Accumulated evidence implicates the Golgi apparatus (GA) in processes associated with several cancer hallmarks. The Transmembrane BAX Inhibitor Motif Containing 4 (TMBIM4) is a GA-localized calcium ion channel that regulates intracellular Ca²⁺ dynamics, inhibits apoptosis, and promotes cell motility. Although cancer cells can survive and proliferate in adverse conditions, such as nutrient-deficient environments, the contribution of the GA to this process is unclear. Here, we present a novel GA-dependent process driving cancer cell survival under starvation conditions.

The impact of TMBIM4 on cell survival was assessed in invasive osteosarcoma U2-OS cells genetically modified to overexpress either the wild-type TMBIM4 or a TMBIM4-null mutant. To mimic a nutrient-deprived environment typical of many tumours, cells were maintained in culture at high confluence for prolonged periods without media replacement. Flow cytometry was used to evaluate intracellular reactive oxygen species levels, cell survival, death, and proliferation, while oxidative damage and lipid peroxidation were analysed via immunofluorescence. A genetically encoded biosensor was employed to measure the intracellular reduced/oxidised glutathione (GSH/GSSG) ratio. Proteomic analysis under normal and prolonged culture conditions were conducted using untargeted mass spectrometry.

In starvation conditions, TMBIM4 overexpression strongly enhanced cell survival. This effect was pH-dependent, occurring only at pH 7.4-7.8, aligning with the proposed pH-dependent gating mechanism of TMBIM family proteins. TMBIM4 overexpressing cells presented less lipid peroxidation and DNA damage despite having higher levels of ROS and maintained a higher GSH/GSSG ratio even in starvation conditions. Cells with higher levels of TMBIM4 displayed increased levels of the two key transsulfuration enzymes. This suggests a role for de novo synthesis of cysteine in this process, and possibly of GSH, since its synthesis is highly dependent on the availability of this amino acid. Supplementation of culture media with N-acetylcysteine or GSH ethyl ester enhanced long-term cell survival in starvation conditions. This supports the importance of cysteine and GSH availability in TMBIM4-mediated protection from cell death and provides a potential mechanism for the observed resistance to oxidative.

Our findings demonstrate that the GA plays a role in cancer cell metabolic adaptation, allowing cancer cells to survive even in starvation conditions. These results also highlight the importance of cysteine availability for cancer cell survival and suggest TMBIM4 and the GA as regulators of cysteine and GSH synthesis. Acknowledgments: FCT (UID/DTP/04567/2016, UIDB04567/2020, UIDP/04567/2020, UI/BD/151424/2021), and COST (CA16112-42814, CA18133-48426).