Abstract B67: Increased CXCR4 expression and its nuclear localization are associated with invasion in sorafenib and sunitinib-tolerant hepatocarcinoma models
Authors: Riveiro Maria Eugenia , de Gramont Armand , Raymond Eric , Faivre Sandrine , Serova Maria , Tijeras-Raballand Annemilai , Dokmak Safi , Neuzillet Cindy , Astorgues-Xerri Lucile , Albert Sebastien , Huet Guillemette , Bieche Ivan 
Published: March 2013

Background: Most patients treated with sunitinib and sorafenib will experience tumor stabilization and eventually develop resistance within 4–6 months after treatment initiation. Increased CXCR4 and VEGF-C expressions were correlated with lymph node metastasis in HCC patients. In addition, high CXCL12 (SDF1α) plasma levels were associated with lack of sunitinib efficacy in HCC.

Objectives: The aim of this work was to investigate the role of CXCR4/CXCL12 expression in human HCC models developing tolerance to sunitinib and sorafenib.

Methods: SK-HEP1 cells were exposed to increasing sunitinib or sorafenib concentrations for more than 6 months to obtain RTKi-tolerant cell lines named as SK-Suni or SK-Sora, respectively. In these cell lines, cell proliferation, clonogenic survival, mRNA levels of selected genes and invasion of cells were evaluated. Western Blot analyzed protein levels. Transient siRNA transfection was carried out to target CXCR4. In vivo sorafenib efficacy was determined in SCID mice bearing SK-HEP1 xenografts and it was administered orally (30 mg/kg/daily) starting when tumors were detectable (100–300 mm3). In tumor samples, CXCR4, vimentin, and E-cadherin expression was evaluated by qRT-PCR and immunohistochemistry.

Results: Sunitinib and sorafenib displayed cytotoxic effects on SK-HEP1 parental cells with IC50 of 4.8 and 8.4 μM. SK-Suni and SK-Sora counterparts tolerated higher concentrations of sunitinib and sorafenib with IC50 of 10.5 and 11.8 μM, respectively. No cross-resistance to sunitinib or sorafenib was observed in SK-Sora or Sk-Suni, respectively. Protracted exposure to sorafenib led to a 3-fold increase mRNA expression CXCL12 and MIF in SK-Sora, whereas after long-term exposure to sunitinib CXCR4, CXCL12, and MIF mRNA levels increased in SK-Suni. Increase in CXCR4 protein levels was confirmed by Western Blot, immunofluorescence, and flow cytometry. Upon CXCL12 treatment, a prompt activation of signaling pathways involved in cytoskeleton reorganization and cell migration such as p-ERK, Cdc42, and STAT-3 were observed in SK-Suni. Interestingly, by immunofluorescence it was observed that CXCL12 treatment induced CXCR4 translocation into the nucleus in SK-Suni cells; associated with an increase Matrigel invasion compared to parental cells. In parental and suntinib/sorafenib-tolerant cells, matrigel invasion was markedly suppressed by 10 μg/ml AMD3100, a selective CXCR4 antagonist and by 2 μM Chalcone-4, a selective direct binding inhibitor of CXCL12. Small interfering RNAs were used to inhibit CXCR4 expression in cultured cells. This experiment showed that cell invasion decreased significantly in SK-Suni transfected cells compared to Sk-Suni. In SK-HEP1 xenografts treated with sorafenib for 25 days, we observed an increased CXCR4 protein expression together with changes usually associated with epithelial-to-mesenchymal transition such as high vimentin and low E-cadherin expression.

Conclusions: Our data show that the CXCL12/CXCR4 axis is up-regulated and functional in sunitinib- and sorafenib- tolerant HCC cells. Inhibiting CXCR4 might counteract the increased invasion associated with tolerance to sorafenib and sunitinib in HCC patients.