Role of tissue differentiation factors in endocrine tumorigenesis

编码脑膜素的MEN1基因的胚系突变易发生内分泌肿瘤，主要发生在甲状旁腺、垂体前叶和胰腺内分泌组织。我们研究了在胰岛细胞肿瘤(胰岛素瘤)的发病机制中，脑膜丢失引起的这种组织特异性肿瘤发生的分子基础。这种组织特异性的原因可能是由于脑膜素对一个或多个组织特异性因子的调节，例如那些在胚胎发育过程中控制分化的因子。因此，我们评估了薄荷素的丢失或增加对已知控制细胞分化的因子表达的影响。我们发现-细胞分化因子HLXB9(Mnx-1)在脑膜素丢失后转录上调。HLXB9在脑膜素存在的情况下引起MIN6胰岛素瘤细胞的凋亡。因此，HLXB9的调节失调预示了胰岛素瘤细胞增殖的可能机制，可能是由于脑膜素丢失时HLXB9的促凋亡活性可能被阻断所致。这些发现促进了人们对薄荷素等普遍表达的蛋白质如何控制胰腺组织特异性肿瘤发生的理解。此外，我们的数据揭示了HLXB9及其靶点在细胞内的作用机制。我们还发现HLXB9被GSK-3磷酸化，磷酸化的HLXB9和GSK-3在小鼠和人胰岛素瘤中都有表达，GSK-3抑制剂(如氯化锂)抑制了小鼠胰岛素瘤细胞系的细胞增殖和延缓了细胞周期的进展。 为了了解磷酸化HLXB9促进肿瘤发生的分子机制，我们在胰岛素瘤细胞中鉴定了磷酸化HLXB9的相互作用蛋白和直接靶基因。我们发现一个生存因子NONO(非POU结构域包含八聚体结合蛋白，也被称为p54nrb，54 kDa核RNA结合蛋白)与HLXB9的磷酸异构体特异地相互作用，解释了为什么磷酸化的HLXB9可能是促癌的。我们通过反磷酸化HLXB9芯片-Seq确定的另一个靶点是c-Met抑制剂Cblb，它被磷酸化的HLXB9下调，从而导致c-Met上调。因此，我们的数据表明，靶向胰岛素瘤中的HLXB9-NONO相互作用和致癌受体c-met可能具有治疗作用。事实上，脑膜素缺失的小鼠模型中的胰岛素瘤表现出致癌的c-Met途径的激活(增加了磷酸化的HLXB9，减少了cblb，增加了c-Met)。对胰岛素瘤和其他胰腺神经内分泌肿瘤的进一步研究将有助于探索这些途径的相关性和c-Met抑制剂治疗的潜力。

Germline mutations in the MEN1 gene encoding menin predispose to endocrine tumors mainly of the parathyroids, anterior pituitary and entero-pancreatic endocrine tissues. We have investigated the molecular basis of this tissue specific tumorigenesis from menin loss in the pathogenesis of tumors of the pancreatic islet -cells (insulinoma). It is possible that the cause of the tissue-specificity is due to menin-mediated regulation of one or more tissue-specific factors such as those that control differentiation during embryogenesis. Therefore, we assessed the effect of menin loss or gain on the expression of factors that are known to control -cell differentiation. We found that the -cell differentiation factor HLXB9 (Mnx-1) is post-transcriptionally upregulated upon menin loss. HLXB9 causes apoptosis in the presence of menin, in MIN6 insulinoma -cells. Thus, dysregulation of HLXB9 predicts a possible mechanism for -cell proliferation in insulinomas resulting from the possible blockade of the pro-apoptotic activity of HLXB9 upon menin loss. These findings advance the understanding of how a ubiquitously expressed protein such as menin controls tissue-specific tumorigenesis in the pancreas. Moreover, our data reveal the mechanisms of action of HLXB9 and its targets in -cells. We also showed that HLXB9 is phosphorylated by the kinase GSK-3, both phospho-HLXB9 and GSK-3 are expressed in mouse and human insulinomas, and GSK-3 inhibitors (such as lithium chloride) reduced cell proliferation and delayed cell cycle progression of mouse insulinoma cell lines. In order to understand the molecular mechanisms by which phospho-HLXB9 promotes tumorigenesis, we have identified interacting proteins and direct target genes of phospho-HLXB9 in insulinoma cells. We found that a survival factor Nono (Non-POU domain-containing octamer binding protein, also known as p54nrb, 54 kDa nuclear RNA binding protein) interacts specifically with the phospho isoform of HLXB9 explaining why phospho-HLXB9 could be pro-oncogenic. Another target that we have identified by anti-phospho-HLXB9 ChIP-Seq is the c-Met inhibitor Cblb, which is downregulated by phospho-HLXB9 that would lead to upregulation of c-Met. Thus our data propose that targeting the HLXB9-NONO interaction and the oncogenic receptor c-MET in insulinomas may be therapeutic. Indeed, insulinomas from the mouse models of menin-loss show activation of the oncogenic c-Met pathway (increased phospho-HLXB9, reduced Cblb and increased c-Met). Further investigations in insulinomas and other pancreatic neuroendocrine tumors will help to explore the relevance of these pathways and the potential of c-Met inhibitor therapy.