Hepatitis B x antigen (HBxAg) is a trans-activating protein that contributes to liver cancer, in part, by altering the expression of cellular genes. However, few natural effectors of HBxAg have been identified. Hence, HBxAg positive and negative HepG2 cells were prepared and analyzed by PCR select cDNA subtraction. The results identified elevated vascular endothelial growth factor receptor-3 short form splice variant (VEGFR-3 S ) expression in HBxAg positive compared to negative cells. Normally, VEGFR-3 activates Akt signaling in lymphatic endothelial cells, resulting in lymphangiogenesis. In contrast, the results here show that the expression of VEGFR-3 S is up-regulated in >75% of HBxAg positive hepatocellular carcinoma (HCC) nodules. VEGFR-3 S up-regulation correlates with the expression of HBxAg, is associated with decreased survival in tumor bearing patients, and when over-expressed in HepG2 cells, strongly stimulated cell growth in culture, in soft agar, and accelerated tumor formation in a ligand independent manner. VEGFR-3 S siRNA partially blocked the ability of HBxAg to promote hepatocellular growth. In conclusion, HBxAg may short circuit VEGFR-3 S signaling in liver cancer. Blocking VEGFR-3 S signaling may be effective in preventing tumor development and/or prolonging survival in tumor bearing patients. (HEPATOLOGY 2007;45:1390-1399.)
C hronic hepatitis B virus infection is associated with the development of hepatitis, cirrhosis and HCC and constitutes a major public health problem worldwide. HBxAg participates in the pathogenesis of HCC. For example, high levels of intrahepatic HBxAg directly correlate with the intensity of liver disease. 5,6 HBxAg also transforms cells in vitro, 7 while sustained high levels of HBxAg in transgenic mice lead to HCC. 8 HBxAg is a trans-activating protein that alters gene expression by binding to nuclear transcription factors, and by stimulating cytoplasmic signaling pathways that promote cell growth and survival. HBxAg also binds to and inactivates or down-regulates tumor suppressors and senescence-related factors. 11 Despite this work, few natural effectors of HBxAg that promote the development of HCC have been identified.
VEGFR-3 is a receptor tyrosine kinase that is expressed in lymphatic endothelial cells. 12 Binding of VEGFR-3 to VEGF-C or VEGF-D stimulate lymphangiogenesis, 13 while in carcinogenesis, the production of VEGFs by tumors promote metastases and result in decreased survival. Elevated VEGF has been found in patients with HCC. 15,16 VEGFR-3 is also expressed in tumor cells from several tumor types, including HCC, 15 implying the existence of an autocrine/paracrine loop that promotes tumor development independent of lymphangiogenesis. In HCC, elevated VEGFR-3 is associated with portal vein invasion of tumors, increased hepatic tumor recurrence, and shorter survival, 15 suggesting that VEGFR-3 is important in the pathogenesis of HCC.