Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX’s role in mediating tumor progression is well-known, the underlying mechanisms remain unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, a proteomic analysis was performed using proximity-dependent biotin identification (BioID) and identified metabolic transporters involved in amino acid transport and pH regulation as high confidence interactors. In my thesis, I show that CAIX interacts with the amino acid transporters, SLC1A5; SLC3A2; SLC7A5, and the bicarbonate transporter, SLC4A7. These findings lay a premise for possible dynamic associations of these metabolic transporters with CAIX in the tumor microenvironment to mediate various functions and support tumor progression. In my thesis, I have focused on investigating the interaction of CAIX with SLC1A5, a glutamine (Gln) transporter crucial for supporting tumor growth. Through in-vitro studies, I show that CAIX associates with SLC1A5 in hypoxic cancer cells and regulates Gln uptake in a SLC1A5-depndent manner. Loss of CAIX expression or CAIX activity results in increased Gln transport. I found that the loss of CAIX activity increases cellular ROS, and cells respond by increasing their Gln utilization to synthesize the antioxidant, glutathione (GSH). This helps the cells to maintain redox homeostasis, preventing the lethal effects of oxidative stress and protecting them from cell death. To identify strategies for overcoming resistance to CAIX inhibition, I found that inhibiting the CAIX activity in combination with blocking Gln metabolism or GSH synthesis induces an iron-dependent, oxidative cell death called ferroptosis. Together, these data reveal an important mechanism by which CAIX cooperatively works with SLC1A5 to protect hypoxic cancer cells from oxidative stress and promotes survival. Furthermore, I have demonstrated four different co-targeting strategies that effectively induce ferroptosis in hypoxic cancer cells. In summary, my work has revealed the co-targeting of CAIX activity and Gln metabolism as a potential strategy to effectively treat aggressive, solid tumors.Medicine, Faculty ofGraduat
