Abstract 4264: CXCL12-CXCR4 axis in sunitinib-sensitive and -resistant hepatocarcinoma cells
Authors: Riveiro Maria E. , Serova Maria , Dokmak Safi , Bieche Ivan , Albert Sebastien , Halini Caroline , Raymond Eric , Faivre Sandrine 
Published: July 2012

Abstract
Background: Even though sunitinib is a multikinase inhibitor with activity in hepatocarcinoma, acquired resistance may eventually develop within 4-6 months. Increased expression of CXCR4 has been associated with poor prognosis and resistance to several anticancer agents. In addition, high plasma levels of CXCL12 (SDF1α) characterize the lack of sunitinib efficacy in patients with hepatocarcinoma. This study aimed to identify the mechanisms associated with acquired resistance to sunitinib in hepatocarcinoma cell lines.
Materials and Methods: SK-HEP1 cells were exposed to increasing sunitinib concentrations for more than 6 months to obtain a resistant cell line named as SK-suni. In these cell lines, cell proliferation, clonogenic survival, mRNA levels of selected genes and invasion of cells were evaluated. Protein levels were analyzed by Western Blot. CXCR4 expression was evaluated by qRT-PCR, Western blot and flow cytometry. Transient siRNA transfection was carried out to target CXCR4.
Results: Exposure to 48-hour sunitinib led to IC50s of 4.4 and 10.5 µM in SK-HEP1 and SK-suni cells, respectively and further confirmed by clonogenic assay. Sk-suni was 4-fold more invasive in matrigel as compared to parental cells. Protracted exposure to sunitinib led to a 3-fold increase mRNA expression of CXCR4, CXCL12, MIF, PDGFRα, RET, IGF1R, VEGFR3, CDH1 in SK-suni as compared to parental SK-HEP1 cells. In addition, a constitutive activation of p-ERK1/2 and p-AKT levels were detected in SK-suni cells. Using 200 ng/ml CXCL12 as chemoatractant, invasion index increased by ∼2.5 and ∼3 times in SK-HEP1 and SK-suni cells, whereas it was markedly suppressed by AMD3100, a CXCR4 antagonist. These findings indicate that sensitive and resistant cells harbour a functional CXCR4, although mRNA and protein levels of CXCR4 are higher in SK-suni. Upon CXCL12 treatment, a prompt activation of p-ERK was observed in SK-suni. However, AMD3100 pretreatment did not inhibit the proliferation and had no effect on the baseline up-regulation of p-ERK levels in SK-suni. To further investigate the function of CXCR4, small interfering RNAs was employed to specifically inhibit CXCR4 expression in vitro, obtaining a 80% inhibition of CXCR4 mRNA expression in SK-HEP1 and SK-suni. siRNA CXCR4 did not modify the IC50 of sunitinib in SK-suni. Matrigel invasion assays showed a significant decrease of CXCL12 induce cell invasion in transfected cells (p&lt;0.05).
Conclusions: In hepatocarcinoma cells, CXCL12/CXCR4 axis is expressed and functional in sunitinib-sensitive and -resistant cells. Inhibiting CXCR4 might counteract increased invasion associated with sunitinib resistance.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4264. doi:10.1158/1538-7445.AM2011-4264