Abstract
Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments.
Highlights
Aggressive tumors are characterized by poorly perfused, hypoxic and acidic niches that are resistant to conventional oncological treatment with cytostatic agents and radiotherapy
Using the well-established anti-carbonic anhydrase IX (CAIX) antibody (α-CAIX) M75 that recognizes the CAIX extracellular domain [11], we could initially confirm that CAIX closely overlaps with the hypoxia marker GLUT1 [12] in patient glioblastoma tumors (Figure 1A), and that CAIX is substantially induced by hypoxia in a patient derived glioma cell-line (U87-MG) in vitro (Figure 1B)
In further support of this notion, we found that cellular acidosis, i.e. another common stress phenomenon of aggressive tumors [34] attenuated the direct association of CAIX with caveolin-1 (Figure 6F), which was accompanied by decreased CAIX co-localization with caveolin-1 in WT-CAIX and hypoxic parental cells (Supplementary Figure 6C and 6D) as well as redistribution of PG-CAIX from low density membrane fractions (Figure 6G)
Summary
Aggressive tumors are characterized by poorly perfused, hypoxic and acidic niches that are resistant to conventional oncological treatment with cytostatic agents and radiotherapy. Recent advances in the understanding of how cancer cells adapt to hypoxic stress have the potential to significantly improve current treatment strategies. The concept of antibody-drug conjugate (ADC) treatment is to repurpose an antibody as a toxin delivery vehicle to kill tumor cells by endocytic uptake and intracellular release of the drug. Tumor specific cell-surface proteins, such as CAIX, with endocytic transport activity are interesting targets for ADCs. Importantly, CAIX and other targets of therapeutic antibodies are plasma membrane resident proteins more or less modified by glycosylation, which is known to have a major impact on the sorting of cell-surface proteins [9], e.g. it was recently reported that tumor resistance to the EGFR-targeting antibody cetuximab correlates with reduced EGFR stability due to deficient glycosylation [10]. While considerable interest is focused on the optimal design of the targeting ADC, it remains unknown how tumor antigen glycosylation may dictate the efficiency of ADC based anti-cancer treatments
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