Targeted inhibition of eIF5Ahpu suppresses tumor growth and M2-like TAM polarization in oral cancer

项目摘要 口腔鳞状细胞癌（OSCC）是头颈部最常见的癌症类型， 全球常见的人类恶性肿瘤。在过去的几十年里，口腔鳞状细胞癌患者的总体生存率 尽管在诊断和治疗方面取得了一些进展，但仍停留在40~55%之间。侵入性生长或 OSCC的进展依赖于癌细胞的侵袭性及其独特的微环境， 癌症干细胞（CSC）和浸润的肿瘤相关巨噬细胞（TAM）起关键作用。先前 研究表明，多胺（PA）通常在肿瘤微环境（TME）中升高， 早已被证明是各种癌症的转化和发展所必需的。eIF5A2， 一种高度保守的翻译因子的同种型，在许多类型的癌症中过表达。值得注意的是， 亚精胺介导的eIF 5A hypusination（eIF 5Ahpu）由两种高度特异性的酶实现， 脱氧羟腐胺赖氨酸合酶（DHS/DHPS）和脱氧羟腐胺赖氨酸羟化酶（DOHH），似乎是大多数人所必需的， 如果不是全部的话，eIF 5A的生物学功能，包括它在调节癌细胞增殖中的重要作用， 上皮-间质转化（EMT）和CSC特性以及免疫细胞功能，因此合理地 成为治疗和预防癌症的潜在靶点。我们对TCGA数据集的分析表明， eIF 5A 2和PA代谢中涉及的几种关键酶的mRNA表达总体上调 免疫印迹和免疫组化结果证实了HNSCC的表达。我们的研究表明， DHPS/eIF 5Ahpu可显著抑制OSCC细胞的增殖和CSC特性，其作用与细胞增殖和CSC特性有关。 下调TWIST 1-BMI 1表达和NOTCH 1/HES 1信号传导。同时，我们发现， DHPS/eIF 5Ahpu强烈抑制OSCC诱导的M2样TAM的极化，并逆转M2样TAM的极化。 OSCC诱导的TAM对体外T细胞活化的免疫抑制作用。更重要的是， 我们发现阻断DHPS/eIF 5Ahpu可显著延缓肿瘤生长和M2样细胞的浸润/极化， TAM在原位同基因小鼠舌SCC模型中的作用。基于这些令人信服的初步研究，我们 推测eIF 5Ahpu可能在口腔鳞癌生长和进展中起关键作用， 调节OSCC细胞的增殖/CSC特性和OSCC诱导的M2样TAM极化。来测试我们 假设，我们提出了三个具体目标：1）阐明eIF 5Ahpu调节增殖的机制 2）确定eIF 5Ahpu是否在OSCC诱导的极化中起关键作用 3）靶向eIF 5Ahpu以抑制体内OSCC中的肿瘤生长和免疫抑制性TAM。 这项应用的新发现不仅可能阐明eIF 5Ahpu激活在细胞内的功能， OSCC的发展和进展，但也有希望确定新的治疗靶点， 口腔鳞状细胞癌的治疗和预防。

PROJECT SUMMARY Oral squamous cell carcinoma (OSCC) is the most common type of head & neck cancer and the 10th most frequent human malignancy worldwide. Over the last several decades, the overall survival rate of OSCC patients has stagnated between 40~55% despite some progress in diagnosis and therapy. The invasive growth or progression of OSCC relies on the aggressiveness of cancer cells and their unique microenvironment, whereby cancer stem cells (CSCs) and infiltrated tumor associated macrophages (TAMs) play pivotal roles. Previous studies have demonstrated that polyamines (PA) are commonly elevated in tumor microenvironment (TME) and have long been proven to be necessary for transformation and progression of various types of cancers. eIF5A2, an isoform of a highly conserved translational factor, is overexpressed in many types of cancer. Remarkably, spermidine-mediated eIF5A hypusination (eIF5Ahpu) that is implemented by two highly specialized enzymes, deoxyhypusine synthase (DHS/DHPS) and deoxyhypusine hydroxylase (DOHH), appears to be essential to most, if not all, of eIF5A’s biological functions, including its important role in regulating cancer cell proliferation, epithelial-mesenchymal transition (EMT), and CSC properties as well as immune cell functions, thus rationally emerging as a potential target for both therapy and prevention of cancer. Our analysis of TCGA dataset indicated an overall upregulation in the mRNA expression of eIF5A2 and several key enzymes involved in PA metabolism in HNSCC, which was confirmed by Western blot and IHC studies. Our studies showed that blocking DHPS/eIF5Ahpu remarkably inhibited proliferation and CSC properties of OSCC cells, which correlated a downregulation of TWIST1-BMI1 expression and NOTCH1/HES1 signaling. Meanwhile, we found that blocking DHPS/eIF5Ahpu robustly inhibited OSCC-induced polarization of M2-like TAMs and reversed the immunosuppressive effects conferred by OSCC-induced TAMs on T cell activation in vitro. More Importantly, we found that blocking DHPS/eIF5Ahpu dramatically retarded tumor growth and infiltration/polarization of M2-like TAM in an orthotopic syngeneic mouse tongue SCC model. Based on these compelling preliminary studies, we hypothesize that eIF5Ahpu might play a critical role in OSCC growth and progression due to its dual functions in regulating proliferation/CSC properties of OSCC cells and OSCC-induced M2-like TAM polarization. To test our hypothesis, we propose three specific aims: 1) Elucidate mechanism by which eIF5Ahpu regulates proliferation and CSC properties in OSCC; 2) Determine whether eIF5Ahpu plays a critical role in OSCC-induced polarization of M2-like TAMs; 3) Target eIF5Ahpu to suppress tumor growth and immunosuppressive TAMs in OSCC in vivo. New findings from this application might not only shed light on elucidating the function of eIF5Ahpu activation in development and progression of OSCC, but also hold promises for identifying novel therapeutic targets for treatment and prevention of OSCC.