Amplification of tumor hypoxic responses by MIF-dependent HIF stabilization

描述(申请人提供)：胰腺导管腺癌(PDAC)是最不利于预后的人类癌症之一。已有研究表明，PDAC肿瘤内的低氧分压与其转移潜能和化疗耐药密切相关。低氧诱导因子(HIF)转录有助于代谢适应、新血管生成潜能、转移扩散和治疗耐药的基因产物。我们最近发现，巨噬细胞移动抑制因子(MIF)是迄今发现的最古老的细胞因子之一，它在PDAC患者的血浆中升高，并由低氧诱导的依赖于HIF-1a的转录在胰腺癌细胞中诱导。有趣的是，MIF被发现可以相互调节缺氧诱导的HIF-1a的稳定。在缺陷缺氧诱导的HIF-1a稳定和随后的HIF依赖转录中，几个人PDAC细胞系被导入针对MIF的siRNA重现MIF-/-成纤维细胞。此外，与含MIF的异种移植相比，MIF缺乏的PDAC细胞显示出有缺陷的异种移植瘤生长，HIF-1a减少，微血管密度显著降低。最后，我们的结果表明，MIF通过功能调节COP9信号体CSN5的一个亚单位来抑制独特的HIF-1特异性泛素E3连接酶，从而促进HIF-1a的稳定性。我们的中心假设是，MIF在胰腺癌病变中的过度表达起到调节游离CSN5的水平或活性的作用，而游离CSN5反过来又起到抑制或掩盖HIF-1a特有的、不依赖氧的泛素E3连接酶的作用。本申请中概述的研究试图确定MIF在PDAC生长、病理生理学和低氧适应中的贡献和需求。此外，我们还将描述MIF依赖的HIF-1稳定的确切机制和分子决定因素。为了实现这一应用的既定目标，提出了以下实验目标：1)阐明MIF对缺氧诱导的HIF-1a稳定的基本机制，2)确定MIF缺陷细胞中HIF-1降解的分子决定因素和效应因子，以及3)确定MIF是否有助于胰腺导管腺癌的生长、病理生理和低氧适应。公共卫生相关性：在整个肿瘤发生过程中，所有实体肿瘤都需要微环境适应。这种适应性反应的特征之一是肿瘤内低氧的发展，这刺激了HIF导向的促血管生成/转移基因产物的表达。我们的发现证实，其中一种基因产物MIF在一部分胰腺癌患者的血浆中升高。我们进一步描述了细胞外细胞因子/生长因子MIF和转录因子HIF-1a在胰腺导管腺癌细胞(PDAC)中的独特功能相互关系。我们的数据证明了这一点，即缺乏MIF的细胞表现出缺陷的缺氧和PHD抑制剂诱导的HIF-1a稳定以及随后代谢和血管生成基因产物的转录。更重要的是，PDAC肿瘤形成的异种小鼠模型显示，与含有MIF的细胞相比，MIF缺陷的肿瘤生长、HIF-1a表达和肿瘤相关血管生成严重受损。由于HIF的表达直接促进了肿瘤的侵袭性，而胰腺癌据报道是非常低氧的，因此MIF在功能上可能有助于胰腺癌患者的肿瘤维持、环境适应和最终预后。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) has the grim distinction of being one of the most prognostically unfavorable human cancers. It has been suggested that low oxygen tensions within PDAC tumors contribute significantly to its metastatic potential and chemotherapeutic resistance. Hypoxia-inducible factors (HIFs) transcribe gene products that contribute to metabolic adaptation, neo-angiogenic potential, metastatic spread and therapeutic resistance. We recently discovered that macrophage migration inhibitory factor (MIF), one of the oldest cytokines ever described, is elevated in PDAC patient's plasma and induced by hypoxia-induced, HIF-1a-dependent transcription in pancreatic cancer cells. Intriguingly, MIF was found to reciprocally modulate hypoxia- induced HIF-1a stabilization. Several human PDAC cell lines transfected with siRNA against MIF recapitulate MIF-/- fibroblasts in defective hypoxia-induced HIF-1a stabilization and subsequent HIF-dependent transcription. Moreover, MIF-deficient PDAC cells display defective xenograft tumor outgrowth, less HIF-1a and significantly reduced microvascular density than MIF-containing xenografts. Finally, our results indicate that MIF promotes HIF-1a stability by functionally regulating a subunit of the COP9 signalosome, CSN5, in repressing a unique, HIF-1-specific ubiquitin E3 ligase. Our central hypothesis is that MIF overexpression in pancreatic adenocarcinoma lesions acts to modulate either the levels or activity of free CSN5 that, in turn, serves to repress or mask a unique, oxygen-independent ubiquitin E3 ligase specific for HIF-1a. Studies outlined in this application seek to identify the contribution and requirements for MIF in PDAC growth, pathophysiology and hypoxic adaptation. We will additionally delineate the precise mechanisms and molecular determinants involved in MIF-dependent HIF-1 stabilization. In order to fulfill the stated objectives of this application the following experimental aims are proposed: 1) Elucidate the basic mechanism by which MIF contributes to hypoxia-induced HIF-1a stabilization, 2) Define the molecular determinants and effectors of HIF-1 degradation in MIF-deficient cells, and 3) Determine if MIF contributes to pancreatic ductal adenocarcinoma growth, pathophysiology and hypoxic adaptation. PUBLIC HEALTH RELEVANCE: All solid tumors require microenvironmental adaptation throughout tumorigenesis. One of the hallmarks of this adaptive response is the development of intratumoral hypoxia that stimulates HIF-directed expression of pro-angiogenic/metastatic gene products. Our findings establish that one of these gene products, MIF, is elevated in the plasma of a subset of pancreatic cancer patients. We further describe a unique functional inter-relationship between the extracellular cytokine/growth factor MIF and the transcription factor, HIF-1a in pancreatic ductal adenocarcinoma (PDAC) cells. This point is demonstrated by our data showing that cells lacking MIF exhibit defective hypoxia and PHD inhibitor-induced HIF-1a stabilization and subsequent transcription of metabolic and angiogenic gene products. More importantly, xenograft mouse models of PDAC tumorigenesis reveal that MIF-deficient tumor outgrowth, HIF-1a expression and tumor-associated angiogenesis are severely impaired when compared to cells containing MIF. Because HIF expression contributes directly to tumor aggressiveness and cancers of the pancreas are reportedly very hypoxic, it is likely that MIF functionally contributes to tumor maintenance, environmental adaptation and ultimately prognosis of pancreatic adenocarcinoma patients.