Role of tissue differentiation factors in endocrine tumorigenesis

编码menin的MEN 1基因的种系突变易患主要发生在甲状旁腺、垂体前叶和肠胰腺内分泌组织的内分泌肿瘤。在MEN 1患者中观察到的其他内分泌肿瘤包括无功能性肾上腺肿瘤、无功能性前肠类癌-胃神经内分泌肿瘤（NET）、胸腺NET和支气管肺NET。MEN 1的非内分泌表现包括皮肤病变-脂肪瘤、面部血管纤维瘤和胶原瘤，中枢神经系统肿瘤-脑膜瘤和室管膜瘤，以及平滑肌肿瘤-食管平滑肌瘤和子宫平滑肌瘤。我们已经研究了这种组织特异性肿瘤发生的分子基础，这种肿瘤发生是由于胰岛β细胞肿瘤（胰岛素瘤）的发病机制中的menin缺失。组织特异性的原因可能是由于menin介导的一种或多种组织特异性因子的调节，例如那些在胚胎发生期间控制分化的因子。因此，我们评估了menin缺失或获得对已知控制β细胞分化的因子表达的影响。我们发现β细胞分化因子HLXB 9（MNX 1）在menin缺失后转录后上调。在存在menin的情况下，HLXB 9在MIN 6胰岛素瘤β细胞中引起细胞凋亡。因此，HLXB 9的失调预测了胰岛素瘤中β细胞增殖的可能机制，该机制可能是由于menin缺失时HLXB 9的促凋亡活性被阻断。这些发现促进了对一种普遍表达的蛋白质如menin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HLXB 9及其在β细胞中的靶点的作用机制。我们还发现，HLXB 9被激酶GSK-3 β磷酸化，磷酸化HLXB 9和GSK-3 β在小鼠和人胰岛素瘤中表达，GSK-3 β抑制剂（如氯化锂）可降低小鼠胰岛素瘤细胞系的细胞增殖并延迟细胞周期进程。 为了了解磷酸化HLXB 9促进肿瘤发生的分子机制，我们鉴定了胰岛素瘤细胞中磷酸化HLXB 9的相互作用蛋白和直接靶基因。我们发现生存因子NONO（非POU结构域八聚体结合蛋白，也称为p54 nrb，54 kDa核RNA结合蛋白）与HLXB 9的磷酸化亚型特异性相互作用，解释了为什么磷酸化HLXB 9可能是促癌的。我们发现，在HLXB 9中的相互作用的最小区域对应于一个未知功能的内部保守区域。该区域可用于设计合成肽以破坏内源性HLXB 9-NONO相互作用并研究对β细胞增殖的影响。我们已经确定了临床相关的HLXB 9和NONO突变的生物学后果：HLXB 9中两种不同的纯合子生殖系突变，（p.F248L和p.F272L），在新生儿糖尿病患者中发现，这是一种功能性β细胞缺乏症;以及内分泌相关肿瘤中的两个体细胞杂合NONO突变，p.H146R（甲状旁腺腺瘤）和p.R293H（小肠神经内分泌肿瘤）。在MIN 6细胞中，HLXB 9/p.F248L突变蛋白定位于细胞核但缺乏磷酸化，NONO/p.R293H突变蛋白结构受损。因此，与β-细胞损失（糖尿病）相关的突变导致的HLXB 9磷酸化的缺乏和过度β-细胞增殖（胰岛素瘤）条件下磷酸化-HLXB 9的丰富水平突出了调节HLXB 9磷酸化作为治疗靶标的重要性。 我们通过抗磷酸化HLXB 9 ChIP-Seq鉴定的另一个靶标是c-MET抑制剂CBLB，其被磷酸化HLXB 9下调，导致c-MET上调。因此，我们的数据表明，在胰岛素瘤中靶向致癌受体c-MET可能是治疗性的。事实上，来自脑膜蛋白缺失小鼠模型的胰岛素瘤显示致癌c-Met途径的激活（磷酸化HLXB 9增加、Cblb减少和c-Met增加）。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和相互作用的相关性，以及c-MET抑制剂治疗的潜力。我们正在进行的工作包括调查各种MEN 1相关的神经内分泌肿瘤的c-MET蛋白表达。 我们还研究了MEN 1相关的胸腺神经内分泌肿瘤（Th-NET）和胸腺瘤（有或没有神经内分泌分化）中组织特异性肿瘤发生的分子基础-统称为胸腺类癌，它们来自胸腺中罕见的内分泌细胞。MEN 1患者中的这些胸腺肿瘤由于其侵袭性、频繁复发和缺乏有效治疗而成为死亡的主要原因。RNA分析揭示了MEN 1相关胸腺肿瘤（胸腺瘤和Th-NET）亚型之间不同的基因表达特征。 MEN 1相关和散发性胰腺NETs的组织特异性分子特征已被审查，以了解遗传，表观遗传和转录组学改变的影响，如MEN 1/DAXX/ATRX基因突变，分化因子PDX 1和ARX的表达，以及端粒的替代延长。我们已经研究了基于血液的签名作为生物标志物预测MEN 1患者组织特异性肿瘤进展的潜力，并在MEN 1相关的胰腺NET中发现了多胺签名。

Germline mutations in the MEN1 gene encoding menin predispose to endocrine tumors mainly of the parathyroids, anterior pituitary and entero-pancreatic endocrine tissues. Other endocrine tumors observed in MEN1 patients include nonfunctioning adrenal tumors, nonfunctioning foregut carcinoids - gastric neuroendocrine tumor (NET), thymic NET, and bronchopulmonary NET. The non-endocrine manifestations of MEN1 include skin lesions - lipomas, facial angiofibromas, and collagenomas, the central nervous system tumors - meningioma and ependymoma, and smooth muscle tumors - esophageal leiomyoma and uterine leiomyoma. We have investigated the molecular basis of this tissue specific tumorigenesis from menin loss in the pathogenesis of tumors of the pancreatic islet beta-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 beta-cell differentiation. We found that the beta-cell differentiation factor HLXB9 (MNX1) is post-transcriptionally upregulated upon menin loss. HLXB9 causes apoptosis in the presence of menin, in MIN6 insulinoma beta-cells. Thus, dysregulation of HLXB9 predicts a possible mechanism for beta-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 beta-cells. We also showed that HLXB9 is phosphorylated by the kinase GSK-3beta, both phospho-HLXB9 and GSK-3beta are expressed in mouse and human insulinomas, and GSK-3beta 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. We showed that the minimal region of interaction in HLXB9 corresponds to an internal conserved region of unknown function. This region could be used to design a synthetic peptide to disrupt the endogenous HLXB9-NONO interaction and to study the impact on beta-cell proliferation. We have determined the biological consequence of clinically relevant HLXB9 and NONO mutations: two different homozygous germline mutations in HLXB9 (p.F248L and p.F272L) that were found in patients with neonatal diabetes, a condition with functional beta-cell deficiency; and two somatic heterozygous NONO mutations in endocrine-related tumors, p.H146R (parathyroid adenoma) and p.R293H (small intestine neuroendocrine tumor). In MIN6 cells, HLXB9/p.F248L mutant protein localized in the nucleus but lacked phosphorylation, and NONO/p.R293H mutant protein was structurally impaired. Thus, the absence of HLXB9 phosphorylation from a mutation associated with beta-cell loss (diabetes) and the abundant level of phospho-HLXB9 in a condition of excessive beta-cell proliferation (insulinoma) highlights the importance of modulating HLXB9 phosphorylation as a therapeutic target. 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 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 interactions, and the potential of c-MET inhibitor therapy. Our ongoing work includes a survey of various MEN1-associated neuroendocrine tumors for c-MET protein expression. We are also examining the molecular basis of the tissue-specific tumorigenesis in MEN1-associated thymic neuroendocrine tumors (Th-NETs) and Thymoma (with or without neuroendocrine differentiation) - collectively known as thymic carcinoids that arise from the rare endocrine cells in the thymus. These thymic tumors in MEN1 patients are a major cause of mortality due to their aggressive nature, frequent recurrence, and lack of effective treatment. RNA analysis has revealed distinct gene expression signatures among the sub-types of MEN1-associated thymic tumors (Thymoma and Th-NETs). Tissue specific molecular signatures in MEN1-associated and sporadic duodenopancreatic NETs have been reviewed to understand the impact of genetic, epigenetic, and transcriptomic alterations, such as MEN1/DAXX/ATRX gene mutations, expression of differentiation factors PDX1 and ARX, and alternative lengthening of telomeres. We have investigated the potential of blood-based signatures as biomarkers to predict tissue-specific tumor progression in MEN1 patients and found a polyamine signature in MEN1-associated duodenopancreatic NETs.