Faculty Bibliography

Ecto-5'-nucleotidase (CD73), encoded by NT5E, is the major enzymatic source of extracellular adenosine. CD73 controls numerous pathophysiological responses and is a potential disease target, but its regulation is poorly understood. We examined NT5E regulation by alternative splicing. Genomic database analysis of human transcripts led us to identify NT5E-2, a novel splice variant that was expressed at low abundance in normal human tissues but was significantly up-regulated in cirrhosis and hepatocellular carcinoma (HCC). NT5E-2 encodes a shorter CD73 isoform we named CD73S. The presence of CD73S protein, which lacks 50 amino acids, was detected in HCC using an isoform-specific antibody. A noncanonical mouse mRNA, similar to human CD73S, was observed, but the corresponding protein was undetectable. The two human isoforms exhibited functional differences, such that ectopic expression of canonical CD73 (CD73L) in human HepG2 cells was associated with decreased expression of the proliferation marker Ki67, whereas CD73S expression did not have an effect on Ki67 expression. CD73S was glycosylated, catalytically inactive, unable to dimerize, and complexed intracellularly with the endoplasmic reticulum chaperone calnexin. Furthermore, CD73S complexed with CD73L and promoted proteasome-dependent CD73L degradation. The findings reveal species-specific CD73 regulation, with potential significance to cancer, fibrosis, and other diseases characterized by changes in CD73 expression and function.

BACKGROUND & AIMS: Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. METHODS: HCCs were collected from 17 patients during surgical resection and single-cell suspensions were analyzed by flow cytometry. CD14(+) TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rg(null) mice from human primary HCC cells or HepG2 cells. RESULTS: CD44(+) cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44(-) cells, indicating that CD44(+) cells are CSCs. Incubation of the CD44(+) cells with TAMs promoted expansion of CD44(+) cells, and increased their sphere formation in culture and formation of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the numbers of CD44(+) cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44(+) cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of signal transducer and activator of transcription 3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. CONCLUSIONS: CD44(+) cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44(-) cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44(+) cells. This drug might be used to treat patients with HCC.

We have characterized herein the heterogeneity of the CD90+ population at each stage of hepatocarcinogenesis using a computer-assisted immunohistochemical staining evaluation method for quantitative analysis on tissue microarrays. We found that CD90 in Hepatocellular carcinoma (HCC) tissues, which has been shown to be a marker for cancer stem cells, is expressed on tumor cells, in the stroma or on endothelial cells. Sub-classification of the CD90+ population was based on morphology and co-expression with known markers including CD45 and CD31. Multiple linear regression suggested that the percentage of CD90+ cancer cells/hepatocyte (p<0.0001), level of overall CD90 expression (p<0.0014), and level of CD90 expression in tumor islands (p<0.0001) increased significantly in each stage of liver disease progression, while the level of stromal CD90 expression (p=0.1129) did not change significantly. Additionally, only the CD90+ cancer cells were positive for other cancer stem cell (CSC) markers including CD24, CD44 and CD133 whereas the other CD90+ cells were negative for these markers. CD90 expression in cirrhosis was observed in hepatocytes, the portal tract area and fibrous septa while CD90 expression in normal liver was limited only to the portal tract area. This study demonstrates the heterogeneity of the CD90+ population in HCC where a small population of the CD90+ cells that expressed other CSC markers are CSCs and are associated with advanced stages of hepatocarcinogenesis. This heterogeneity should be emphasized in further studies where other methods may not be able to discriminate these distinct types of CD90+ cells.

Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as pancreatic intraepithelial neoplasias (PanIN). PanIN formation is accompanied by the establishment of an immunotolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we genetically eliminate CD4+ T cells in the iKras* mouse model of pancreatic cancer, in the context of pancreatitis, to determine the functional role of CD4+ T cells during mutant Kras-driven pancreatic carcinogenesis. We show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. Furthermore, we show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation and provide a rationale to explore immunomodulatory approaches in pancreatic cancer.