Menin promotes hepatocellular carcinogenesis andepigenetical

　　　Menin promotes hepatocellular carcinogenesis andepigenetically up-regulates Yap1 transcription

　　Menin is a scaffold protein encoded by the multiple endocrineneoplasia type 1 (MEN1) gene in humans, and it interacts with
a variety of transcriptional proteins to control active or repressivecellular processes. Here, we show that heterozygous ablation of
Men1 in female mice reduces chemical carcinogen-induced livercarcinogenesis and represses the activation of the inflammation pathway. Using ChIP-on-chip screens and ChIP assays, we find that menin occupancy frequently coincides with H3K4me3 at the promoter of many liver cancer-related genes, such as Yes-associated protein (Yap1). Increased menin and Yap1 expression in human hepatocellular carcinoma specimens was associated with poor prognosis. Our findings reveal that menin plays an important epigenetic role in promoting liver tumorigenesis, and support the notion that H3K4me3, which is regulated by the menin–mixedlineage leukemia complex, is a potential therapeutic target in hepatocellular carcinoma.

　　H3K4 methylation | interleukin 6

　　Menin, the product of the mice), is responsible for the inherited tumor syndrome, MEN1 gene in humans (Men1 in multiple endocrine neoplasia type 1 (MEN1) (1). The phenotype of mice with a heterozygous loss of Men1 (Men1+/-) is markedly similar to the human MEN1 disorder (2). Both MEN1 alleles areinactivated somatically in human endocrine tumors, which suggests that menin is a tumor suppressor (3). Supporting this notion, menin interacts with the JUN family transcription factor JUND and inhibits its transcriptional activity (4). Interestingly,as an activator, menin interacts with the trithorax group (trxG) proteins (Drosophila) and the mixed-lineage leukemia (MLL) (humans) histone methyltransferase (HMTase) complex, and it alters histone tail modifications and the transcription of target genes (5–7). The methylated H3K4 can be recognized by other specific binding proteins, including WDR5, RbBP5, and Ash2L,that alter chromatin structure and lead to the activation of gene transcription (5). Menin is a highly specific partner for the MLL complex, and it links the MLL complex to target gene promoters in MLL-associated leukemogenesis (5–7). To date, the putative biological function of menin in the liver is not well defined. Homozygous loss of Men1 (Men1-/-) in mice leads to embryonic lethality at embryonic day (E) 11.5–13.5 and is associated with defective growth and organogenesis, including the liver (8).Microarray screenings indicate that menin is a key regulator of the gene networks activated in fibrogenesis and associated with hepatocellular carcinoma (HCC) (9). The potential link between menin and liver disease offers a fresh opportunity to uncover the function of menin in the liver.

　　HCC is the main type of liver cancer, and it is governed by cumulative genetic and epigenetic alterations (10). Research in the past several years has greatly advanced our understanding of the essential role of key regulators and their roles in HCC. For example, the essential functions of the Mst1/2, HNF4a–miRNA,IKKβ–NF-κB, and Ptpn11–Shp2 pathways in liver tumorigenesis have been demonstrated in genetically engineered mice (11–14).Central to the Hippo pathway is a kinase cascade leading from the tumor suppressor Hippo (Mst1/2) to Yes-associated protein (Yap1), an oncogene in various tumors (11). Inactivation of Mst1/2 induces Yap1-mediated activation of various target genes functionally involved in control of liver organ size and tumorigenesis (15). In accord with the repressive nature of Mst1/2, the genome-wide analysis of HCC identified Yap1 as an important“driver oncogene” (16). Although excellent studies have demonstrated that Yap1 is a major downstream target of the Hippo pathway (11, 15), the transcriptional regulation of Yap1 by critical effectors in HCC has not been defined to date. It is important to uncover how Yap1 activation is associated with other well-defined molecular alterations in HCC.This study is designed to investigate the possible tumor promoter function of menin in the liver.

　　Results

　　　　Menin Is Up-Regulated in HCC and Is Correlated with Poor Prognosis.

　　To unravel the functional significance of menin in HCC, wemeasured the expression of menin in 89 patients’ primary HCCs.The HCC specimens exhibited robust expression and exclusive nuclear staining of menin (42.7%) compared with adjacent tissues (Fig. 1A). Kaplan–Meier survival analysis showed that the 3-y overall survival (Fig. 1B, P = 0.000) and the recurrence-free survival (Fig. 1C, P = 0.002) were significantly lower in the menin–(+) HCC patients than in the menin–(–) patients. The serum level of alpha-fetoprotein (AFP) was dramatically elevated Significance Epigenetic changes commonly occur in hepatocellular carcinoma (HCC) and are associated with aberrant gene expression. Most studies have focused on epigenetic gene-silencing events;therefore, the mechanism that promotes gene activation in HCC is not well established. We identify an epigenetic activation mechanism whereby menin promotes Yes-associated protein (Yap1) transcription, which is associated with a poor prognosis for HCC patients. Substantial overexpression of the menin–mixed-lineage leukemia complex is associated with increased histone 3 lysine 4 trimethylation at certain loci of the tumor promoter in HCC. Heterozygous ablation of multiple endocrine neoplasia type 1 (Men1) in mice reduces diethylnitrosamine-induced development of HCC. Our findings reveal that menin plays an important epigenetic role in up-regulating Yap1 transcription, leading to liver tumorigenesis.Author contributions: G.-H.J. designed research; B.X., S.-H.L., R.Z., S.-B.G., L.-H.D., Z.-J.F.,and S.Z. performed research; Z.-Y.Y., S.Z., and X.-M.W. contributed new reagents/analytic tools; B.X., S.-H.L., X.L., and G.-H.J. analyzed data; and G.-H.J. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.1B.X. and S.-H.L. contributed equally to this work.2To whom correspondence should be addressed. E-mail: ghjin@xmu.edu.cn.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1312022110/-/DCSupplementa

　　Fig. 1. Up-regulation of menin in HCC patients correlates with poor prognosis. (A) H&E staining and IHC staining of menin in HCC paraffin sections (n = 89). The dotted lines indicate juncture of tumor (T) and adjacent tissue (N). Menin was easily detectable in the nucleus of certain HCC. Original magnification, 100× or 400×, respectively. (B and C) Kaplan–Meier curves for overall and tumor-free survival in menin-hyperexpressing (+) and -underexpressing (–) HCC patients (P = 0.000 and P = 0.002, respectively, log rank test). (D) Serum AFP level of HCC patients with menin–(+) (n = 37) or (–) (n =50). In each panel, the line indicates the median (P = 0.001).
　　in the menin–(+) group compared with the menin–(–) group (Fig.1D, P = 0.001). Further correlation analyses in both cohorts showed that the robust expression of menin was associated with the more aggressive phenotype of HCC, including tumor multiplicity and neoplasm staging (Table S1).To explore how menin influences liver cancer cells, the HepG2 cells were stably transfected with either a control (shLuc) or shMEN1 (17). MEN1 shRNA substantially repressed HepG2 cell proliferation and colony forming activity (CFA) (Fig. 2 A–C).Conversely, ectopic expression of menin increased the CFA in HepG2 and PLC/PRF5 HCC cell lines (Fig. 2 D–F). Furthermore, reduced expression of menin significantly suppressed tumor volume and weight in two independent pairs of MEN1 shRNA knockdown (KD) HepG2 xenografts (Fig. 2 G and Hand Fig. S1). Overall, these findings suggest a potential tumorpromoting function of menin in HCC.