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. Author manuscript; available in PMC: 2018 Jan 15.
Published in final edited form as: Cancer Res. 2016 Nov 21;77(2):401–411. doi: 10.1158/0008-5472.CAN-16-1567

Menin and Daxx interact to block neuroendocrine tumors through epigenetic control of the membrane metallo-endopeptidase MME

Zijie Feng 1,2,3, Lei Wang 2,3,6, Yanmei Sun 1, Zongzhe Jiang 1, John Domsic 3,4, Chiying An 2,3,7, Bowen Xing 1, Jingjing Tian 1, Xiuheng Liu 6, David C Metz 3,5, Xiaolu Yang 2,3, Ronen Marmorstein 3,4, Xiaosong Ma 1,*, Xianxin Hua 2,3,*
PMCID: PMC5243199  NIHMSID: NIHMS829704  PMID: 27872097

Abstract

Neuroendocrine tumors (NET) often harbor loss-of-function mutations in the MEN1 and DAXX tumor suppressor genes. Here we report that the products of these genes, menin and Daxx, interact directly with each other to suppress the proliferation of NET cells, to a large degree by inhibiting expression of the membrane metallo-endopeptidase MME. Menin and Daxx were required to enhance histone H3 lysine9 trimethylation (H3K9me3) at the MME promoter, as mediated partly by the histone H3 methyltransferase SUV39H1. Notably, the menin T429K mutation associated with a NET syndrome abolished Daxx binding, MME repression and proliferation of NET cells. Conversely, inhibition of MME in NET cells repressed proliferation and tumor growth in vivo. Our findings reveal a previously unappreciated crosstalk between two crucial tumor suppressor genes thought to work by independent pathways, focusing on MME as a common target of menin/Daxx to treat NET.

Keywords: Neuroendocrine tumor (NET), menin, Daxx/ATRX, Mme, epigenetic regulation

Introduction

Neuroendocrine tumors (NETs), including insulinoma, can produce excessive hormones and lead to metastatic lesions and morbidity. The prevalence and incidence have increased over the past few decades (1,2). Multiple endocrine neoplasia type1 (MEN1), an inherited autosomal dominant syndrome characterized by the development of endocrine tumors including NETs, results from mutation in the MEN1 gene that encodes the protein menin (3,4). In mouse models, heterozygous loss of Men1 leads to multiple endocrine tumors with loss of heterozygocity at the Men1 locus (5). Over 40% of patients with sporadic NETs, including insulinomas, harbor somatic mutations in the MEN1 gene in tumors (6,7), indicating that menin acts as a crucial tumor suppressor for both inherited and sporadic NETs.

Menin can up-regulate anti-proliferative genes via recruiting mixed lineage leukemia (MLL) methyltransferase complex and enhancing histone H3 lysine 4 trimethylation (H3K4me3) (8) and also down-regulate expression of pro-proliferative genes by up-regulating protein arginine methyltransferase 5 (PRMT5)-mediated histone H4 arginine 3 dimethylation (H4R3me2) (9) and SUV39H1-mediated histone H3 lysine 9 trimethylation (H3K9me3) (10).

Recently, it was reported that death-domain-associated protein (Daxx) and its interacting partner, α-thalassemia X-linked mental retardation protein (ATRX) (11,12), are also frequently mutated in NET samples (7). Mutations in Daxx and ATRX are mutually exclusive in these tumors, suggesting that Daxx/ATRX function as tumor suppressors in the same pathway to regulate NETs. In addition, the expression of Daxx/ATRX is reduced in NETs and loss of their expression correlates with advanced stages of tumors and metastasis in patients (13). Daxx is also an H3.3-specific histone chaperone and deposits H3.3 at specific chromatin regions in cooperation with ATRX (11,12). Furthermore, Daxx interacts with several other proteins involved in transcriptional repression, such as histone deacetylase 2 (HDAC2) and DEK (14), implicating that Daxx can function as a co-repressor to repress gene transcription. In Daxx/ATRX-mutated NET cells, alternative lengthening of telomere (ALT) is often observed (13,15). However, if Daxx regulates growth of NET cells is poorly understood.

Notably, MEN1 and Daxx/ATRX are the most frequently mutated genes in NETs and mutations of menin and Daxx/ATRX are not mutually exclusive in the same tumor (7), seemingly suggesting that menin and Daxx/ATRX are involved in distinct pathways in regulating NETs. However, mutations in Daxx, ATRX and H3.3 were found in the same paediatric glioblastomas (16) and yet Daxx/ATRX and H3.3 are known to act in the same chromatin remodeling pathway, indicating that different genes harboring simultaneous mutations may function in the same pathway. It is not yet known whether menin and Daxx act separately or functionally interact to regulate NETs. Here we show that menin and Daxx directly interact with each other and crosstalk to regulate proliferation of NET cells at least partly via inhibiting Mme and its underlying mechanisms.

Materials and Methods

Viral infection and cell proliferation assay

pMX-puro-menin, pMX-puro-Daxx or indicated shRNAs for retroviral packaging were co-transfected with psi-2 helper plasmid into 293 cells using the calcium chloride precipitation method, and the resulting recombinant virus were used to transduce Men1-null MEFs, Daxx-null MEFs, STC-1 or INS-1 cells, as previously reported (9). Stable Mme-expressing INS-1 cells were established by transduction with pBOB-GFP or pBOB-Mme-derived lentiviruses, as previously reported (17). The MTT assay and colony formation assay were used to quantify cell proliferation (18). Three independent experiments were performed.

Microarray analysis

Daxx−/− and Daxx+/+ or Men1−/− and Men1+/+ MEFs samples were hybridized on an Affymetrix Mouse Gene 1.0 ST chip. The data analysis was done in the statistical environment R for the quality analysis of the affyPLM library, as previously described (19). Microarray data were deposited to GEO and the accession numbers are GSE87732 for Daxx and GSE87733 for Men1 knockout microarray.

Quantitative real time PCR (qRT-PCR) and Western blotting

Total RNA was extracted from cultured cells with Trizol and an RNeasy extraction kit from Qiagen. 1 µg RNA was used as template to synthesize cDNA for qRT-PCR using a 7500 fast real-time PCR from Applied Biosystems, as previously reported (9), with primers listed in Table S4. Western blotting was performed as previously described (20).

Co-immunoprecipitation

293 cells were transiently transfected with indicated plasmids and were suspended in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5% glycerol, 1% NP-40, 1 mM EDTA) supplemented with protease inhibitors (sigma). Co-immunoprecipitation was performed as previously described (9).

In vitro binding assays

E. coli BL21 transformed with GST-WT menin, GST-T429K menin mutant, His-menin, His-Daxx fragments or GST-Daxx fragments alone or co-transformed with Daxx (183–398) and H3.3/H4 were used to express and purify the fusion proteins, as previously described (20). The purified proteins or peptides were incubated with either control IgG or anti-menin in buffer with 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% NP-40, 10% glycerol at 4°C for 2 hrs, followed by IP and immunobloting.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed as previously described using a Quick ChIP Kit from Imgenex (21). Briefly, 106-107 formaldehyde cross-linked cells were lysed and sonicated to obtain sheared DNA for ChIP assays. Oligos used for ChIP real-time PCR are provided in Table S2.

Tumor Xenografts

INS-1 or STC-1 cells stably transfected with control shRNA or Mme shRNAs were collected, and 5 × 106 viable cells in 100 µL PBS were injected subcutaneously into the flanks of 5-wk-old female athymic nude mice. The tumor volume was estimated every 3 days by measuring the tumor diameter in two dimensions with a Vernier caliper to calculate tumor volume in mm3 (volume = width2 ×length × 0.52) (22). All animal experiments were conducted with approval from Animal Ethical and Welfare Committee of Medical College at Shenzhen University.

Statistical analysis

Statistical analyses were performed using Graphpad Prism (version 5.0; Graphpad Software). The data are presented as the mean ± S.D. of n determinations unless noted otherwise. A two-tailed Student's t test was used for measuring statistical differences.

Cell Authentication

Authentication of cell lines was done by comparing STR sequences obtained from the actual cell lines as determined by IDEXX Bioresearch (Columbia, USA). Recent STR analysis has been performed within 6 months before the beginning or in the course of the experiments for all cell lines.

Results

Both menin and Daxx suppress proliferation of neuroendocrine cells

Genetic ablation of the Men1 gene in pancreatic β cells results in development of insulinoma in mice (23). As expected, we showed that ectopic expression of menin repressed proliferation of INS-1 cells, a rat insulinoma cell line (Fig. 1A and Supplemental Fig. S1A). In contrast, knockdown of menin by shRNAs increased growth of INS-1 cells (Fig. 1B and Supplemental Fig. S1B), indicating the crucial role for menin in regulating the proliferation of neuroendocrine cells. On the other hand, Daxx, a protein with multiple functions including epigenetically regulating chromatin and associating with histone H3 variant H3.3 (11,12), and its partner ATRX are somatically mutated in ~40 % of the patients with NETs (7). To explore whether Daxx also plays a role in regulating proliferation of NET cells, we knocked down Daxx expression using two distinct shRNAs (Fig. 1C, lanes 2–3) and the proliferation assay clearly showed that Daxx knockdown led to increased growth of INS-1 cells (Fig. 1C and Supplemental Fig. S1C). Together, our findings indicate that both menin and Daxx can potently suppress proliferation of the same type of neuroendocrine cells.

Figure 1. Menin and Daxx each suppress proliferation of insulinoma cells and Mme expression.

Figure 1

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(A) INS-1 cells transduced with control retroviral vector and retroviruses expressing menin were examined for menin expression and cell growth (n=3). (B and C) Western blot and growth curve of the INS-1 cells transduced with either control retroviruses or retroviruses expressing two shRNAs against menin (B) or Daxx (C) (n=3). (D and E) Detection of protein and mRNA levels of menin and Mme (D), or Daxx and Mme (E), with Western blot and qRT-PCR, respectively, in Men1 or Daxx-null MEFs complemented with indicated retroviruses expressing either WT menin or Daxx (n=3). (F) Daxx and Mme protein and mRNA levels were determined in Daxx overexpressed Men1-null MEFs (n=3). Data are shown as mean ± SD.

Menin and Daxx co-regulate expression of membrane metallo-endopeptidase, Mme (CD10)

Next, we sought to determine whether menin and Daxx interact to control certain common genes and thus regulate cell proliferation. We performed cDNA microarray analysis using Men1- or Daxx-null mouse embryonic fibroblasts (MEFs), which are sensitive to menin-mediated suppression (24), and their menin- or Daxx-complemented counterparts (Fig. 1D, E). We found that a number of genes, including Mme, DCN, and Gria3, were up-regulated in either Daxx or menin knockout MEFs (Supplemental Tables S1 and S2; and Supplemental Fig. S2A). Further, we confirmed that either Daxx or menin knockout cells have increased expression of these genes (Supplemental Fig. S2B). Among these genes co-regulated by both menin and Daxx, Mme, also called Neprilysin, common acute lymphoblastic leukemia antigen (CALLA) or cluster of differentiation 10 (CD10), is a zinc-dependent metalloprotease that cleaves peptides at the amino side of hydrophobic residues and inactivates a number of signaling peptides (25). Expression of Mme is increased in tumors such as colon cancer (26) and controversially in NETs (27,28), and its increased expression is also correlated with a high proliferative index, large tumor size and metastasis (29). As such, we chose Mme for further analysis.

We found that complementation of the MEFs with either menin or Daxx cDNA reduced Mme expression (Fig. 1D, E). Notably, ectopic expression of Daxx failed to reduce Mme expression when the Men1 gene was deleted (Fig. 1F) and vice versa (Supplemental Fig. S2C), indicating that menin and Daxx are required for each other to repress Mme expression. Together, our results unravel a common target gene co-regulated by both menin and Daxx.

Mme is required for proliferation of the neuroendocrine cells co-repressed by menin and Daxx

We further showed that overexpression of menin led to reduced Mme expression at the protein and mRNA levels in INS-1 cells (Fig. 2A). In contrast, knockdown of either menin or Daxx with two distinct shRNAs resulted in enhanced expression of the Mme protein and mRNA (Fig. 2B, C). While Mme was up-regulated in NET cells, whether it affects proliferation of the cells remains unclear. To evaluate the potential role of Mme in controlling NET cell proliferation, we generated two shRNAs targeting Mme and stably transfected them into INS-1 cells. Mme knockdown (KD) substantially reduced the proliferation of INS-1 cells (Fig. 2D, right panel).

Figure 2. Menin and Daxx-regulated Mme is crucial for optimal proliferation of insulinoma cells.

Figure 2

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(A–C) Mme protein and mRNA levels were determined in menin overexpressed (A), menin knockdown (B) or Daxx knockdown (C) INS-1 cells (n=3). (D) The INS-1 cells were stably transduced with vector or two distinct Mme-targeting shRNA-expressing retroviruses, and the Mme protein and mRNA levels were confirmed with Western blot or qRT-PCR. Number of the cells was counted (right) (n=3). (E) The INS-1 cells were treated with a Mme inhibitor, Thiorphan, followed by MTT assay (n=6). (F) Control (vector) and menin knockdown INS-1 cells (left), or control and Daxx knockdown cells (right) were treated with Thiorphan for 4 days, followed by MTT assay (n=6). Data are shown as mean ± SD.

Moreover, we treated the INS-1 cells with a small molecule Mme inhibitor, Thiorphan (30), and found that Thiorphan repressed INS-1 cell growth at different time points in a dose-dependent manner (Fig. 2E and Supplemental Fig. S3A). Next, we treated the menin or Daxx KD INS-1 cells with various concentrations of Thiorphan, and found that the Mme inhibitor repressed INS-1 cell proliferation up-regulated by either menin or Daxx KD (Fig. 2F, left and right panels). We further showed that Mme overexpression partially rescued proliferation inhibition that was mediated by ectopic expression of menin (Supplemental Fig. S3B, C). Together, these findings indicate that menin and Daxx co-regulated Mme is crucial for menin-Daxx-mediated suppression of insulinoma cell proliferation.

To further confirm our findings, we extended our studies to an additional neuroendocrine tumor cell line, STC-1 (31). As expected, overexpression of menin or Daxx reduced, while knockdown of menin increased, Mme expression and proliferation of STC-1 cells (Supplemental Fig. S4A–H), indicating a crucial role for menin in repressing the proliferation of another neuroendocrine cell line. Furthermore, both Mme knockdown and Thiorphan treatment repressed growth of STC-1 cells (Supplemental Fig. S5A–C). However, Thiorphan had little effect on growth of human 293 cells and BT474 breast cancer cells (Supplemental Fig. S5D). Together, these findings are consistent with the notion that menin and Daxx axis inhibited proliferation of neuroendocrine tumor cells at least partly via regulating Mme.

Menin interacts with Daxx via its C-terminal region and a menin point mutation from human MEN1 patients abolishes its ability to bind Daxx

It has been unclear how menin and Daxx crosstalk in controlling Mme expression, while Menin and Daxx did not regulate each other’s expression in MEFs (Supplemental Fig. S6A, B). Thus, we sought to determine whether menin and Daxx interact with each other. Co-immunoprecipitation (co-IP) results showed that ectopically expressed Daxx or menin interacted with each other (Fig. 3A, lane 4 and Supplemental Fig. S7A, lane 4). As Daxx binds to ATRX (Fig. 3A, lanes 3–4) via its N-terminal region (32), we next determined if menin also binds to ATRX. Ectopically expressed ATRX brought down menin in the co-IP assay (Fig. 3B, lanes 3–4), and interestingly, binding of ATRX to menin was further increased by ectopically expressed Daxx (Fig. 3B, lane 4), suggesting that menin, Daxx and ATRX co-exist in a complex. Furthermore, endogenous Daxx and menin also interacted in 293, INS-1 or STC-1 cells (Fig. 3C, lanes 3–5 and Supplemental Fig. S7B, C).

Figure 3. Menin interacts with Daxx via the C-terminal part of menin.

Figure 3

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(A) 293 cells were co-transfected with untagged menin and Flag-Daxx, followed by co-IP and blotting with indicated antibodies. (B) Untagged menin and Flag-ATRX were co-transfected with or without HA-Daxx into 293 cells, followed with co-IP and Western blot with indicated antibodies. (C) Detection of interaction between endogenous menin, Daxx and ATRX in 293 cells using co-IP. (D) Schematic representation of the menin fragments (top) that bound (+) or failed to bind (−) Daxx, as shown in (E), denotes minimal Daxx Binding Domain (DBD). The crystal structure of menin 396–450 amino acid residues (DBD) comprises two helixes linked by a loop (bottom, and ref. 34). (E) 293 cells were co-transfected with the indicated constructs, followed by co-IP and Western blot using the indicated antibodies. (F) 293 cells were co-transfected with either Flag tagged WT menin or the indicated disease-related menin mutations and HA-Daxx, followed by co-IP and Western blot with indicated antibodies. (G) His-Daxx (183–398) was incubated without or with biotinylated menin peptide GTCK422WEEGSPTPVLHVGWATQSE, or T429K mutant, followed by co-IP and blot with indicated antibodies.

To further identify which part of menin mediates interaction with Daxx/ATRX, we generated multiple constructs that express various menin fragments (Fig. 3D, top panel). The co-IP showed that the C-terminal part of menin bound to Daxx and that amino acid (aa) residues 396–450 (Daxx binding domain, DBD) were required for Daxx binding to menin (Fig. 3E, lanes 5 and 7 and Supplemental Fig. S8A, lane 3). The menin DBD sequence is highly conserved among different species ranging from zebrafish to human (Supplemental Fig. S9). In addition, analysis of the crystal structure of menin showed that residues of 396–450 form two helixes linked by a surface loop containing G426SPTPVLH sequence (Fig. 3D, bottom panel and ref. 34).

To explore whether menin mutations in this region affect the interaction between menin and Daxx, wild type (WT) menin or inherited MEN1 tumor syndrome-related menin mutations, W423S, T429K and W436R (33), were co-expressed with HA-Daxx in 293 cells, followed by co-IP assay. While W423S and W436R mutations, which were close to the helical parts of the menin structure (Fig. 3D, bottom), remained capable of binding to Daxx (Fig. 3F, lanes 3 and 5), the menin mutation T429K, which protrudes toward the surface of menin protein (Fig. 3D, bottom) (34), completely lost its ability to bind to Daxx (Fig. 3F, lane 4). Consistently, menin mutations A242V and H139D, which failed to bind to another partner, MLL (34), remained capable of binding to Daxx (Supplemental Fig. S8B, lanes 3–4). Furthermore, in vitro IP showed that WT menin bound to both MLL peptide and Daxx (Supplemental Fig. S8C, D). While T429K menin mutant failed to bind to Daxx, it remained capable of binding to a biotinylated MLL peptide (Supplemental Fig. S8C, lane 7 and D, lane 6), indicating that distinct domains of menin are involved in mediating interaction with Daxx and MLL.

Further, a WT biotinylated peptide (422–438) composing the surface loop did pull-down Daxx (Fig. 3G, lane 3). Notably, the menin T429K mutant peptide failed to bind to Daxx (Fig. 3G, lane 4). These findings indicate that the menin surface loop (422–438) linking the two helixes in the menin DBD is not only required for optimal Daxx binding, and is also sufficient for detecting Daxx binding.

Menin binds to the Daxx domain without partners H3.3/H4

To determine the region of Daxx that interacts with menin, we generated various Flag-tagged Daxx truncations (Fig. 4A). Co-IP results showed that the minimal examined Daxx fragment containing aa 1–160 (ATRX binding domain, ABD) interacted with ATRX (Fig. 4B, lane 8), as expected (32), but not with menin. Notably, all the Daxx fragments containing residues 157–260, which are distinct from Daxx’s ABD, were sufficient for menin binding (Fig. 4B, lane 4). To determine whether menin directly interacts with Daxx, we expressed and purified His-menin (Supplemental Fig. S10A) and GST-Daxx fragments in E. coli. In vitro co-IP showed that WT Daxx and Daxx fragments containing residues 157–260 bound to menin (Fig. 4C, lanes 3–4), whereas the Daxx fragments that did not contain these residues failed to do so (Fig. 4C, lane 5), indicating that residues 157–260 of Daxx are sufficient for directly binding to menin.

Figure 4. Daxx directly binds to menin.

Figure 4

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(A) Schematic diagram of the Daxx truncations for the study. (B) Flag-Daxx was co-expressed with untagged menin in 293 cells, followed by co-IP and blot with the indicated antibodies. Asterisk indicates the non-specific binding. (C) GST-Daxx or GST-Daxx fragments were incubated with His-menin, and pulled down with glutathione-sepharose beads, followed by Western blot using the indicated antibodies. (D) The crystal structure of Daxx fragment in complex with histone H3.3/H4 (ref. 35), the circle highlights residues 183–260 of Daxx that directly binds menin. (E) His-Daxx (183–398) alone or complexed with H3.3/H4 were incubated with His-menin, followed by co-IP and Western blot with indicated antibodies. Asterisk indicates the heavy chain of IgG. (F) Similar to (E), various indicated His-Daxx fragments were incubated with His-menin, followed by co-IP and Western blot with the indicated antibodies. (G) Lysates from WT or Daxx-null MEFs were immunoprecipitated with anti-menin or anti-ATRX antibodies, followed by Western blot using indicated antibodies. Indirect interaction between menin and ATRX through Daxx is indicated by the arrows.

Recently, a crystal structure of Daxx in complex with a histone H3.3-H4 dimer was reported (Fig. 4D and ref. 35), revealing that Daxx interacts with H3.3 and H4 through residues 183–398 (Histone binding domain, HBD) (35). Interestingly, residues 157–260 of Daxx overlap with the HBD. We found that purified Daxx fragment (183–398) (Supplemental Fig. S10B) interacted with menin (Fig. 4E, lane 6), while menin pulled down less Daxx fragment assembled with H3.3/H4 complex (Fig. 4 E, lane 7). However, we cannot rule out the probability that menin can also bind Daxx-H3.3/H4 under certain conditions in vivo.

It has been reported that aa 183–398 of Daxx form 6 helixes in association with H3.4/H4 dimer (Fig. 4D and ref. 35). A series of Daxx truncations purified from E. coli (Supplemental Fig. S10C) were incubated with menin, followed by Co-IP. The results showed that 183–260 (menin binding domain, MBD) of Daxx remained able to bind to menin (Fig. 4F, lane 4), but further deletion of 241–260 or 221–260 from Daxx abolished the binding ability (Fig. 4F, lanes 5–6). Further experiment showed that 183–260 was the minimal Daxx domain that binds to menin (Supplemental Fig. S11A, lane 4).

We further tested whether menin binds to endogenous ATRX through Daxx using Daxx-null MEFs. As expected, endogenous menin or ATRX pulled down each other, albeit modestly (Fig. 4G, lanes 3–4, as indicated by arrowhead), but menin or ATRX failed to bind each other when Daxx was deleted (Fig. 4G, lanes 6–7). In contrast, deletion of menin had no effect on the interaction between Daxx and ATRX (Supplemental Fig. S11B, lanes 2–3 vs 5–6). Together, these results indicate that menin directly binds to Daxx via the MBD, indirectly binding to ATRX through Daxx, and forms a complex with Daxx/ATRX.

Menin and Daxx are required for each other in their binding to the promoter of the Mme gene and enhancing H3K9me3 via recruiting SUV39H1

Menin interacts with various proteins, such as MLL, PRMT5 and SUV39H1 to influence gene transcription by regulating histone modifications (8–10). To investigate how menin and Daxx regulate Mme expression, we performed chromatin immunoprecipitation (ChIP) assays, and found that menin bound to the Mme promoter (Fig. 5A). Ectopic menin expression increased binding of Daxx and ATRX to the Mme promoter, but not to the β-actin promoter in INS-1 cells (Fig. 5A and Supplemental Fig. S12A). Notably, ectopic menin expression robustly enhanced the H3K9me3 level, consistent with menin-mediated increase of SUV39H1 binding to the Mme promoter (Fig. 5B). However, H3K4me3, histone H3 lysine 9 acetylation (H3K9ace) or total H3 level was not changed upon menin overexpression (Fig. 5B).

Figure 5. Menin and Daxx are required for each other in their binding to the promoter of the Mme gene, for enhancing H3K9me3, and for detecting SUV39H1, a H3K9 methyltransferase, at the Mme promoter.

Figure 5

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(A and C) ChIP assays were performed using the indicated antibodies in menin-overexpressing (A) or menin knockdown INS-1 cells (C) (n=3). Control IgG served as a negative control for ChIP assays. (B and D) ChIP assays for detecting H3K4me3, H3K9me3, H3K27me3, H3K9ace, or H3 levels at the Mme promoter, using the cognate antibodies, in menin-overexpressing (B) or menin knockdown INS-1 cells (D) (n=3). (E and F) ChIP assays for detecting menin, Daxx, ATRX, SUV39H1 (E), and for detecting H3K4me3, H3K9me3 or total H3 (F), at the Mme promoter, using the indicated antibodies, in control or Daxx knockdown INS-1 cells (n=3). Control IgG served as a negative control for ChIP assays. Data are shown as mean ± SD.

Consistently, menin KD reduced binding of Daxx, ATRX and SUV39H1 to the Mme promoter, decreased detection of H3K9me3, which can be modified by SUV39H1 (Fig. 5C, D), but did not affect the level of H3K4me3, histone H3 lysine 27 trimethylation (H3K27me3), H3K9ace or total H3 (Fig. 5C, D). As a control, no difference in binding was observed at the β-actin promoter in menin KD INS-1 cells (Supplemental Fig. S12B). Consistently, we found that binding of Daxx/ATRX, SUV39H1, and H3K9me3 (but not H3K4me3 or total H3) at the Mme promoter were markedly increased in Men1-null MEFs complemented with menin (Supplemental Fig. S13B–D).

Daxx interacts with several crucial proteins involved in transcriptional repression at epigenetic level, such as HDAC1 and HDAC2 through histone deacetylation (14,36), but whether Daxx regulates other histone modifications, like repressive methylation H3K9me3, is not yet clear. Interestingly, Daxx KD reduced binding of menin, ATRX and SUV39H1 at the Mme promoter (Fig. 5E). Notably, reduced binding of menin and SUV39H1 caused by Daxx KD at the Mme promoter was correlated with reduced H3K9me3 level, but not with reduced H3K4me3, or total histone H3 level (Fig. 5F). Moreover, ectopic Daxx expression-induced binding of menin and ATRX to, as well as H3K9me3 at, the Mme locus was also observed in Daxx- null MEFs (Supplemental Fig. S14A–D). As Daxx KD reduced binding of SUV39H1 and H3K9me3, but not H3K4me3 at Mme promoter (Fig. 5E, F), we examined whether Daxx binds to SUV39H1. Indeed, ectopic Daxx robustly bound to SUV39H1 even more than menin binding (Supplemental Fig. S15).

A menin point mutation compromises Daxx binding, H3K9me3, suppression of Mme expression and proliferation of NET cells

To determine whether the interaction of menin and Daxx/ATRX is required for inhibiting Mme expression and cell proliferation, we stably transduced INS-1 cells with vector, WT menin or menin disease-related point mutation, T429K, which we showed lost the ability to bind to Daxx (Fig. 3F, G). As expected, WT menin repressed proliferation of INS-1 cells and Mme expression (Fig. 6A, B); notably, T429K mutant lost the function of repressing either INS-1 cell proliferation or Mme expression (Fig. 6A, B). Furthermore, ChIP results showed that T429K mutation markedly reduced menin’s ability to bind to the Mme promoter and to recruit Daxx to the promoter (Fig. 6C) to increase H3K9me3 level at the promoter, but did not affect the levels of H3K4me3 or histone H3 (Fig. 6D). Similarly, ectopic expression of WT menin also repressed proliferation of STC-1 cells and Mme expression, but T429K mutant was compromised to do so (Fig. 6E, F). Consistently, ectopic menin expression increased menin binding and the level of H3K9me3, at the Mme promoter, but T429K mutant reduced the function (Fig. 6G, H). These results strongly indicate that menin/Daxx interaction is crucial for regulating neuroendocrine cells.

Figure 6. MEN1 tumor-related point mutation lost the ability to inhibit Mme expression as well as proliferation of insulinoma cells.

Figure 6

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(A) Growth curve for INS-1 cells expressing WT menin or menin disease-related point mutation, T429K (n=3). (B) Mme protein and mRNA levels were determined by Western blot and qRT-PCR (n=3). (C) ChIP assays for menin or Daxx in the cells described in (A) (n=3). (D) ChIP assays for H3K4me3 and H3K9me3 (left) or histone H3 (right) (n=3). (E) Growth curve for STC-1 cells expressing vector or WT menin and menin disease-related point mutation, T429K (n=3). (F) Mme protein and mRNA levels determined by Western blot or qRT-PCR (n=3). (G) ChIP assays in the cells described in (E) (n=3). (H) ChIP assays for H3K4me3 and H3K9me3 or histone H3 (right) (n=3). Error bars are mean ± SD.

Next, We transduced retroviral WT menin, T429K, W423S, and NLS1,2pm into menin-null MEFs for further analysis, and found that menin mutants T429K and NLS1,2pm, both of which could not bind to Daxx, had reduced ability to decrease Mme expression (Supplemental Fig. S16A, B). In contrast, WT menin and W423S mutant, which remains capable of binding to Daxx, were still able to repress Mme expression (Supplemental Fig. S16A, B). Consistently, T429K and NLS1,2pm, but not W423S, failed to bind to the Mme promoter, recruit Daxx, and further increase the H3K9me3 level (Supplemental Fig. S16C).

Knockdown of Mme suppresses tumor growth in a mouse model

To determine whether Mme has an impact on growth of NET cell-derived tumors in vivo, INS-1 or STC-1 cells stably transfected with either control or two shRNAs against Mme were subcutaneously transplanted into nude mice (n=9 per group). We found that Mme KD significantly reduced the growth of xenografted tumors (Fig. 7A, p= 0.0042 or 0.0012, respectively and Supplemental Fig. S17A). Mice were sacrificed 17 days after transplantation and tumors were harvested, weighed and photographed, and tumor weight from Mme KD tumor cells was markedly reduced (Fig. 7B and Supplemental Fig. S17B). Mme KD in the tumor cells was confirmed by qRT-PCR (Fig. 7C). Furthermore, we treatment of the tumor-harboring mice with Mme inhibitor, Thiorphan, with 2.5 or 5 mg/kg Thiorphan (n=8 per group), significantly reduced the tumor growth in mice (Figure 7D, p= 0.0001 or 0.0001, respectively), but did not affect the body weight of the mice (Figure 7E), indicating that Mme can be a potential target for treating NETs with high Mme expression.

Figure 7. Menin and Daxx-regulated Mme is crucial for tumor growth in mice.

Figure 7

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(A) Control (vector) and two lines of INS-1 cells knockdown with two distinct Mme shRNAs were transplanted into nude mice subcutaneously (n=9), and the tumor volume (mean ± SD) was since measured every 3 days. (B) The tumor weight and size. (C) Mme knockdown in the xenograft tumors as shown by qRT-PCR (#P < 0.005). (D) Effect of Thiorphan treatment on the growth of INS-1 xenografts in nude mice treated with vehicle (n=8), 2.5 (n=8) or 5 mg/kg Thiorphan (n=8) i.p every 3 days. The tumor volume was measured as in (A). (E) The tumor weight, size and body weight (#P < 0.005). Error bars are mean ± SD. (F) A model for crosstalk between menin and Daxx/ATRX in epigenetic regulation of Mme expression and NETs.

Discussion

In human NETs, several genes including MEN1 and Daxx/ATRX frequently undergo loss-of-function mutations (7). Unlike mutually exclusive mutations of Daxx and its partner ATRX in the tumors, a sign indicating that the two genes work together in the same pathway to regulate tumors, mutations of menin and Daxx in the NETs are not mutually exclusive (7). This seemingly suggests that menin and Daxx act in separate pathways. Unexpectedly, we found that menin and Daxx, seemingly unrelated to each other in suppressing NETs, interplay to suppress NETs. Menin directly interacts with Daxx via amino acids 422–438, which form a loop on the surface of the C-terminal part of menin (Fig. 3E, G). A menin point mutation derived from MEN1 patients, T429K, abolishes menin’s ability to interact with Daxx and fails to suppress proliferation of NET cells, suggesting that menin and Daxx functionally interact and that this previously unappreciated interaction between menin and Daxx is crucial for suppressing NETs. These findings are also consistent with a recent report that mutations of Daxx/ARTX are correlated with more aggressive NETs (13).

Our results are consistent with a model in which menin and Daxx each utilize their own separate signaling pathways to control NETs, as menin represses endocrine tumor growth by up-regulating anti-proliferative genes such as p18 and p27 via recruiting MLL and enhancing H3K4me3 (8), and Daxx/ATRX can also regulate chromosomal stability by regulating ALT (15). Notably and unexpectedly, menin and Daxx also closely crosstalk beyond their separate and distinct roles to suppress gene expression and tumor growth/progression at least partly via suppressing expression of Mme (Fig. 7F). It is likely that each of menin and Daxx has its own downstream effectors to suppress NETs. However, they may work together to regulate certain common genes, such as CD10. The importance of the interplay between menin and Daxx in suppressing NETs is underscored by the fact that a point mutation, T429K, failed to repress Mme expression and the tumor cell proliferation. While we observed the specific menin/Daxx interaction, the functional interaction may be more applicable to the tumor types that harbor the MEN1 mutations.

The crystal structure of menin itself, or in complex with an MLL peptide, or with a peptide from JunD, has recently been reported (34,37,38). Our domain mapping data indicates that menin binds to Daxx via amino acids 396–450 which is distinct from the MLL binding domain (34). Amino acid residues 396–450 of menin form two α helixes linked by a loop (GSPT429PVLH). MEN1 tumor-related point mutation T429K, which was found in a human insulinoma (39), is located in the middle of the loop (GSPK*PVLH) and fails to interact with Daxx.

Daxx is a histone-specific chaperone and deposits H3.3 at PML-NBs in cooperation with ATRX (11,12). Daxx/ATRX dependent H3.3 deposition at telomeres is crucial for maintaining chromosome stability. Daxx and ATRX are mutated in NETs and their mutations are correlated with features of alternative lengthening of telomeres (ALT) and chromosomal instability (13,15,16,40). Our results show that Daxx suppresses proliferation of NET cells and Daxx-mediated repressing of NETs requires the presence of menin. Unlike Daxx mutation, the menin mutation is not correlated with ALT in NET cells (7,40). The crosstalk between menin and Daxx may control NETs beyond the function of Daxx/ATRX in regulating ALT.

Daxx alone can bind to menin, but once Daxx forms a complex with H3.3/H4, its ability to bind menin is attenuated. This finding suggests that the menin/Daxx axis can work separately beyond the Daxx/H3.3 pathway in controlling H3.3 deposition (11,12), but we cannot rule out the possibility that menin/Daxx/H3.3/H4 also plays an additional role in suppressing NETs. Future studies remain to determine the detailed mechanisms regarding how menin and H3.3/H4 influence each other in their interaction with Daxx.

Mme is also correlated with metastasis of colorectal cancer and the expression of Mme in colorectal tumors is associated with higher risk of liver metastasis (41,42). Abnormal expression of Mme is found in numerous solid tumors such as NETs and expression of Mme is significantly correlated with large tumor size and the presence of metastasis (29). However, the role of Mme in NETs has not previously been explored. Our results show that Mme is a common target gene co-regulated by both menin and Daxx. Interaction of menin and Daxx is crucial for inhibiting expression of Mme. Thiorphan, a specific inhibitor of Mme, antagonizes enhanced proliferation of NET cells that was induced by knockdown of either menin or Daxx. It is not yet clear how Mme promotes proliferation of NET cells.

In summary, we uncovered a previously unappreciated mechanism whereby two crucial tumor suppressors, menin and Daxx, seemingly unrelated, crosstalk to suppress NETs by interacting with each other and epigenetically inhibiting a pro-proliferative gene in endocrine tumors, Mme, via enhancing H3K9me3 modification, which is distinct from their separate individual pathways (Fig. 7F). This mechanism is also supported by a recent report that the Daxx mutation is correlated with more malignant phenotypes of NETs (13). Our findings may pave the way to develop target-based therapy against MEN1 or Daxx mutated NETs by inhibiting their common target Mme.

Supplementary Material

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Acknowledgments

We thank Prof. Yong Liu (Shanghai Institute for Nutritional Sciences of Chinese Academy of Sciences) for the INS-1 cells, and Prof. Dinshaw Patel (Rockefeller University) for Daxx, H3.3/H4 plasmids.

Grant Support

This work was supported by grants in part from NIH/NCI R01CA178856 (X. Hua) and NIH/NIDDK R01DK097555 (X. Hua), a grant from Caring for Carcinoid Foundation 2014-565669 (X. Hua), AACR-NETRF grant (X. Hua), Natural Science Foundation of China (81402333 to Z. Feng), Shenzhen Peacock plan KQTD20140630100746562 (X. Ma), and a Commonwealth of PA grant (040-0427-4-561074-XXXX-2433-8341 to D. C. Metz).

Footnotes

Disclosure of Potential Conflicts of Interest

The authors disclose no potential conflicts of interest.

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Supplementary Materials

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NIHMS829704-supplement-1.docx (2MB, docx)
2
NIHMS829704-supplement-2.docx (45.2KB, docx)
3
NIHMS829704-supplement-3.docx (37.4KB, docx)

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