2014PIGF1R inhibition affects the survival of HT29 cancer cells by alterations of the TLR9- and autophagy signaling

Abstract Background In HT29 cells, an interplay between self-DNA-induced TLR9-signaling and autophagy response was found with significant effects on cell survival and kinetic parameters. The IGF/IGF1R system plays a determinant role in the pathogenesis and progression of colorectal cancer. This pathway is upstream of mTORC1, a negative regulator of autophagy. I mammalian systems chronic IGF1R inhibition was shown to attenuate autophagosome formation. The interrelated action of IGF1R inhibition and TLR9/autophagy signaling in cancer cells has not yet been clarified. The present study was designed to assess this complex network using HT29 colon carcinoma cells. Methods HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, IGF1R, mTORC, Akt, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM). Results In case of autophagy g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of mTORC, and Akt, verified by immunocytochemistry, as well. IGF1R-inhibition alone altered inversely the autophagy-associated gene- and protein-expressions. Upon combined inhibition of autophagy, TLR9 and/or IGF1R-signaling varying degree of autophagy was detected in all groups of tumor cells according to NanoString and TEM. Incubation with IGF1R inhibitor and with m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were found. Moreover, IGF1R inhibition affected negatively the cell-protective effect f-DNA on macroautophagy and lipophagy. Conclusions Our study provided evidence for an interaction between the inhibited IGF1R and TLR9/autophagy signaling with major influences on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. (Funded by StartUp.). Legal entity responsible for the study Ferenc Sipos. Funding StartUp. Disclosure All authors have declared no conflicts of interest.