D. Chirumbole1, M. Xu1, X. Wang1, G. A. Upadhya1, Y. Lin1, W. C. Chapman1 1Washington University,Transplant Section, General Surgery,St. Louis, MO, USA
Introduction:
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and studies suggest that CD47mAb immunotherapy could be an effective therapeutic option. CD47 ligation has also been shown to inhibit vascular endothelial growth factor (VEGF) signaling in various cell types. However, few studies have considered the effects of CD47mAb treatment on VEGF signaling and tumor progression in HCC. The purpose of this study is to investigate the effects of CD47mAb treatment on VEGF signaling in the Huh-7 HCC cell line.
Methods:
The Huh-7 HCC cell line was treated with Control IgG, CD47mAb1 (VX1000R), or CD47mAb2 (VX1004R) at a concentration of 10 µg/mL for 24 hours. Cells were then collected for study. ELISA, qRT-PCR, Western blot, and immunofluorescent staining analyses were performed to investigate the expression of VEGFs, VEGFRs, epidermal growth factor receptor (EGFR), and downstream markers related to cell growth and proliferation at the mRNA or protein level.
Results:
VEGFR-1 and -2 expression was significantly reduced in VX1004R-treated cells compared to control in terms of relative density to β-actin (VEGFR-1: 0.181±0.105 vs. 0.618±0.064, p=0.004; VEGFR-2: 0.127±0.071 vs. 0.259±0.038, p=0.048). Phosphorylated ERK1/2 and total p38 MAPK were also significantly reduced after VX1004R treatment (phospho-ERK1/2: 0.582±0.020 vs. 0.736±0.088, p=0.042; p38 MAPK: 0.857±0.077 vs. 1.120±0.014, p=0.004), as was the expression of proliferating cell nuclear antigen (PCNA) (0.457±0.044 vs. 0.546±0.003, p=0.047). On the other hand, there was no significant change in phosphorylated p38 MAPK expression after treatment. VX1000R-treated cells showed a trend toward decreased expression of most markers, but there were no significant differences at p<0.05. Expression of vegf-a mRNA was significantly reduced in both CD47mAb-treated groups (VX1004R: fold change=0.593±0.061, p=0.002; VX1000R: fold change=0.775±0.046, p=0.010). Expression of vegf-b mRNA was reduced in VX1004R-treated cells only (fold change=0.693±0.083, p=0.010). There was a non-significant trend toward decreased EGFR expression in both CD47mAb-treated groups.
Conclusion:
The results of this study suggest that CD47mAb treatment may inhibit VEGF signaling, downstream kinase activation, and cell proliferation in the Huh-7 cell line and therefore could inhibit tumor progression. VX1004R appears to be a more effective treatment than VX1000R. Further study is needed to investigate the effects of CD47mAb treatment on VEGF signaling in other HCC cell lines and in an in vivo system.
