In this study, we showed that YAP could be found at all levels of the intrahepatic and extrahepatic biliary tract. We found nuclear (active) YAP expression was significantly elevated in BECs of ICC tumors compared to BECs in normal interlobular bile ducts. YAP activity is regulated by the Hippo signaling pathway, a tumor suppressor pathway. Conditional deletion of the Hippo signaling pathway tumor suppressors Nf2
in mouse livers increased YAP activity levels (6
) and resulted in aberrant proliferation of BECs which can lead to ICC (6
). In contrast, Yap
deficiency in mouse livers compromised bile duct development, leading to bile duct paucity in the adult phase (8
). Furthermore, deletion of Yap
is able to suppress the BEC over-proliferation in Nf2
-deficient livers (8
). Taking these studies together with our current observations, elevated YAP activity may contribute to the malignant transformation of BECs in ICC tumors. Thus YAP may be a promising therapeutic target in this devastating cancer.
Our findings further support YAP’s potential role in the development of HCC (4
). Specific deletion of the Hippo signaling pathway tumor suppressors Mst1
increased activity of YAP and resulted in the development of HCC (7
). We have previously shown that Yap
-deficient primary hepatocytes have much lower viability compared to wild type primary hepatocytes when cultured in vitro
). The elevated nuclear active form of YAP in some HCC tumors could explain enhanced hepatocyte proliferation and survival in these patients. This study and previous reports showed that increased nuclear YAP protein is present in about 50% of human HCCs (10
). However, the amplification of the Yap
locus appears to be relatively uncommon, restricted to about 5% to 10% of tumors, which prompted us to define the mechanisms of YAP overexpression in HCC. Through measuring DNA, RNA and protein levels of YAP in HCC tumors, we found that the YAP protein upregulation may due to gene amplification, mRNA upregulation and post-transcription mechanisms. However, gene amplification and mRNA upregulation do not necessarily result in increased YAP protein levels. Therefore, the levels of YAP protein may be controlled by multiple mechanisms.
Our data showed that YAP and Survivin levels significantly correlate in both ICC and HCC patients and mice. Since Survivin is a known prognostic marker (25
), our observation provides the first clinically relevant target that is regulated by YAP. Survivin is a member of the inhibitor of apoptosis protein (IAP) family which inhibits caspase activity and cell death in response to apoptotic stimuli. Survivin is primarily found in developmental and cancerous tissues, but not in normal, terminally differentiated tissues (33
). The molecular function of Survivin is to inhibit apoptosis while at the same time promote cell division by interfering with Aurora-B kinase (34
), exhibiting similar cellular roles of YAP (3
). Since the expression of both YAP and Survivin increased in a wide spectrum of human cancers (3
), it is particular interest to determine if YAP correlates with Survivin in non-hepatic cancer types. Several direct and indirect Survivin inhibitors have undergone investigations and clinical trials (35
) and these inhibitors may be useful in targeting YAP-overexpressed hepatic cancer cells.
Although Glypican-3 mRNA levels do not perfectly match YAP protein levels in the ApoE/rtTA-YAP mice, Glypican-3 mRNA levels were indeed elevated significantly after transgenic YAP induction, so we are not able to exclude Glypican-3 from the list of YAP target genes. However, our correlation studies indicate there is no positive correlation between nuclear YAP expression and Glypican-3 expression in 25 HCC tumors. There are several possibilities for this contradiction. First, Glypican-3 expression can be regulated by multiple mechanisms during HCC formation; or secondly, YAP may regulate Glypican-3 expression in special subsets of HCC or during early stages of tumor formation. Although Gypican-3 is an important diagnostic and prognostic marker and potential therapeutic target of HCC, the mechanisms of its regulation in HCC have not been illustrated. Further investigation on relationship of YAP and Glypican-3 is needed to explore the regulatory mechanism for Glypican-3.