Eukaryotic cells undergo a continuous cycle of regeneration and degeneration of macromolecules (Tettamanti et al., 2019). This recycling aids in the elimination of unwanted and damaged macromolecules, thus protecting the cells and producing components that act as cellular ‘building blocks’. Typically, the breakdown of intracellular dysfunctional constituents is delivered to the lysosomes through the autophagy pathway (Gatica et al., 2018). Autophagy, a cytoprotective pathway, is initiated by AMPK activation and mTOR inactivation owing to cellular stress, leading to autophagosome-sequestered cargo breakdown in the autolysosome (Panwar et al., 2023, Park et al., 2023). Efflux due to autolysosome cargo degradation is sufficient to replenish the nutrient pool and reactivate mTOR (Panwar et al., 2023). Simultaneously, continuous autophagy leads to lysosomal pool depletion, hence requiring the cell to maintain lysosomal homeostasis (Yim and Mizushima, 2020). On mTOR reactivation, the cells initiate autophagic lysosome reformation (ALR) to produce autophagic lysosomes through the development of ‘reformation tubules’, which undergo scission to form nascent lysosomes and mature into active lysosomes (Chen and Yu, 2017). The lysosomes, often called the ‘suicidal bag’ of the cell, are prime organelles for degradation, and maintaining their balance is vital for healthy cellular functionality (Patra et al., 2023). Lysosomes have a crucial role in tumor progression and pathogenesis, as shown by modifications in the quantity and composition of lysosome hydrolases of cancerous tissue (Zamyatnin et al., 2022, Sudhan and Siemann, 2015). The importance of lysosomal hydrolases in therapy has been recognized for decades, with fundamental research describing the enhanced lysosomal activity in solid tumor compared to tissues of their origin, leading to enhanced tumor aggression (Seo et al., 2022, Dheer et al., 2019). Usually, the lysosomes in cancerous cells are modified and mislocalized compared to their standard counterparts due to increased hydrolytic enzymes and fundamental changes to the membrane composition of lysosomes (Machado et al., 2021). Nevertheless, such discrepancy may provide a better foundation for therapeutic intervention.
In cancer cells, glutamine serves as a vital source of nitrogen for amino acid and nucleotide biosynthesis as well as a carbon source to restore the tricarboxylic acid cycle, lipid biosynthesis pathways (Yoo et al., 2020, Li et al., 2021), and autophagy activation (Sakiyama et al., 2009), thus addicting the cancer cells to glutamine. Additionally, blocking glutamine metabolism inside the tumor microenvironment (TME) has gained much significance due to the use of widespread immune checkpoint inhibitors for cancer therapy (Ma et al., 2022, Shi et al., 2022). In several malignancies, autophagy has been consistently marked as a response to glutamine deficiency, establishing a complex interaction between glutamine and autophagy to determine cancer fate in TME (Sakiyama et al., 2009, Fares et al., 2022). For example, glutamine starvation triggers the general amino acid control pathway, resulting in the upregulation of amino acid transporters, thus enhancing amino acid uptake. This increases the intracellular amino acid concentration, subsequently reactivating mTOR and inhibiting autophagy (Chen et al., 2014). On the other hand, glutamine supplementation during prolonged amino acid starvation in cancer reactivates mTORC1 through the autophagy-dependent pathway, establishing glutamine and autophagy signaling coordination for cancer cell survival (Tan et al., 2017, Bodineau et al., 2022). In another study, a high glutamine concentration creates a nutrient imbalance in cancer cells in response to amino acid deprivation. This results in mTORC1 activation, which inhibits autophagy and induces apoptosis, limiting cell viability (Bodineau et al., 2022, Bodineau et al., 2021). In this setting, the role of autophagy and mTOR reactivation in replenishing the lysosomal pool and function under glutamine starvation is yet to be identified. Our study aims to establish the role of mTOR reactivation in maintaining the lysosomal pool during glutamine starvation in oral cancer cells. Moreover, we documented that prolonged glutamine starvation reactivates mTOR, halting autophagy and initiating ALR, which leads to the development of reformation tubules that subsequently form proto-lysosomes from existing autolysosomes to maintain lysosomal homeostasis. Further, we established that autophagy is essential for glutamine starvation-induced ALR, which sustains oral cancer cell viability.
