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。在MEN1患者中观察到的其他内分泌肿瘤包括无功能肾上腺肿瘤、无功能前肠类癌-胃神经内分泌肿瘤(NET)、胸腺网和支气管肺网。MEN1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，以及平滑肌瘤-食道肌瘤和子宫肌瘤。我们研究了在胰岛β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β细胞分化的因子表达的影响。我们发现，β细胞分化因子HLXB9(MNX1)在脑膜素丢失时转录后上调。HLXB9在脑膜素存在的情况下引起MIN6胰岛素瘤β细胞的凋亡。因此，HLXB9的失调预示了胰岛素瘤中β细胞增殖的可能机制，这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β细胞中的作用机制。我们还发现，HLXB9被GSK-3β磷酸化，磷酸化的HLXB9和GSK-3β在小鼠和人胰岛素瘤中都有表达，GSK-3β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可能是致癌的。我们证明了HLXB9中的最小相互作用区域对应于未知函数的内部保守区。这一区域可以用来设计一种合成肽来破坏内源性HLXB9-NONO相互作用，并研究其对β细胞增殖的影响。我们已经确定了临床相关的HLXB9和NONO突变的生物学后果：在新生儿糖尿病患者中发现了两种不同的HLXB9纯合子胚系突变(p.F248L和p.F272L)，这是一种功能性β细胞缺陷的情况；在内分泌相关肿瘤中发现了两种体细胞杂合性NONO突变，p.H146R(甲状旁腺腺瘤)和p.R293H(小肠神经内分泌肿瘤)。在MIN6细胞中，HLXB9/p.F248L突变蛋白定位于细胞核内，但缺乏磷酸化，NONO/p.R293H突变蛋白结构受损。因此，在β细胞过度增殖(胰岛素瘤)的情况下，缺乏与β细胞丢失(糖尿病)相关的突变中的HLXB9磷酸化，以及大量的磷酸化HLXB9，突出了调节HLXB9磷酸化作为治疗靶点的重要性。 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的上调。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与MEN1相关的神经内分泌肿瘤的c-met蛋白表达。 我们还在研究MEN1相关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)的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。MEN1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分析已经显示了MEN1相关胸腺肿瘤(胸腺瘤和Th-Net)亚型之间的不同基因表达特征。 为了了解遗传、表观遗传和转录改变的影响，如MEN1/DAXX/ATRX基因突变、分化因子PDX1和ARX的表达以及端粒交替延长(ALT)，我们对MEN1相关和散发性十二指肠胰脏网络中的组织特异性分子特征进行了综述。我们研究了基于血液的标记作为预测MEN1患者肿瘤特异性事件的生物标记物的可能性，并在MEN1相关的十二指肠胰腺网络中发现了多胺标记，以及与肿瘤进展相关的蛋白质组标记。

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 (ALT). We have investigated the potential of blood-based signatures as biomarkers to predict tumor specific events in MEN1 patients and found a polyamine signature in MEN1-associated duodenopancreatic NETs, and a proteomic signature associated with tumor progression.