Abstract B80: Targeting CXCR4/CXCL12 axis as strategy to inhibit tumor cell invasion in RTKi-tolerant HCC models.
Authors: Riveiro Maria Eugenia , Serova Maria , Tijeras-Raballand Annemilai , Huet Guillemette , Dokmak Safi , Couvelard Anne , Albert Sebastien , Martinet Matthieu , Bieche Ivan , Raymond Eric , Faivre Sandrine 
Published: June 2012

Background: Sunitinib and sorafenib are multi-targeted tyrosine kinase receptor inhibitors (RTKi) with activity in hepatocellular carcinoma (HCC). However, disease-stabilizing effects are eventually followed by emergence of resistance in patients within 4–6 months. Several studies point out that CXCR4 overexpression may be associated to metastasis and poor survival in HCC patients. Furthermore, high plasma levels of CXCL12 (SDF1, CXCR4 ligand) characterize the lack of sunitinib efficacy in HCC patients. Objectives. The aim of the present work was to characterize the mechanisms involved in the invasive phenotype of sunitinib and sorafenib-tolerant HCC cell lines.

Methods: SK-HEP1 cells were exposed to increasing sunitinib or sorafenib concentrations for more than 6 months to obtain RTKi-tolerant cell lines named SK-Suni and SK-Sora, respectively. In these cell lines, cell proliferation, clonogenicity and mRNA levels of selected genes were evaluated. Invasiveness of cells was determined by Matrigel invasion assay Transient siRNA transfection (siRNAs) was carried out to target CXCR4. Total and phospho-protein levels of signaling pathways were analyzed by Western blot In vivo sorafenib efficacy (10 and 30 mg/kg daily) was determined in SCID mice bearing SK-HEP1 xenografts. 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. These findings were further confirmed by clonogenic assays. 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. SK-Suni was 4-fold more invasive in matrigel as compared to parental cells, similar results were obtained for SK-Sora. In parental and RTKi tolerant-HCC cell lines, matrigel invasion was markedly suppressed by 10 g/ml AMD3100, a selective CXCR4 antagonist and 2 M Chalcone-4, a selective inhibitor of CXCL12 via direct binding. 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. siRNA targeted CXCR4 in SK-suni did not modify the sunitinib-IC50 in the tolerant cell line, whereas, matrigel invasion assays showed a significant decrease of cell invasion in transfected cells despite the presence of CXCL12 (p&lt;0.05). Sorafenib significantly suppressed SK-HEP1 xenograft tumor growth (34.6%) after 25 days of treatment. CXCR4 expression increase and changes associated with epithelial-to-mesenchymal transition were detected in SK-HEP1 xenographs after sorafenib treatment.

Conclusions. Our data show that long term exposure to RTKi is associated with changes in the invasion properties of HCC cells. In addition, CXCL12/CXCR4 axis is up-regulated and functional in RTKi- tolerant HCC cells. Inhibiting CXCR4 might counteract increased invasion associated with RTKi tolerance in HCC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B80.