Improved sorafenib activity on hepatocellular carcinoma in Notch3 silenced in vivo and in vitro models.

Abstract

3061 Background: Sorafenib is the only approved drug for the treatment of the advanced stage of HCC. Although its efficacy has been proven with randomized clinical trials, the clinical benefit seen in overall survival for advanced HCC patients treated with sorafenib could be improved. New molecules or combination of novel targeted agents to improve sorafenib efficacy for advanced stage HCC patients are needed. Notch genes are a family of receptors involved in many cell fate regulations, their expression has been found altered in many tumors, including HCC. The aim of this study was to study the combination of sorafenib and Notch3 signaling inhibition to improve sorafenib’s therapeutic effect. Methods: HepG2 and Huh7 cellular models were used for Notch3 stable silencing by retroviral introduction of specific interfering RNAs Xenograft models in both Notch3 stable shRNA cell lines have been developed using NOD/SCID mice. Animals bearing tumors were treated with 60 mg/kg of sorafenib for 21 consecutive days. Notch3, p21 and pGSK3βSer9 protein expression were also analyzed in 20 human HCCs. Results: Notch3 silencing (shN3) in HuH7 and HepG2 cell lines treated with sorafenib showed an increase in cell death (3.8 to 5 fold increase) when shN3 cell lines were compared with their relative controls (GL2). A difference in tumor growth was observed between GL2 negative control vs shN3 xenografts in both HepG2 and Huh7 after 21 days of sorafenib treatment. Two tailed student’s t test (shN3 vs GL2) P=0,04 and P=0,01 for Huh7 and HepG2 respectively. Molecular investigations have shown the involvement of p21 and GSK3β in the enhanced response to sorafenib in Notch 3 silenced models. A significant inverse correlation between Notch3 and pGSK3βSer9 proteins accumulation (Spearman ρ= -0.666) (p < 0.01) and a direct correlation between Notch3 and p21 proteins expression (Spearman ρ= 0.681) (p < 0.01) was found in HCC samples obtained from patients. Conclusion: Our preclincal findings outline the effect of combined Notch3 inhibition and sorafenib. The molecular mechanisms responsible for sorafenib induced cell death associated with Notch3 silencing relays on inhibition of p21/CDKN1A and GSK3βSer9. This study is supported by a grant from Bayer Healthcare, Italy.