Adaptive mechanisms in the hypoxic tumor microenvironment. Functional role of proteoglycans and identification of potential treatment targets

Abstract: Cancer cells reside in a complex microenvironment comprising stromal cells and immune cells embedded in an extracellular matrix (ECM). Early on in tumor progression, cells reach a critical volume beyond which blood supply is impaired, leading to a decrease in oxygen levels (hypoxia) and nutrient supply. This situation triggers a stress response characterized by a metabolic switch to glycolysis, which will in turn induce acidification of the extracellular environment. Under hypoxia and acidosis, cancer cells adapt their integration of nutrients, signaling molecules, and more generally their exchanges with the extracellular environment not only to survive but also enhance tumor aggressiveness, metastasis and treatment resistance. The overall aim of this thesis was to gain a better understanding of cancer cell adaptive mechanisms in the tumor microenvironment under hypoxic and acidic stress, that could be exploited therapeutically. We focused here on proteoglycan (PG)-dependent uptake mechanisms and cell surface proteins as targets of drug delivery. In paper I, we provided evidence for a role of heparan sulfate PGs in increased uptake of lipoproteins linked to enhanced pro-tumorigenic signaling and acquisition of a lipid droplet loaded phenotype under hypoxia and acidosis, associated with increased tumor-forming capacity. In the follow-up paper II we investigated the functional effects of this phenotype during post-hypoxic stress and found increased tumor aggressiveness, macrophage recruitment and angiogenesis in glioma mouse models, correlating with enhanced expression of pro-angiogenic and pro-tumorigenic factors (e.g. VEGF, HGF, CAIX, VIM) in vitro. In paper III, the global effects of hypoxia on tumor cell surface proteome internalization were studied. We showed downregulation of the surface and internalized proteome at hypoxia, involving caveolin-1 negative regulation of endocytosis. Importantly, we identified several surface proteins that were actively internalized at hypoxia, including CAIX. We then provided proof of concept that these proteins could be hypoxia-specific target candidates of antibody drug conjugate (ADC) treatment. In paper IV, the CAIX target protein was identified as a part-time PG. Mechanistically, glycosaminoglycan (GAG) conjugation of CAIX negatively regulated its internalization through increased association with caveolin-1 membrane domains, which was partially alleviated in acidic conditions. We then showed that CAIX GAG depletion enhanced its internalization and that this could be used to potentiate the cell killing effect of anti-CAIX ADC treatment. Altogether, these studies further demonstrate the important role of PGs in adaptive mechanisms to tumor microenvironmental stresses at different levels, and pave the way for the evaluation of novel, potential therapeutic strategies.