RASSF1A-Mediated Regulation of AREG via the Hippo Pathway in Hepatocellular Carcinoma

Abstract

Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor that is methylated in many human cancers, including hepatocellular carcinoma (HCC). RASSF1A has been shown to suppress tumors via activation of the Hippo tumor suppressor pathway, including mammalian STE20-like kinase (MST). Amphiregulin (AREG), a target gene for Yes-associated protein (YAP), is a known oncogenic component of the Hippo pathway; however, the tumor-suppressive effect of RASSF1A on AREG in regard to regulation of the Hippo pathway remains unclear in HCC. Overexpression of RASSF1A in HCC cells, which lack functional RASSF1A, significantly inhibited cell proliferation and induced apoptosis by activating the Hippo pathway. Consequently, overexpression of RASSF1A inhibited the oncogenic functions of YAP, leading to a significant reduction in AREG secretion via regulation of the Hippo pathway. In human specimens, greater expression of RASSF1A was observed in chronic hepatitis/cirrhosis than in HCC, whereas expression of YAP and AREG was higher in 81% and 86% of HCC than in corresponding chronic hepatitis/cirrhosis, respectively. Furthermore, RASSF1A protein gradually decreased as multistep hepatocarcinogenesis progressed from chronic hepatitis/ cirrhosis dysplastic nodules toward HCC, whereas the protein expression of YAP and AREG gradually increased. These findings provide mechanistic insight into the regulation of YAP and AREG by RASSF1A in human multistep hepatocarcinogenesis. Mol Cancer Res; 11(7); 748-58. Ó2013 AACR. IntroductionHepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related death (1). Recently, several genetic and epigenetic alterations were shown to be related with hepatocarcinogenesis, including frequent promoter hypermethylation of several tumor suppressor genes, such as Ras association domain family 1 isoform A (RASSF1A; ref. 2). In HCC, epigenetic alterations, such as global DNA hypomethylation or CpG island hypermethylation, are critical mechanisms that affect the loss and gain of DNA methylation, respectively (3). Accordingly, inactivation of RASSF1A by DNA methylation has been shown to be involved in the development of many human cancers,

WWC2 is an independent prognostic factor and prevents invasion via Hippo signalling in hepatocellular carcinoma

“…Activated LATS1/2 phosphorylate YAP, which inhibited cell proliferation in mammalian cells 38, 39, 40. In confirmation that WWC2 activates the Hippo pathway in HCC, overexpression of WWC2 in SMMC‐7721 cells (which express low levels of WWC2) increased phosphorylation of both LATS and YAP, which has been previously shown to negatively regulate the Hippo pathway 11, 41, 42. The latter study also revealed HCC cells transfected with the WWC2 overexpression plasmid decreased the mRNA level of Cyr61 and CTGF, both which are the target genes of Hippo pathway.…”

Yes-associated protein in the liver: Regulation of hepatic development, repair, cell fate determination and tumorigenesis

“…Consistently, most research groups observed more than 50% of human liver cancer tumors show elevated nuclear YAP expression [9,51,[54][55][56][57][58][59][60]. Multiple M a n u s c r i p t mechanisms were attributed to the elevated nuclear YAP expression, including gene amplification [40,56], increased Yap mRNA levels [51,55,56] and the dysregulation of the Hippo signaling with reduced expression of MST1 [47], LATS [57] and RASSF1A [59].…”

“…Several genes were proposed as YAP downstream targets in driving liver cancer development, including Axl (AXL receptor tyrosine kinase) [61], Survivin [56] and Amphiregulin [59]. Combining human and mouse data together, YAP is a very promising therapeutic target for liver cancer.…”

“…As a scaffold protein, Menin partners with mixed-lineage leukemin (MLL) histone methyltransferase to regulate the transcription of target genes by altering histone tail modifications [67]. In an effort to Domain Family Member 1) increased YAP phoshporylation [59].…”

“…Among these candidate YAP target genes, Axl is a direct target of YAP and has been shown to contribute to YAP-dependent oncogenic functions in HCC cell lines [61]; Ctgf [54] and Amphiregulin [90] were shown to be direct targets of YAP using non-liver cell lines and found to be induced by YAP in mouse liver [91] and HCC cell lines [59], respectively; in human liver cancer survey studies, expression levels of CTGF, Survivin and Glypican 3 were found to correlate with YAP activity levels [56,57]. Two independent studies have put YAP upstream of the Notch signaling pathway [23,24].…”