Induction of autophagy represents an effective survival strategy for nutrient-deprived or stressed cancer cells. Autophagy contributes to the modulation of communication within the tumor microenvironment. Here, we conducted a study of the metabolic and signaling implications associated with autophagy induced by glutamine (Gln) and serum starvation and PI3K/mTOR inhibitor and autophagy inducer NVP-BEZ235 (BEZ) in the head and neck squamous cell carcinoma (HNSCC) cell line FaDu. We compared the effect of these different types of autophagy induction on ATP production, lipid peroxidation, mitophagy, RNA cargo of extracellular vesicles (EVs), and EVs-associated cytokine secretome of cancer cells. Both BEZ and starvation resulted in a decline in ATP production. Simultaneously, Gln starvation enhanced oxidative damage of cancer cells by lipid peroxidation. In starved cells, there was a discernible fragmentation of the mitochondrial network coupled with an increase in the presence of tumor susceptibility gene 101 (TSG101) on the mitochondrial membrane, indicative of the sorting of mitochondrial cargo into EVs. Consequently, the abundance of mitochondrial RNAs (mtRNAs) in EVs released by FaDu cells was enhanced. Notably, mtRNAs were also detectable in EVs isolated from the serum of both HNSCC patients and healthy controls. Starvation and BEZ reduced the production of EVs by cancer cells, yet the characteristic molecular profile of these EVs remained unchanged. We also found that alterations in the release of inflammatory cytokines constitute a principal response to autophagy induction. Importantly, the specific mechanism driving autophagy induction significantly influenced the composition of the EVs-associated cytokine secretome.

1st Faculty of Medicine BIOCEV Charles University Prumyslova 595 Vestec CZ 252 50 Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 Brno CZ 613 00 Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 5 Brno CZ 625 00 Czech Republic

Department of Otorhinolaryngology and Head and Neck Surgery 1st Faculty of Medicine University Hospital Motol Charles University 5 Uvalu 84 Prague CZ 15006 Czech Republic

Department of Pathological Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 625 00 Czech Republic

Department of Pathology and Molecular Medicine 2nd Faculty of Medicine Charles University University Hospital Motol 5 Uvalu 84 Prague 5 CZ 15006 Czech Republic

Department of Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 625 00 Czech Republic

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