In HT29 colon cancer cells a close interplay between self-DNA-induced TLR9-signaling and autophagy response was found with remarkable effects on cellular survival and differentiation. Pathogenesis and progression of colorectal cancer is deeply influenced by the insulin-like growth factor 1 receptor (IGF1R) pathway. This system is upstream of mTORC1, a negative regulator of autophagy. Chronic IGF1R inhibition was shown to attenuate autophagosome formation in mammalian systems. The interrelated role of IGF1R inhibition and TLR9/autophagy signaling in cancer cells has not yet been clarified. Our present study was designed to assess this complex interplay using HT29 cells.

HT29 cells were incubated for 72 h with intact genomic (g), arteficially hypermethylated (m), and fragmented (f) tumoral self-DNAs, with or without IGF1R inhibitor (picropodophyllin), autophagy inhibitor (chloroquine) and TLR9-inhibitor (ODN 2088), respectively. Cell viability was measured using alamarBlue. Transcriptional changes of TLR9-signaling and the autophagy process were assayed by Human v3 mRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry. Morphologic features of apoptosis and autophagy were examined by transmission electron microscopy (TEM).

Regarding autophagy g- and f-DNAs caused significant upregulation of Atg16L1, Beclin1, and LC3 mRNAs, and downregulation of mTORC, and Akt as verified by immunocytochemistry. IGF1R-inhibition in alone altered inversely the autophagy-associated gene- and protein-expressions. By using combined inhibition of autophagy, TLR9 and/or IGF1R-signaling according to NanoString and TEM varying degree of autophagy was observed in each group of tumor cells. In case of simultaneous incubation with IGF1R inhibitor and m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were detected. Further, IGF1R inhibition negatively altered the cell-protective effect f-DNA on macroautophagy and lipophagy.

Our study provides evidence for a close relationship between IGF1R inhibition and TLR9/autophagy signaling with profound influence on survival, proliferation and death of HT29 cells subjected to intact or modified self-DNA treatments. (Funded by StartUp).