Menin promotes hepatocellular carcinogenesis and epigenetica

　　　　the Essential Role of the Menin–MLL Complex in Yap1 Transcription and Function.

　　Currently, it is poorly understood whether alterations of active histone modifications, including those of H3K4,are associated with liver tumorigenesis. We further identified the genomic occupancy of menin and two associated transcriptionally active histone modifications, H3K4me3 and H3K79me2 in HepG2, using ChIP-on-chip screens. Our data showed that menin occupied the promoter regions of thousands of human genes (Fig. S2 A and B and Dataset S1). Menin occupancy frequently coincides with H3K4me3 and H3K79me2; however,menin can also target promoters independently of H3 modifications (Fig. S2B). Menin occupancy at the HOXA clusters is extensive with broad footprints that spanned intergenic and intragenic portions of the HOXA genes in chromosome 7 (Fig.S2A), which is similar to the epigenetic-regulating features of menin in leukemia systems (5–7). Hyperexpression of HOXA genes, including HOXA5, 7, 10, and 13, was observed in a series of HCC specimens, and deregulation of HOXA13 led to cell-cycle alterations (18). Fig. S2C shows that genes (HOXA7, 9, and 13) with promoters bound by menin were correlated with higher mRNA levels in menin-overexpressing PLC/PRF5 cells (Fig.S2C). We previously found that menin represses cell proliferation and transcription of pleiotrophin (PTN) through polycomb group (PcG)-mediated H3K27me3 in lung adenocarcinoma and melanomas (19, 20). Nevertheless, the PTN mRNA level is augmented by menin in HepG2 cells (Fig. S2D), which further implies a tumor-promoting function of menin in HCC in a tissue-specific manner. Unsupervised hierarchical clustering analysis indicated that the top 26 genes with menin promoter occupancy coincide with H3K4me3 and H3K79me2 in ChIP-on-chip, and these genes are closely related with tumor development and progression, including the proto-oncogene Yap1 (Fig. S2E). These findings indicate an interesting epigenetic mechanism by which menin regulates gene expression through H3 modification in HCC.We explored the relationship between menin and Yap1 expression. The substantial MEN1 siRNAs reduced Yap1 mRNA and protein expression in HepG2 cells (Fig. 3A), whereas the expression and nuclear localization of Yap1 were increased in menin-overexpressing HepG2 cells (Fig. 3B and Fig. S3 A and B). Following the increased total Yap1 protein level resulting from menin expression, phospho-Yap1 (Ser127) was also slightly elevated in PLC/PRF5 cells (Fig. S3C). Nevertheless, there are no obvious effects on Yap1 protein expression by menin overexpression or MEN1 siRNA KD in other types of cell lines, including MCF-7, Wilm’s, and A549 (Fig. S3 D and E), suggesting a liver-specific function of menin in regulating Yap1. Correlated

　　Fig. 2. Menin promotes HCC cell proliferation and HepG2 xenograft growth. (A) The HepG2 cells were stably transfected with either control shLuc or shRNA against MEN1 retrovirus, and the expression of menin was confirmed by Western blot. (B and C) The proliferation and CFA assaysperformed by shLuc and shMEN1 HepG2 cells (n = 3). (D) HepG2 and PLC/ PRF5 cells were stably transfected with either empty vector or MEN1-overexpressing pLNCX2–MEN1 retrovirus, and the expression of menin was confirmed by Western blot. (E and F) The transfected cells were used to perform CFA assays. The above data are mean ± SD (n = 3). (G) Both shLuc and shMEN1 KD HepG2 cells were s.c. transplanted into nude mice (n = 13 or 14, respectively), and the tumor volume (mean ± SEM) was measured every 2 d at the indicated time points after transplantation (P < 0.05). (H) Thetumor weight and middle tumor pictures (P = 0.000).

　　Fig. 3. The essential role of the menin–MLL complex in Yap1 transcription and function. (A) The siLuc and each of the three distinct siRNAs (siMEN1-1, 2, and 3) that specifically target MEN1 were transfected into HepG2 cells, and the MEN1 and Yap1 mRNA and protein expression were determined by quantitative reverse transcription (qRT)-PCR and Western blotting, respectively. The P1 and P2 are two distinct Yap1 primers. (B) Immunofluorescent detection of Yap1 (green), DAPI (blue), and the merged signals in control and menin-overexpressing HepG2 cells. (C) The siLuc and each of the three distinct siRNAs (siYap1-1, 2, and 3) specifically targeting Yap1 were transfected into HepG2 cells. The generated cells were used to perform CFA assays (n = 3). (D) Either siLuc or siYap1-1 was transfected into control and menin-overexpressing HepG2 cells, and the generated cells were used to perform CFA assays. (E) Western blot analysis of 9–11 paired samples of HCC (T) and adjacent tissues (N) from the same patients. (F) A schematic representation of the human Yap1 and PPs used for ChIP assays. ChIP assays were performed using the antibody against menin in shLuc and shMEN1 HepG2 cells. (G) ChIP assays using antibody against H3K4me3 were performed in siRNAtargeting Luc, MEN1, or MLL HepG2 cells. The rabbit IgG served as a negative control for ChIP assays. (H) The siLuc and each of the three distinct siRNAs specifically targeting either MLL or MEN1 were transfected into HepG2 cells, and the transfected cells were used to perform CFA assays.

　　with reduced Yap1 expression, the specific targeting of Yap1 by siRNA dramatically reduced the HepG2 CFA (Fig. 3C and Fig.S3F). Furthermore, the promoted CFA by ectopic expression of menin was reduced by the Yap1 KD in HepG2 cells (Fig. 3D and Fig. S3G).

　　Yap1 is an important “driver gene” for HCC (16), however the transcriptional regulation of Yap1 by critical effectors in HCC has not been defined to date. In our ChIP-on-chip analyses, we found that menin occupancy coincides with the transcriptional activation of H3 modification at Yap1 promoter loci (Fig. S2E),which raises an interesting hypothesis of whether menin regulates Yap1 transcription through epigenetic mechanisms, such as H3K4me3. First, we assessed the expression of the MLL–HMTase complex in HCC using Western blotting. Associated with immunohistochemistry (IHC) observation, menin (6/11), Ash2L (9/11),WDR5 (6/11), RbBP5 (5/11), and MLL (5/8) expression were robustly activated in HCC compared with the surrounding tissues (Fig. 3E). We further performed ChIP assays using three distinct pairs of primers for the Yap1 promoter loci (Fig. 3F). As expected,the ChIP assays clearly showed that menin bound to the Yap1 promoter, and MEN1 KD notably reduced binding of menin at Yap1 loci but not at the GAPDH locus (Fig. 3F). Further, either MEN1 or MLL KD by siRNA reduced the H3K4me3 level at Yap1 loci, especially in primer pair 1 (PP1) and PP2 (Fig. 3G).Nevertheless, although menin bound to the promoter of Yap1,there are no obvious effects on H3K4me3 levels at Yap1 loci by menin overexpression in other types of cell lines, including MCF-7, Wilm’s, and A549 (Fig. S3 H and I), which further suggests the liver-specific function of menin in regulating Yap1 was dependent on H3K4 histone remodeling. In addition, the substantial KD of menin and MLL clearly reduced Yap1 expression (Fig. 3A and Fig. S3J) and CFA (Fig. 3H and Fig. S3K) in HepG2 cells, respectively. It appears that a pivotal biological role of Yap1 in the liver is attributable partly to the menin–MLL complex.Heterozygous Men1 Ablation Reduced Diethylnitrosamine-Induced HCC Development in Mice. The diethylnitrosamine (DEN)-induced HCC mouse model is widely used to investigate the features of human HCC (21). Because Men1-/- causes embryonic lethality in mice,we used Men1+/- mice to address the tumor-promoting function of menin in development of primary HCC. We treated Men1WT and Men1+/- mice with DEN at 15 d of age. After 8 mo, the Men1WT and Men1+/- mice developed visible hepatic tumor foci (Fig. 4A), and histological examination revealed that the liver tumors were highly aggressive HCCs (Fig. 4B). We did not observe typical pancreatic islet tumors in either Men1WT or Men1+/- mice at 8 mo (Fig. S4A). These observations ruled out the possibility that metastatic tumors came from pancreatic islets to the livers. As shown in Fig. 4C, 47.6% of the Men1WT female mice developed HCC; however, a significantly lower HCC incidence of only 8.33% was observed in Men1+/- female mice (P < 0.05). Furthermore, Men1+/- dramatically reduced the tumor multiplicity and maximal tumor size in female mice (Fig. 4 D and E). Compared with Men1WT mice, the tumor–liver ratio was reduced to almost onethird in Men1+/- female mice, and it was correlated with a slightly reduced liver–body weight ratio (Fig. 4 F and G). Men1+/- females displayed significantly less hepatic injury after DEN administration, which was determined by the reduced serum AFP, IL-6, and TGF-β (Fig. 4 H–J).Unexpectedly, all male mice developed HCC with no difference in tumor incidence (Fig. 4C) between Men1WT and Men1+/-.To elucidate the contribution of menin in hepatocarcinogenesi in male mice, 6-wk-old male mice were exposed to DEN. Administration of DEN resulted in the stimulation of the Akt, also known as protein kinase B (PKB), STAT3, and MAPK pathways in the liver; however, Men1+/- reduced their activation (Fig. S4B).Further, the serum IL-6 and TNF-α concentrations up-regulated by DEN exposure were remarkably reduced in Men1+/- male mice (Fig. S4 C and D), which indicates that menin participates in the liver injury response of male mice at early stages. The carbon tetrachloride (CCl4)–induced mouse model incorporates chronic injury, inflammation, and fibrogenesis and shares several features with the microenvironment in which the majority of human HCCs arise (22). In this model, we found that expression of menin, Yap1, pSTAT3, and pAKT was increased in the livers of males and females exposed to CCl4 (Fig. S4E, lanes 1–3 and

　　Fig. 4. Heterozygous loss of Men1 reduces DEN-induced development of HCC. Men1WT (n = 42) and Men1+/- (n = 23) mice were injected with DEN (25mg/kg, i.p.) at 15 d of age and killed 8 mo after DEN injection. Shown are representative liver images (A) and H&E staining sections (B). (C) Shown are different tumor incidences in male and female mice with DEN exposure. (D–G) The compared results of tumor multiplicity, maximal tumor diameter,tumor–liver, and liver–body weight ratios between Men1WT and Men1+/-mice. (H–J) Circulating serum levels of AFP, IL-6, and TGF-β were measured by ELISA. Data of D–J are represented as mean ± SEM. (K) Western blot analysis of menin, Yap1, pYap1 (ser127), Ash2L, RbBP5, and WDR5 in tumor (T) and normal (N) liver tissues of mice. (L) H3K4me3 ChIP assays performed with indicated PPs of mouse Yap1 promoter in female mice liver.

　　Xu et al. PNAS | October 22, 2013 | vol. 110 | no. 43 | 17483 MEDICAL SCIENCES