To determine the status of the Hippo pathway in HCC, we adopted an experimental protocol

wherein mice were given an initiating dose of DENA, followed by repeated injections of TCPOBOP for 27 weeks (Fig. 4A). Control mice were given DENA or TCPOBOP alone. As shown, the livers of mice treated with 27 injections of TCPOBOP were only twice that of controls, BMN 673 molecular weight confirming the existence of a strict regulation of liver size (Fig 4B,C). Whereas at the time of sacrifice, control mice and animals treated with TCPOBOP alone were completely devoid of tumors, livers from all mice exposed to DENA+TCPOBOP exhibited multiple tumors (Fig. 4C), which on histological examination showed nuclear atypia, cellular pleomorphism, and increased trabecular size and were therefore classified as medium- to high-grade HCCs (Fig. 4D). All tumors showed a high proliferative rate as detected by way of BrdU immunohistochemistry (Fig. 4E); conversely,

only a negligible proliferative activity was observed in nontumoral areas of the liver or in the liver from mice treated with TCPOBOP or DENA alone (Fig. 4E). Western blot analysis on total cellular lysates (Fig. 5A) of 21 HCCs, revealed in most of the tumors a significant increase in the levels of YAP compared with those of mice treated with TCPOBOP alone or DENA alone. Notably, a remarkable increase of YAP levels was observed in the nuclear fraction of randomly selected HCCs (5B, top). medchemexpress Accordingly, immunohistochemical staining revealed the presence of several YAP-positive cells in the tumors (Fig. 5C), Roxadustat nmr whereas no positive hepatocytes were observed in the livers of mice treated with DENA or TCPOBOP (data not shown). Notably, YAP was localized mainly in the nucleus of tumoral hepatocytes, although a cytoplasmic localization was also observed. No major changes of phosphorylated YAP were detected in the cytosolic fractions between tumors and normal or hyperplastic livers (Fig. 5B, bottom). To prove that YAP was indeed more active

in HCCs, we evaluated the level of expression of two other genes that are direct transcriptional targets of YAP, namely AFP and CTGF.15, 17 As shown in Fig. 6A,B, we found that the expression of these two genes was up-regulated in HCCs, 100% of the tumors showing increased expression of AFP and 60% exhibiting increased levels of CTGF. It was shown recently that miR-375 regulates the expression of YAP and is down-regulated in human HCC.29 To verify whether down-regulation of miR-375 is associated with increased YAP expression in mouse HCC, we performed a real-time PCR analysis of miR-375 expression in 21 HCCs developed in DENA+TCPOBOP–treated mice and in livers from animals treated with DENA or TCPOBOP alone. Fig. 6C shows that miR-375 was significantly down-regulated in HCC (17/21) (P < 0.01) and was inversely correlated with the protein levels of YAP (Fig. 5A).