Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4

Abstract

In different tumour entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumour dissemination and poor prognosis. Therefore, we evaluated, if the expression of CXCR4 exerts similar effects in human hepatocellular carcinoma (HCC). Expression analysis and functional assays were performed in vitro to elucidate the impact of CXCL12 on human hepatoma cells lines. In addition, expression of CXCR4 was evaluated in 39 patients with HCC semiquantitatively and correlated with both, tumour and patients characteristics. Human HCC and hepatoma cell lines displayed variable intensities of CXCR4 expression. Loss of p53 function did not impact on CXCR4 expression. Exposure to CXCL12 mediated a perinuclear translocation of CXCR4 in Huh7/Hep3B cells and increased the invasive potential of Huh7 cells. In HCC patients, CXCR4 expression significantly correlated with progressed local tumours (T-status; P=0.006), lymphatic metastasis (N-status; P=0.005) and distant dissemination (M-status; P=0.009), as well as with a decreased 3-year-survival rate (P=0.01). In summary, strong expression of CXCR4 is significantly associated with progressed hepatocellular cancer.

Figure 1

(A) Expression and immunohistochemical staining of CXCR4 in diverse human hepatoma cell lines. Huh7 and Hep3B cells revealed a weak CXCR4 expression, whereas HepG2 cell lines depicted medium CXCR4 staining, independent from p53 status. (B) Exposure to CXCL12 mediated a rapid perinuclear translocation of CXCR4 from the cytoplasma and membrane (inlet patch). This translocation was strongly evident in Huh7 and also in Hep3B cells, but absent in HepG2 cells. (C) Fusion of nuclear staining (blue) and CXCR4 staining (green). Exposure to CXCL12 mediated a rapid cytoplasmatic clearance and perinuclear translocation of CXCR4 in HT29. (D) FACS analysis revealed a significantly decreased amount of positive HT29 cells for membrane-bound-CXCR4 upon CXCL12 exposure.

Figure 2

(A and B) Exposure to CXCL12-induced proliferation and invasion of Huh7, but not of Hep3B or HepG2 cells. While the impact of CXCL12 on invasion was highly significant, it was only marginally significant on proliferation.

Figure 3

(A) Depicts the respective cytoplasmatic expression grades of CXCR4 (weak; medium and strong) as well as a rare sample of a membranous CXCR4 staining. (B) The probability of survival of HCC patients is given in relation to time after histological confirmation. Patients with grade 3 CXCR4 expression showed a significantly reduced 3-year-survival rate as compared to all other patients (P=0.01).