 7 human cancer cell lines derived from the pancreas (PaCa3, Suit-2, PANC1 and MDA Panc3) and colon (HCT116, HKe3 and HKh2)
Summary
Autophagy is an evolutionarily conserved biological process that degrades long-lived proteins and cytoplasmic organelles. Oncogenic RAS mutations, which activate both the RAFMAPK/ERK (mitogen-activated protein kinase) and phosphoinositide 3-kinase (PI3K)-AKT signalling pathways, are among the most prevalent genetic changes found in human cancers, occurring in approximately 20% of human tumors [13,14]. Yang and colleagues demonstrated that the inhibition of autophagy in pancreatic tumor cells suppressed their growth by reducing the production of reactive oxygen species, limiting effective metabolism via decreased mitochondrial oxidative phosphorylation and increased DNA damage [8]. This finding provides an example of a positive role of autophagy in pancreatic tumorigenesis [8]. There are several phase I/II clinical trials in progress using the autophagy inhibitors chloroquine or hydroxychloroquine in combination with chemotherapy for the treatment of a range of tumors, including pancreatic cancer [15]
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