Roles of HLXB9 in insulinoma cells

Pancreatic islet !-cells that lack the MEN1 encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the !-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting !-cell tumors (insulinomas). In this study, we investigated the binding-partners and target-genes of phosphoHLXB9 in mouse insulinoma MIN6 !-cells. Coimmunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54 kDa nuclear RNA binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatinimmunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phosphoHLXB9 as a pro-oncogenic factor by interacting with a survival factor, and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing !-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono, and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in !-cell expansion strategies to alleviate !-cell loss in diabetes. Human or mouse pancreatic islet !-cells that lack the MEN1 encoded tumor suppressor protein menin develop into pancreatic neuroendocrine tumors (PanNETs or PNETs) (13). Inactivating MEN1 mutations have been http://www.jbc.org/cgi/doi/10.1074/jbc.M115.661413 The latest version is at JBC Papers in Press. Published on September 4, 2015 as Manuscript M115.661413 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from Roles of HLXB9 in insulinoma cells 2 observed in 40% of human sporadic nonfunctioning PNETs (not associated with hormone hypersecretion) (4). However, sporadic functioning PNETs, the insulin-secreting pancreatic islet !-cell tumors (insulinomas), rarely show MEN1 mutations (5-7). Thus, although menin loss/inactivation specifically in the !-cells can lead to tumor formation, there are many PNETs such as the insulinomas that can form without menin loss/inactivation. Various roles of menin, a predominantly nuclear protein, in transcriptional regulation and other critical cellular processes have been reported (8). However, it is unclear why menin loss leads to tumors in only specific cell types, for instance, menin loss in the whole pancreas of mice results in only insulinomas (9). In order to decipher the pathogenesis of insulinomas, we recently investigated the targets of menin in !-cells as potential candidates in the tumorigenesis process. Menin deficiency coincided with upregulation of a !-cell differentiation factor HLXB9, particularly its phospho isoform, in cell culture experiments and in tumors (10,11). Also, insulinomas without menin loss expressed phospho-HLXB9 underscoring the importance of meninindependent regulation of this protein in such tumors (10). However, how HLXB9 elicits prooncogenic activities in insulinomas is not known. HLXB9 (also known as HB9, MNR2, and MNX1) is a transcription factor with a dual expression profile during pancreas and !-cell development in embryogenesis, and later in adult !-cells (12). The expression of HLXB9 in the adult pancreas is !-cell specific (13). Hlxb9 mice are viable but show dorsal pancreas agenesis and small islets in the rest of the ventral pancreas (13,14). HLXB9 is phosphorylated by GSK-3! at Ser-78 and Ser-80, and the level of phosphoHLXB9 is increased upon menin knockdown in mouse insulinoma MIN6 !-cells (10). HLXB9 is pro-apoptotic in MIN6 cells; however, phosphoHLXB9 is not pro-apoptotic because HLXB9 expression together with menin knockdown in MIN6 cells does not cause apoptosis (11). This indicates unique activities of phospho-HLXB9 in insulinoma cells. c-MET (also known as HGFR or MET) is a receptor for hepatocyte growth factor (HGF). Aberrant activation of HGF/MET signaling (through c-MET mutation or increased expression) has been observed in a variety of tumors (15). Although activating c-MET mutations have not been reported in PNETs, they show increased cMET expression (16,17). c-Met knockout mice are embryonic lethal but conditional loss of c-Met in !-cells results in smaller islets and impaired insulin secretion (18-20). Mouse studies have also shown that c-Met expression or signaling could be utilized for !-cell regeneration strategies in diabetes (21). Thus, proper regulation of c-MET is critical for normal growth and proliferation of !cells. CBLB (Casitas B-lineage lymphoma b) is an E3 ubiquitin ligase that belongs to the CBL protein family (CBL, CBL-b and CBL-c) (22). CBL proteins can ubiquitinate activated receptor tyrosine kinases (RTK), such as c-MET, and target them for lysosomal or proteasomal degradation, thus negatively regulating various signaling pathways (22,23). Loss of CBL protein function predicts increased RTK levels and activity that could lead to increased growth and cancer. Studying the activities of HLXB9 in insulinomas can have dual benefits for understanding !-cell tumorigenesis and also to unravel endogenous !-cell replication mechanisms to replace !-cells in conditions of !-cell loss such as in diabetes. In order to study the mechanisms by which HLXB9 functions in insulinomas, we investigated the binding partners and direct target genes of phospho-HLXB9 in mouse insulinoma MIN6 !-cells. Phospho-HLXB9 interacted with Nono (Non-POU domain-containing octamerbinding protein) in the nucleus. Nono, also known as p54nrb (54 kDa nuclear RNA binding protein), is associated with RNA processing, DNA repair and transcriptional regulation; and it has been shown to act as a survival factor in melanoma cells (24-26). Using Phospho-HLXB9 ChIP-Seq we found that the c-Met inhibitor, Cblb, is a direct target of phospho-HLXB9, another possible reason for pro-oncogenic effects from HLXB9. We also found an inverse correlation between Cblb and cMet expression in mouse insulinomas. Our findings support therapeutic implications from modulating phospho-HLXB9 or its targets in insulinomas. EXPERIMENTAL PROCEDURES Plasmids, shRNA, siRNA, and antibodies The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) by gest on M arch 4, 2020 hp://w w w .jb.org/ D ow nladed from Roles of HLXB9 in insulinoma cells 3 (Invitrogen), mouse-HLXB9 (pcDNA3.1-mhHB9-WT and pcDNA3.1-mh-HB9-AA (phosphodead mutant of HLXB9 with alanine substitution at Serine-78 and Serine-80)) (11), pcDNA3.1-mhmenin (27), pCMV6-XL4-CBLB (Origene, SC107022), pFlag-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA (28), and control and Nono shRNA (29). The following siRNA were used: negative control (Qiagen, 1027280), and mouse HLXB9 (Dharmacon, L049859-01). For luciferase reporter assays the promoter-less pEZX-PG02 vector, and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-CblbSDM2 was constructed by site-directed mutagenesis of the two HLXB9-binding motifs at -735 and -722 in the mouse Cblb promoter (Agilent, quick-change site-directed mutagenesis kit). The following antibodies were used: Mouse anti-HB9 (DSHB, 81.5C10), Rabbit anti-HB9 (Bethyl, A303183A), Rabbit anti-HB9-PO4 (10), Rabbit anti-menin (Bethyl, A300-105A), Mouse anti-myc-tag (Millipore, 05-724), Rabbit anti-myctag (Millipore, 06-549), Mouse anti-p54 (Nono) (Millipore, 05-950), Mouse anti-Cblb (Santa Cruz, sc-8006), Rabbit anti-c-Met (Santa Cruz, sc-10), Rabbit anti-H3K4me3 (Active Motif, 39159), Rabbit anti-H3K27me3 (Millipore, 07-449), Mouse anti-Flag-tag (Sigma, F3165), Mouse antiHA-tag (Cell Signaling, 2367), Mouse anti-!-actin (Sigma, A1978), Rabbit anti-Histone H3 (Millipore, 06-755), Rabbit anti-HSP90 (Cell Signaling, 4877), Mouse anti-p84 (Gene Tex, GTX70220), Chicken anti-Insulin (Abcam, Ab14042), Mouse secondary antibody (HRP) (Santa Cruz, sc-2055), Rabbit secondary antibody (HRP) (Santa Cruz, sc-2054), Alexa Fluor 594 goat anti-chicken secondary antibody (Life technologies, A11042), Alexa Fluor 594 goat antimouse secondary antibody (Life technologies, A21132), Alexa Fluor 488 goat anti-rabbit secondary antibody (Life technologies, A11008). Cell culture and transfection A mouse insulinoma cell line, MIN6-4N was cultured in low glucose DMEM supplemented with 15% FBS and 1X antibiotic/antimycotic (Invitrogen, Gemini) (complete DMEM) at 37 ̊C and 5% CO2. MIN6-4N is a tetraploid (4N) cell line that was generated by isolating single cell clones from the mixed ploidy (2N and 4N) MIN6 cell line (10,30). Plasmids or siRNA were transfected using Lipofectamine 2000 (Invitrogen) or nucleofection (AMAXA/Lonza). RNA, chromatin or protein was isolated 48h or 96h post-transfection. For menin knockdown combined with HLXB9 overexpression, shRNA plasmids were transfected by nucl