Tumor‐Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma

Abstract

CD73, a cell‐surface N‐linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti‐CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N‐linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N‐linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor‐associated CD73 resulted in a 3‐fold decrease in 5′‐nucleotidase activity (P < 0.0001). Biochemically, tumor‐associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C‐terminal catalytic domain. Blocking N311/N333 glycosylation by site‐directed mutagenesis produced CD73 with significantly decreased 5′‐nucleotidase activity in vitro, similar to the primary tumors. Glycosylation‐deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N‐linked glycoprotein‐encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell‐surface CD73 in solid tumors and small‐molecule adenosine receptor agonists that are in clinical development for HCC.

CD73's Potential as an Immunotherapy Target in Gastrointestinal Cancers

“…Similarly, recent studies by Alcedo et al (169) report CD73 enzyme activity in HCC is significantly limited by aberrant glycosylation (169). The authors discovered that in HCC cells, unlike normal hepatocytes, CD73 carries abnormal N-linked glycosylation in its C-terminal catalytic domain, which greatly impairs the enzyme activity of CD73 (169). Aberrantly-glycosylated CD73 also showed to remain partially localized to the cytoplasm with golgi structural protein, GM130 (169).…”

“…Currently, NT5E-2 remains unstudied in other human tumors despite possible clinical implications. Similarly, recent studies by Alcedo et al (169) report CD73 enzyme activity in HCC is significantly limited by aberrant glycosylation (169). The authors discovered that in HCC cells, unlike normal hepatocytes, CD73 carries abnormal N-linked glycosylation in its C-terminal catalytic domain, which greatly impairs the enzyme activity of CD73 (169).…”

“…In recent years, HCC has been a platform for the discovery of novel biology for CD73 in human tumors ( Table 2) (168,169). Studies by Snider and colleagues (168) identified an alternative splicing variant of NT5E, NT5E-2 expressed in liver cirrhosis and HCC.…”

“…The authors discovered that in HCC cells, unlike normal hepatocytes, CD73 carries abnormal N-linked glycosylation in its C-terminal catalytic domain, which greatly impairs the enzyme activity of CD73 (169). Aberrantly-glycosylated CD73 also showed to remain partially localized to the cytoplasm with golgi structural protein, GM130 (169). Importantly, these studies show that CD73 protein expression levels may not necessarily reflect its ability to generate extracellular adenosine.…”

“…Importantly, these studies show that CD73 protein expression levels may not necessarily reflect its ability to generate extracellular adenosine. Studies by Snider et al (168) and Alcedo et al (169) are significant in that they demonstrate CD73 overexpression in human tumors can be misleading. Thus, CD73 immunohistochemistry may fall short in identifying patients likely to benefit the most from CD73 blockade therapy.…”