Investigation of the interplay between autophagy and therapy-induced senescence in thyroid cancer

Therapy-induced senescence (TIS) is a potential outcome of anti-cancer treatments, characterized by a stable cell cycle arrest. Besides this feature, senescent cells are highly metabolically active, and present a specific secretory phenotype. In cancer, senescence acts as a double-edged sword: in facts, although unable to proliferate, senescent cells are active drivers of cancer relapse, aggressiveness and metastasis, through the release of pro-inflammatory factors and the ability to resume proliferation. For these reasons, selectively targeting TIS cells is crucial to avoid their harmful effects. This strategy may become particularly important for those aggressive tumors that currently lack effective therapeutic options, like aggressive dedifferentiated thyroid tumors, such as poorly differentiated and anaplastic thyroid carcinomas. To this purpose, identifying targetable characteristics of TIS cells, conserved across different tumors and inducers, is essential for the development of new senotherapeutics. TIS is often associated with variations and abnormalities of autophagy, a catabolic process exploited by cells to degrade and repurpose macromolecules and organelles, and to rewire their metabolism under stress. The aim of this PhD project was to investigate the interplay between autophagy and therapy-induced senescence in thyroid cancer cells. To induce TIS, several thyroid cancer cell lines of different histotypes were treated with either CDK4/6 inhibitor palbociclib or ionizing radiation (IR). We demonstrated that TIS thyroid cancer cells develop highly heterogenous senescent phenotypes, based both on the cell line and the senescence inducer. Further, TIS thyroid cancer cells do not always exhibit a sufficient enlargement of the lysosomal compartment to sustain autophagy function. Indeed, we identified two distinct phenotypes, autophagy-proficient and autophagy-deficient TIS cells, based on lysosomal content. The deficiency in lysosomal biogenesis is driven by the inability of transcription factor EB (TFEB), this process’ master regulator, to properly enter and remain inside the nucleus. The disruption of the autophagic flux leads to the accumulation of SQSTM1/p62, not only the most widely known selective autophagy receptor, but also a signaling hub for the regulation of pathways like mTOR, NFκB and Nrf2. In autophagy-deficient models, SQSTM1/p62 accumulation is associated with the activation of both NFκB and Nrf2. In contrast to autophagy-proficient cells, that are highly sensitive to the senolytic potential of chloroquine (CQ), these Nrf2-activated cells display a higher tolerance to oxidative stress, making them resistant to the senolytic activity of CQ. Despite these contrasting results, CQ treatment shows promising effects also as a senomorphic agent, as shown by its ability to prevent senescence escape and diminish stemness markers in the CQ-insensitive senescent cell line. Moreover, CQ effectively changes the secretory phenotype of TIS cells, as shown by the significant changes in their paracrine effects on the parental cell line. Finally, to deepen the characterization of the autophagy/lysosomal-related secretory routes during TIS, we analyzed proteomics datasets and uncovered a coordinated upregulation of proteins involved in secretory autophagy and lysosomal exocytosis, particularly in IR-TIS cells, regardless of autophagic functionality. In conclusion, these findings suggest that during therapy-induced senescence the functionality of the autophagic flux varies due to significant differences in the lysosomal biogenesis machinery. This influences the accumulation of several protein receptors that may also act as signaling hubs, such as SQSTM1/p62, and shapes both cell-intrinsic survival pathways and, likely, the senescence-associated secretory phenotype. Together, these results indicate multiple targetable features of TIS thyroid cancer cells and highlight the senolytic/senomorphic potential of lysosomal inhibition in these tumors.