Characterization of CA IX interactome: new insights on a highly versatile carbonic anhydrase

CA IX is a member of the carbonic anhydrase family of enzymes. It is a well known marker of hypoxia and is involved in pH regulation, migration/invasion and survival in hypoxic cancer cells. It is indicative of a poor prognosis in many cancer types and is associated with resistance to conventional therapy. So, there is a diffuse interest in inhibiting its function. Unfortunately, small molecule inhibitors that are available to inhibit CA IX demonstrated to be not much selective because of a high degree of homology amongst the catalytic sites of the various CA isoforms. The main aim of my PhD project was to identify molecular interactors of CA IX, and through them, to contribute to clarification of CA IX biological mechanisms. These molecules may drive design and development of peptide mimetics interfering with CA IX function. A complex protein network of novel CA IX interactors has been highlighted: several proteins belong to the family of the ARM and HEAT-repeat containing proteins and several members of the nucleocytoplasmic transport machinery have been identified as CA IX interactors under hypoxia, including importins and exportins. XPO1 and TNPO1 have been chosen as representative members of the nucleocytoplasmic transport machinery. Cullin-associated NEDD8-dissociated protein 1 (CAND1) is a nuclear HEAT/ARM-containing protein that is involved in gene transcription and assembly of the SCF E3 ubiquitine ligase complex. It interacts with CA IX under both normoxic and hypoxic conditions. Immunofluorescence (IF) analysis further proved complex subcellular localization of CA IX in human cell lines, highlighting a nuclear accumulation of CA IX in hypoxic cells. Nuclear presence of CA IX was also observed in two out of seven cases of clear cell renal cell carcinoma (ccRCC). Putative NLS/NES sequences have been identified in CA IX protein sequence; IF analysis showed that they are able to affect distribution of reporter protein GFP inside the cell. Collectively these data suggest that subcellular localization and functions of CA IX are more complex than previously thought. CA IX may have intracellular functions different from those already known at the plasma membrane. Investigation on this emerging scenario may prove useful to highlight unsuspected features in CA IX biology and its involvement in molecular mechanisms of cancer.