Abstract
Pancreatic islet β-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the β-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting β-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 β-cells. Co-immunoprecipitation 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 chromatin immunoprecipitation 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 phospho-HLXB9 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 β-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 β-cell expansion strategies to alleviate β-cell loss in diabetes.
Highlights
Pancreatic-islet -cell tumors that lack menin express the phospho-isoform of the differentiation factor HLXB9
Whole cell extract (WCE) were prepared from mouse insulinoma MIN6 -cells (MIN6-4N is a tetraploid (4N)) transfected with empty vector or plasmids expressing normal myc-Histagged HLXB9 or its phospho-dead mutant
In vivo interaction of HLXB9 with Nono was confirmed by co-IP and Western blot analysis of WCE prepared from MIN6-4N cells transfected with mh-HB9-WT (Fig. 1D) and from untransfected cells (Fig. 1E)
Summary
Pancreatic-islet -cell tumors (insulinomas) that lack menin express the phospho-isoform of the differentiation factor HLXB9. 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. 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 findings support therapeutic implications from modulating phospho-HLXB9 or its targets in insulinomas
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