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

项目总结 口腔鳞状细胞癌(OSCC)是头颈癌中最常见的一种，居第十位。 世界范围内频发的人类恶性疾病。在过去的几十年里，口腔鳞癌患者的总体存活率 尽管在诊断和治疗方面取得了一些进展，但仍停滞不前40%~55%。侵入性增长或 口腔鳞状细胞癌的进展依赖于癌细胞的侵袭性及其独特的微环境，因此 肿瘤干细胞(CSCs)和浸润性肿瘤相关巨噬细胞(TAMs)起着关键作用。上一首 研究表明，多胺(PA)通常在肿瘤微环境(TME)和 早已被证明是各种类型癌症转化和发展所必需的。EIF5A2， 一种高度保守的翻译因子的亚型，在许多类型的癌症中过度表达。值得注意的是， 亚精胺介导的eIF5A激活(EIF5Ahpu)是由两种高度专门化的酶实现的， 脱氧亚硫氨酸合成酶(DHS/DHPS)和脱氧亚精氨酸羟基酶(DOHH)似乎是大多数生物所必需的， 如果不是全部，eIF5A的生物学功能，包括它在调节癌细胞增殖方面的重要作用， 上皮-间充质转化(EMT)和CSC特性以及免疫细胞功能，从而合理地 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 EIF5A2及参与PA代谢的几种关键酶的mRNA表达全面上调 免疫印迹和免疫组织化学研究证实了HNSCC的表达。我们的研究表明，阻止 DHPS/eIF5Ahpu显著抑制口腔鳞状细胞癌细胞的增殖和CSC特性，这与 Twist1-BMI1表达下调和NOTCH1/HES1信号转导。与此同时，我们发现阻止 DHPS/eIF5Ahpu强效抑制M2样TAM的OSCC诱发极化并逆转 口腔鳞状细胞癌诱导的TAMS体外对T细胞活化的免疫抑制作用更重要的是， 我们发现，阻断DHPs/eIF5Ahpu显著抑制肿瘤生长和M2样细胞的渗透/极化 在同种异体原位小鼠舌鳞状细胞癌模型中的应用。基于这些令人信服的初步研究，我们 推测eIF5Ahpu可能在口腔鳞状细胞癌的生长和发展中起关键作用，因为它在 口腔鳞癌细胞增殖/干细胞特性的调控及口腔鳞状细胞癌诱导的M2样极化。测试我们的 假设，我们提出了三个具体的目标：1)阐明eIF5Ahpu调控细胞增殖的机制 和口腔鳞状细胞癌中的CSC特性；2)决定eIF5Ahpu是否在口腔鳞癌诱发极化中起关键作用 3)靶向eIF5AhPU抑制口腔鳞癌体内肿瘤生长和免疫抑制TAMs。 这一应用的新发现可能不仅有助于阐明eIF5AhPU激活在 口腔鳞癌的发展和进展，但也为寻找新的治疗靶点提供了希望 口腔鳞癌的治疗和预防。

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.