Targeting the PHD/HIF Pathway to Mitigate Radiation Induced Gl Toxicity

对胃肠道（GI）的正常组织毒性经常发生在放化疗中，是一个主要的临床挑战。不幸的是，目前还没有有效的治疗方法来解决这一重大的临床问题。胃肠道的腔上皮细胞存在于生理性缺氧中，因此缺氧信号对其存活和正常功能至关重要。缺氧诱导因子-1（HIF-1）是细胞缺氧反应的核心转录因子，其水平与细胞氧张力成反比波动。稳定的HIF-1表达已被证明是至关重要的正常肠道稳态以及在炎症的挑战期间的生存。HIF-1是维持肠的几种基本功能所必需的，例如屏障和吸收功能。增加肠中HIF-1的表达可以改善上皮屏障功能，增加营养吸收，减少对感染和炎症应激的细胞凋亡。相反，肠特异性敲除HIF-1导致结肠炎小鼠模型中上皮细胞凋亡增加。HIF的氧依赖性调节主要通过含脯氨酰羟化酶结构域（PHD）的蛋白介导，所述蛋白使HIF上的脯氨酸部分羟化，并充当von Hippel Lindau（VHL）蛋白的识别位点，所述VHL蛋白靶向HIF以进行蛋白酶体破坏。迄今为止，已经鉴定了三种氧依赖性脯氨酰羟化酶（PHD 1 -3），然而，每种异构体在肠内稳态中的作用尚不清楚。由于PHD蛋白调节HIF水平，而HIF反过来又保护肠道免受炎症应激，因此我们假设PHD蛋白调节胃肠道中的放射敏感性，使得特定PHD同种型的缺失将通过HIF介导的对上皮完整性和隐窝再生的作用来提供肠道的放射保护。在本提案中，我们将：1）用项目2和核心B研究PHD 1-3对低分次和分次辐射的胃肠道辐射保护的特异性贡献，2）用项目3确定PHD抑制的辐射保护机制; 3）用项目2探讨PHD 1-3在肠干细胞辐射反应中的作用，4）用项目2确定小分子PHD抑制剂保护胃肠道的功效;和5）测定PHD 1- 3敲除和PHD活性的小分子抑制剂对肿瘤生长的响应相对于正常组织的辐射防护与项目4。该项目代表了辐射防护剂发展的一个新的范式变化，我们正在研究通过改变上皮屏障功能来实现组织辐射防护的生理基础。

Normal tissue toxicity to the gastrointestinal (Gl) tract occurs frequently with chemoradiotherapy and represents a major clinical challenge. Unfortunately, no effective treatments exist to combat this significant clinical problem. The luminal epithelia of the Gl tract exist in physiologic hypoxia, and thus hypoxic signaling is crucial to the their survival and normal functions. Hypoxia-inducible factor-1 (HlF-1) is a transcription factor at the heart of the cellular hypoxia response whose levels fluctuate inversely with cellular oxygen tension. Stabilized HIF-1 expression has been shown to be critical for normal intestinal homeostasis as well as survival during inflammatory challenges. HIF-1 is required to maintain several basic functions of the intestine, such as barrier and absorptive functions. Augmenting HIF-1 expression in the intestine improves epithelial barrier functions, increases nutrient absorption and reduces apoptosis in response to infection and inflammatory stress. Conversely, intestinal specific knockouts of HIF-1 cause increased epithelial apoptosis in murine models of colitis. The oxygen dependent regulation of HIF is chiefly mediated through the prolyl hydroxylase domain (PHD)-containing proteins which hydroxylate proline moieties on HIF, and serve as a recognition site for the von Hippel Lindau (VHL) protein, which targets HIF for proteasomal destruction. To date, three oxygen-dependent prolyl hydroxylases have been identified (PHD1-3), however, the roles of each isoform in intestinal homeostasis is not clear. Because PHD proteins regulate HIF levels, and HIF, in turn protects the gut from inflammatory stress, it is our hypothesis that the PHD proteins regulates radiosensitivity in the gastrointestinal tract, such that deletion of specific PHD isoforms will afford radioprotection of the gut through HIF-medlated effects on epithelial integrity and crypt regeneration. In this proposal, we will: 1) investigate the specific contributions of PHD 1-3 on radioprotecting the Gl tract from hypofractionated and fractionated radiation with Project 2 and Core B, 2) determine the mechanisms of radioprotection by PHD inhibition with Project 3; 3) explore the role of PHD 1-3 in the radiation response ofthe intestinal stem cell with Project 2, 4) determine the efficacy of a small molecule PHD inhibitor to protect the Gl tract with Project 2; and 5) determine the therapeutic efficacy of PHD 1-3 knockouts and small molecule inhibitors of PHD activity on tumor growth in response to radiation relative to normal tissue radioprotection with Project 4. This project represents a new paradigm change in the development of radioprotectors in that we are investigating the physiological basis of tissue radioprotection through alterations in epithelial barrier function.