Menin promotes hepatocellular carcinogenesis and epigenetica

　　7–9). Nevertheless, the Men1+/- effectively reduced their effect in female mice but not in males (Fig. S4E, lanes 4–6 and 10–12).In addition, administration of DEN significantly increased the menin levels in the livers of C57BL/6 males compared with females at 4 mo (Fig. S4 F and G). These results suggest that although the function of menin is involved in inflammation response at an early stage, the heterozygous ablation of Men1 was easily compensated in liver injury and inflammation in male micecompared with female mice.To evaluate the potential relationship of the menin–Yap1 axis and liver tumorigenesis, we collected liver tumors and surrounding tissues. Because the primary tumors resulting from DEN exposure of Men1+/- female mice were not large enough for Western blotting analysis, we used tumors from Men1WT female mice.We detected elevated menin, Yap1, MLL, Ash2L, RbBP5, and WDR5 levels in the liver tumors (Fig. 4K), as well as pAKT,pSTAT3, pERK1/2, and p65 (Fig. S4H). In ChIP assays, following the increase of menin at the Yap1 loci (Fig. S4I), the H3K4me3 levels increased nearly threefold in tumors compared with surrounding tissues (Fig. 4L). The H3 antibody ChIP was used as a loading control (Fig. S4I).

　　The Menin–Yap1 Axis Responded to CCl4-Induced Liver Inflammation.

　　HCC promotion depends on the microenvironment, including inflammation pathways (12, 13). To determine the relationship between menin–Yap1 axis and inflammation pathways, we used CCl4-induced inflammation mouse model. Exposure to CCl4 significantly stimulated the expression of menin, MLL, and Yap1 in the livers of male C57BL/6 mice at 24 and 48 h (Fig. 5A). This response is associated with elevated mRNA levels of IL-6 and TGF-β and serum concentrations of IL-6 (Fig. S5 A and B). An increased binding of menin and accumulation of H3K4me3 at the Yap1 promoter were revealed by ChIP assays (Fig. 5B and Fig. S5C). In accord with the reduction of Men1 or Yap1 expression by siRNA, the IL-6 mRNA level was markedly reduced in primary isolated liver Kupffer cells (KCs) (Fig. 5 C and D). Yap1 KD dramatically reduced IL-6 mRNA expression in two independent pairs of Yap1 siRNA KD HL-7702 and HepG2 cells (Fig. 5 E and F and Fig. S5 D and E). Furthermore, the upregulation of IL-6 by ectopic expression of menin was reduced by the Yap1 KD in HepG2 cells (Fig. 5G), which suggests that menin regulates IL-6 expression at least partly through Yap1. Finally, the protein or mRNA expression of menin and Yap1 was increased in HL-7702 or HepG2 cells exposed to IL-6 (Fig. 5 H and I). These results point to an interesting HCC-related positive feedback loop between menin–Yap1 and IL-6.

　　Yap1 Is Epigenetically Regulated by Menin and Correlated with Poor Prognosis in Human HCCs. To confirm the clinical significance of the menin–Yap1 axis, we sought to address the deregulation of Yap1 in HCCs. The HCC specimens exhibited robust expression and exclusive nuclear staining of Yap1 compared with the adjacent tissues (Fig. 6A). Kaplan–Meier survival analysis showed that the 3-y overall survival rate of patients with Yap1– (+) HCCs was significantly lower than that of patients with Yap1–(–) HCCs (Fig. 6B, P = 0.000). A significantly lower recurrence-free survival rate was found in HCC patients in the Yap1–(+) group compared with Yap1–(–) HCC patients (Fig. 6C, P = 0.000). Furthermore, serum AFP levels were dramatically higher in the Yap1–(+) group than in the Yap1–(–) group (Fig. 6D, P = 0.004). Yap1 hyperexpression was significantly associated with more aggressive phenotypes of HCC, including tumor multiplicity, vascular invasion, and neoplasm staging (Table S2). In 23.75% of HCCs, menin and Yap1 were collectively up-regulated (Fig. 6E, P = 0.014). In HCC tissue ChIP assays, the binding of menin and accumulation of H3K4me3 at the Yap1 promoter were markedly increased in HCC specimens compared with adjacent tissues (Fig. 6 F and G and Fig. S6). These results support the mechanistic and clinical significance of the menin– Yap1 axis as an effective biomarker for HCC diagnosis and prognosis evaluation.

　　Discussion

　　The findings of this study advance our knowledge of the epigenetic activation mechanism of H3K4me3 and indicate that menin plays an important, yet previously unappreciated, role in promoting the development of HCC. Menin, as a scaffold protein, lacks identifiable functional motifs, and the putative function of menin is not well defined. Menin interacts with a variety of transcription factors and is involved in a variety of cellular processes, including gene activation and repression (7). In this notion, the crystal structure shows that menin contains a deep pocket that interacts similarly with MLL1 and JUND, indicating

　　Fig. 5. Menin–Yap1 axis is responsive to CCl4-induced liver inflammation. The Men1WT C57BL/6 mice were treated with CCl4 (2 mL/kg, i.p.) at 6 wk of age and killed 24 and 48 h after CCl4 injection, respectively. (A) The expression of menin, MLL, and Yap1 in liver was detected by Western blot. (B) The ChIP assays of liver tissues were carried out with antibodies either against menin or H3K4me3 (n = 3). (C and D) The primary KCs were isolated from WT C57BL/6 mice, and each of the three distinct siRNAs specifically targeting either Men1 (Men1-1, 2, and 3) or Yap1 (siYap1-1, 2, and 3) were delivered to KCs.The mRNA expression of Men1, Yap1, and IL-6 in KCs was determined by qRT-PCR (n = 3). (E and F) Either HL-7702 or HepG2 cells were transfected with each of the three distinct siYap1s, and the mRNA level of Yap1 and IL-6 was determined by qRT-PCR (n = 3). The relative mRNA levels were normalized to β-actin. (G) The siLuc or siYap1-1 was transfected to control or menin-overexpressing HepG2 cells, respectively, and the mRNA level of Yap1 and IL-6 was quantified by qRT-PCR (n = 3).(H) The HL-7702 cells were exposed to IL-6 (50 ng/mL), and the protein levels of menin and Yap1 were detected by Western blot. (I) The HepG2 cells were exposed to IL-6 (50 ng/mL), and the mRNA level of MEN1 and Yap1 was determined by qRT-PCR at the indicated times.
　　that the diverse functions may be largely attributed to the crucialrole of menin as a core scaffold molecule (7). MLL, an epigenetic regulator, plays a critical role in acute leukemias, but theputative biological function of MLL in the liver is not well defined. Supporting our notion, HGF-MET (hepatocyte growth factor-mesenchymal epithelial transition factor) signals were reported to promote the expression of matrix metallopeptidases (MMPs), and the development of HCC through the ETS2–MLL complex mediated H3K4me3 (23). Strikingly, whole-genome sequencing analysis revealed that the MLL family of methyltransferases for H3K4 was somatically mutated in HCCs (24), which further supports the biological relevance of MLL in HCC. In this report, we propose that the unique biological role of menin in promoting HCC depends on the oncogenic activity of MLL. Although we have identified a unique tumor-promoting action of menin that is essential for liver tumorigenesis, significant work remains to identify the functions of MLL in HCC. It is clear that better definitions of active histone modification in HCC are important steps in the design of improved therapeutic strategies. HCC is the sixth most prevalent cancer, and systemic chemotherapy has marginal activity and frequent toxic effects (10). MI-2 (menin inhibitor-2), a small molecule inhibitor that is based on the menin structure, has been shown to effectively inhibit leukemia cell proliferation by disrupting the menin–MLL

　　Fig. 6. Yap1 is epigenetically regulated by menin in human HCCs, and it is correlated with poor prognosis. (A) IHC staining of Yap1 in HCC paraffin sections (n = 80). (Scale bar, 500 μm and 100 μm, respectively.) (B and C) Kaplan–Meier curves for overall and tumor-free survival in Yap1–(+) and (–) HCC patients (P = 0.000 and P = 0.000, respectively, log rank test). (D) Serum AFP level of HCC patients with Yap1–(+) (n = 32) or (–) (n = 46). In each panel,the line indicates the median (P = 0.004). (E) Hyperexpression of menin and Yap1 in overlapping HCC patients (P = 0.014, χ2 test). (F and G) ChIP assays using antibodies against menin or H3K4me3 in four cases of matched HCC and adjacent tissue from the fresh specimens (n = 3). (H) A model for the menin–MLL complex up-regulates Yap1 transcription through H3K4me3 in HCC.

　　complex, highlighting the potential therapeutic application of inhibitors targeting the menin–MLL complex in human diseases　(25). Similarly, considering the importance of the menin–MLL complex in controlling the aggressive nature of HCCs, we propose a unique therapeutic strategy for HCC by targeting the menin–MLL complex. Further studies will be required to evaluate the therapeutic effect of epigenetic-targeting drugs, such as MI-2, on drug-resistant, aggressive HCC. Site-specific histone modifications are major epigenetic mechanisms for maintaining stable gene transcription, which is fundamental to the development of most cancers (26). Here, we present the profile of genomic occupancy of menin and two associated transcriptionally active histone methylation markers, H3K4me3 and H3K79me2, in liver cancer cells. These methylation markers are associated with many liver cancer-related genes, including Yap1. Notably, Hippo-dependent or -independent mechanisms of Yap1 transcription remain to be identified. Recently, Wu et al. demonstrated that the Ets family member GABP binds to the Yap promoter and activates YAP transcription in liver cancer (27). Our results indicate that menin binds to Yap1 promoter loci and upregulates H3K4me3. The menin–MLL complex and Yap1 are constitutively activated in primary HCC patients and DENinduced liver cancer, and they serve as critical HCC tumor promoters. The aberrant expression of the menin–Yap1 axis is strongly correlated with poor prognosis, suggesting the clinical significance of the menin–Yap1 axis as a biomarker for HCC diagnosis and prognosis evaluation. Epidemiological surveys indicate that HCC occurs mainly in men and that men are about three to five times more likely to develop HCC than women (28). Similar sex disparity is seen in mice given DEN (29). Estrogen suppresses IL-6 production in liver KCs and reduces liver cancer risk in females but not in males (29). Our results suggest that Men1+/- dramatically repressed the development of HCC in female mice but not in male mice. We also observed that menin participates in the liver injury response of male mice at early stages, and menin activation was more readily compensated upon liver injury in males than in females. It is reported that menin expression, which contributes to gestational diabetes in females, is repressed by increased prolactin and progesterone (30). So we suppose that the expression of menin was regulated by estrogen only in female mice. This is likely one of the reasons for sex biases of menin in our liver cancer animal model. It will be interesting to determine whether the homozygous deletion of Men1 can repress development of HCC in males and determine how menin expression is regulated by estrogen in the liver. Conditional knockout mice that are viable and have the Men1 gene inactivated in their livers would allow further assessment of menin in liver disease processes, which would advance the understanding of the precise mechanism by which menin contributes to HCC. Here, we did not find a significant sex bias ofhyperexpression of menin in HCC specimens. However, we cannot rule out the possibility that the result was limited by an insufficient number of female samples.Altogether, we propose that augmented menin activation during chronic liver injury plays a crucial role in promoting hepatocarcinogenesis (Fig. 6H).

　　Materials and Methods

　　Human HCC Samples. The study was approved by the Xiamen University Medical Ethics Committee. Frozen and paraffin-embedded primary HCC tissues and corresponding adjacent nontumorous liver samples were obtained from the Chronic Liver Disease Biological Sample Bank, Department of Hepatobiliary Surgery, Zhongshan Hospital Xiamen University. These samples were from male and female patients histopathologically diagnosed as having stage I–IV HCC. The demographic data and clinicopathological features of the HCC patients are listed in Tables S1 and S3. The ages of the cases ranged from 25 to 75 y. In total, we examined 89 HCC samples together with adjacent normal tissues. Sections from paraffin-embedded samples were stained with affinitypurified antimenin or anti-Yap1 (Cell Signaling) antibodies (Table S4) for IHC. The specificity of the antimenin antibody was verified in menin-null and menin-expressing cells (19). The method and procedure of IHC are as described previously (19). The expression of menin and Yap1 in livers was determined from the IHC results by three independent pathologists. ACKNOWLEDGMENTS. We thank Dr. Francis Collins at National Human Genome Research Institute for providing the heterozygous Men1 locus (Men1+/-) mice and Dr. Xianxin Hua for critical reading of the manuscript.This research was supported by grants from the Natural Science Foundation of China (91229111 and 81272719 to G.-H.J., 81101924 to S.-H.L., and 81101763 to S.-B.G.), the Natural Science Foundation of Fujian Province (2011J06016 to G.-H.J.), and the Natural Science Foundation of Xiamen(3502Z20104001 to G.-H.J.).