Apoptosis-induced ectodomain shedding of hypoxia-regulated carbonic anhydrase IX from tumor cells: a double-edged response to chemotherapy

Abstract

BackgroundCarbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin.MethodsPresence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay.ResultsWe found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness.ConclusionsThese findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

Evaluation of soluble carbonic anhydrase IX as predictive marker for efficacy of bevacizumab: A biomarker analysis from the geparquinto phase III neoadjuvant breast cancer trial

“…Similarly, presurgery sCAIX levels in early-stage primary nonsmall cell lung cancer patients did not correlate with immunohistochemical quantification of intratumoral CAIX. 42 This could explain why in our patient cohort low sCAIX levels predict worse outcome in patients treated with NCT alone, since they might be less prone to chemotherapy-induced apoptosis. The mechanisms underlying our finding have yet to be elucidated.…”

“…21 This finding shows that sCAIX levels do not necessarily reflect intratumoral CAIX expression. 42 Considering that intratumoral, membrane-bound CAIX protects the tumor cells from hypoxia and acidosis, a reduction of CAIX by extracellular domain shedding might render tumor cells more sensitive to chemotherapy and environmental stress factors. 41 We observed a divergent effect of baseline sCAIX level in the NCT arm compared to the NCT-B arm.…”

“…42 It was demonstrated that hypoxia leads to expression of a plethora of different pro-angiogenic factors besides the VEGF axis, thus hypoxic tumors become independent of VEGF to maintain angiogenesis. One reason could be the association of increased sCAIX shedding in hypoxic conditions.…”

“…40 Interestingly, recent in vitro studies showed that extracellular domain shedding of CAIX is increased after chemotherapy-induced apoptosis. 42 Considering that intratumoral, membrane-bound CAIX protects the tumor cells from hypoxia and acidosis, a reduction of CAIX by extracellular domain shedding might render tumor cells more sensitive to chemotherapy and environmental stress factors. 42 This could explain why in our patient cohort low sCAIX levels predict worse outcome in patients treated with NCT alone, since they might be less prone to chemotherapy-induced apoptosis.…”

“…42 Considering that intratumoral, membrane-bound CAIX protects the tumor cells from hypoxia and acidosis, a reduction of CAIX by extracellular domain shedding might render tumor cells more sensitive to chemotherapy and environmental stress factors. 42 This could explain why in our patient cohort low sCAIX levels predict worse outcome in patients treated with NCT alone, since they might be less prone to chemotherapy-induced apoptosis. In line with this hypothesis, one study reported that an increase in sCAIX level during neoadjuvant chemotherapy in locally advanced colorectal cancer predicted outcome, as shown by improved 5-year PFS in patients with a sCAIX increase (94% 5-year PFS for patients with an sCAIX increase vs. 56% 5-year PFS in patients with no increase, 66 patients).…”