The aberrant expression of miR-221 is a hallmark of human cancers, including hepatocellular carcinoma (HCC), and its involvement in drug resistance, together with a proved efficacy of anti-miR-221 molecules, strengthen its role as an attractive target candidate in the oncologic field. The discovery of biomarkers predicting the response to treatments represents a clinical challenge in the personalized treatment era. This study aimed to investigate the possible role of miR-221 as a circulating biomarker in HCC patients undergoing sorafenib treatment as well as to evaluate its contribution to sorafenib resistance in advanced HCC. A chemically induced HCC rat model and a xenograft mouse model, together with HCC-derived cell lines were employed to analyze miR-221 modulation by Sorafenib treatment. Data from the functional analysis were validated in tissue samples from surgically resected HCCs. The variation of circulating miR-221 levels in relation to Sorafenib treatment were assayed in the animal models and in two independent cohorts of patients with advanced HCC. MiR-221 over-expression was associated with Sorafenib resistance in two HCC animal models and caspase-3 was identified as its target gene, driving miR-221 anti-apoptotic activity following Sorafenib administration. Lower pre-treatment miR-221 serum levels were found in patients subsequently experiencing response to Sorafenib and an increase of circulating miR-221 at the two months assessment was observed in responder patients. MiR-221 might represent a candidate biomarker of likelihood of response to Sorafenib in HCC patients to be tested in future studies. Caspase-3 modulation by miR-221 participates to Sorafenib resistance. .

To successfully target Notch receptors as part of a multidrug anticancer strategy, it will be essential to fully characterize the factors that are modulated by Notch signaling. We recently reported that Notch3 silencing in HCC results in p53 up-regulation in vitro and, therefore, we focused on the mechanisms that associate Notch3 to p53 protein expression. We explored the regulation of p53 by Notch3 signalling in three HCC cell lines HepG2, SNU398 and Hep3B.We found that Notch3 regulates p53 at post-transcriptional level controlling both Cyclin G1 expression and the feed-forward circuit involving p53, miR-221 and MDM2. Moreover, our results were validated in human HCCs and in a rat model of HCC treated with Notch3 siRNAs. Our findings are becoming an exciting area for further in-depth research toward targeted inactivation of Notch3 receptor as a novel therapeutic approach for increasing the drug-sensitivity, and thereby improving the treatment outcome of patients affected by HCC. Indeed, we proved that Notch3 silencing strongly increases the effects of Nutilin-3. With regard to therapeutic implications, Notch3-specific drugs could represent a valuable strategy to limit Notch signaling in the context of hepatocellular carcinoma over-expressing this receptor.

Sorafenib (Nexavar), a multiple kinase inhibitor, is the only clinically approved drug for patients with advanced HCC. However, its therapeutic success is limited by the emergence of drug resistance. Here we found that p21 and pGSK3βSer9 are major players in the resistance to sorafenib. We recently reported that aberrant Notch3 expression in HCC contributes to doxorubicin resistance in vitro and, therefore, we focused on the mechanisms that associate Notch3 to acquired drug resistance. In this study we first found that Notch3 inhibition significantly increased the apoptosis inducing effect of sorafenib in HCC cells via specific down-regulation of p21 and up-regulation of pGSK3βSer9. Using a mouse xenograft model we further found that Notch3 depletion combined with 21 days of sorafenib treatment exerts a substantial antitumor effect in vivo. Interestingly, we showed that, upon exposure to sorafenib treatment, Notch3 depleted xenografts maintain lower levels of p21 and higher levels of pGSK3βSer9 than control xenografts. Thus, this study demonstrated that inhibition of Notch3 signaling prevents HCC-mediate drug resistance and sensitizes HCC cells to sorafenib. Finally, we validated our in vitro and in vivo results in primary human HCCs showing that Notch3 protein expression positively correlated with p21 protein expression and negatively correlated with pGSK3βSer9 expression. In conclusion, the results presented in this study demonstrated that Notch3 silencing enhances the effect of sorafenib by overcoming drug resistance. Notch3 inhibition in combination with sorafenib can be a promising strategy for treatment of HCC.