OPN expressions in patients with HCC
OSTEOPONTIN PROTEIN WAS immunohistochemically labeled in 40–55% of HCC,[11, 44-46] and OPN mRNA was overexpressed in 55% of HCC. The immunohistochemical staining and RNA in situ analysis were observed in the cytoplasm of HCC cells, but not in nuclei.[11, 12, 44] OPN positive HCC cells were scattered in the periphery of cancer nodules adjacent to stromal cells, or dispersed in the cancer nodules.[11, 46, 47] OPN protein and/or mRNA overexpression was significantly associated with large size,[12, 45] late tumor stage,[12, 48] poor differentiation,[12, 45, 46, 48] capsular infiltration,[11, 44, 45] vascular invasion,[44, 46] lymph node invasion and intrahepatic metastasis[12, 13, 46] of HCC.
Plasma OPN levels were significantly higher in patients with HCC than in patients with chronic liver diseases without HCC, and healthy controls.[47, 49] In patients after curative resection of hepatitis B virus (HBV)-related HCC, plasma OPN levels significantly decreased after a transient fluctuation, and increased again at the time of tumor recurrence.
As a marker for the diagnosis of HCC in patients with cirrhosis, plasma OPN level had a greater area under curve (AUC) value than α-fetoprotein (AFP)[47, 49, 51] and protein induced by vitamin K absence/antagonist-II by receiver–operator curve (ROC) analysis. Furthermore, the combination of OPN and AFP levels enhanced sensitivity and specificity in detecting HCC.
Moreover, plasma OPN levels were reported to be useful as a prognostic factor after liver resection or transplantation in patients with HCC of tumor–node–metastasis (TNM) stage I, II or III.[48, 52] In a prospective study, TNM stage and plasma OPN level measured prior to tumor resection were highly significant predictors of overall survival (OS) and disease-free survival (DFS) in patients with HCC in China. Preoperative plasma OPN level and Edmondson's grade were also identified as independent predictors for prognostic factor for OS and DFS in patients with TNM stage I of HBV-related HCC.
Increased expression of OPN protein in HCC was also shown to be an independent predictor of poor OS and/or poor DFS in patients undergoing liver transplantation and resection of HCC.[44, 54, 55] Finally, a meta-analysis revealed high OPN expression in HCC predicted poor OS (hazard ratio, 1.37; 95% CI, 1.21–1.55) and DFS (hazard ratio, 1.62; 95% CI, 1.24–2.11) of HCC after liver resection, liver transplantation or transarterial chemoembolization.
OPN roles in growth, invasion and metastasis of HCC in vitro and in vivo
It has been reported that OPN plays significant roles in the metastasis of HCC in vivo and in vitro. However, the effects of OPN on the growth of HCC cells were controversial. In nude mice, s.c. injected with human HCC derived cell line HCCLM3 which showed high metastatic potential and expressed high level of OPN, simultaneous injection of anti-OPN antibody significantly inhibited lung metastasis of the cells, but the antibody did not affect tumor growth. In contrast, i.v. injection of anti-OPN antibody twice per week significantly inhibited tumor growth and angiogenesis as well as lung metastasis in nude mice implanted with HCCLM3 cells. Downregulation of OPN by shRNA inhibited tumor growth and lung metastasis of HCCLM3 cells in the implanted nude mice. OPN antisense oligonucleotides significantly inhibited lung metastasis in mice bearing orthotopic xenografts with the human metastatic HCC cell line HCCLM6, although tumor weight was not reduced. Transfection of OPN significantly enhanced migration and invasion, but not proliferation of the human HCC SMMC-7721 cell line, which was weakly tumorigenic and non-metastatic, and expressed a low level of OPN. On the other hand, overexpression of OPN via transfection significantly stimulated proliferation of Huh-7 cells in vitro and growth of tumors in nude mice injected with the cells.
These discrepancies may be explained by the following observation, suggesting the necessary level of OPN for tumor growth was much lower than that for metastasis of HCC cells. Each of the Lentiviral-mediated miRNA against OPN, Lenti.OPNi-2 and Lenti.OPNi-3, significantly suppressed the migration of HCCLM3 cells in vitro and lung metastasis of HCCLM3 xenografts in nude mice. On the other hand, Lenti.OPNi-3, but not Lenti.OPNi-2, significantly inhibited proliferation of cultured HCCLM3 cells and tumor growth in nude mice. The downregulation degrees of OPN expression were 78% and 95% by Lenti.OPNi-2 and Lenti.OPNi-3, respectively. Suppression of tumor growth may be more difficult compared with prevention of metastasis during the OPN-targeting HCC therapy.
The molecular mechanisms of tumor progression and metastasis, influenced by tumor cell-derived OPN, have been investigated especially in breast cancer, but they are still poorly explored in HCC. OPN silencing by shRNA resulted in an increase of Bax expression, inhibition of Bcl-2/Bcl-xL and XIAP expressions and nuclear factor-κB activation, and induction of mitochondria-mediated apoptosis in HCCLM3 cells. Specific suppression of OPN inhibited MMP-2[58, 61, 63] and urokinase-type plasminogen activator (uPA) expressions[58, 61] in HCC cells. Addition of OPN to the medium or transfection of OPN enhanced expressions of MMP-2[59, 61] and uPA in HCC cells.