Role of EphB4-ephrin-B2 interaction in regulating crosstalk between cancer cell and vascular tumor microenvironment in head and neck cancer

1高危HPV阴性头颈部鳞状细胞癌（HNSCC）的结局仍然很差 2尽管积极治疗。一种可能的解释是，由于异常的细胞毒性， 3肿瘤血管生成的调控。受体酪氨酸激酶受体EphB 4及其膜结合蛋白 4配体ephrinB 2（EFNB 2）既可以发出信号，又可以发挥促肿瘤发生和促血管生成作用。 5在许多恶性肿瘤和早期胚胎发育中的作用。我们的数据显示出很强的相关性 HPV阴性HNSCC中EphB 4和EFNB 2基因表达升高与生存结局之间的关系 7例患者。我们发现EphB 4主要在癌细胞上表达，EFNB 2主要在癌细胞上表达。 8存在于内皮细胞（EC）上。我们的数据还表明，用重组融合蛋白靶向HNSCC肿瘤， 阻断EphB 4-EFNB 2相互作用的蛋白质增强癌细胞凋亡， 10血管生成/淋巴管生成，并减少患者来源的异种移植模型中的肿瘤生长。这个我们 11证明，与Fas R、Bax、Bim和半胱天冬酶3裂解凋亡标志物的增加相关， 12以及VEGFR 2/3、JAK 2和Stat 3信号传导组分的减少。基于这些数据，我们假设 EphB 4和EFNB 2在癌-EC连接处的相互作用同时导致EphB 4和EFNB 2的激活。 14癌细胞的存活和抗凋亡途径以及EC的血管生成途径。所以我们 15提出，其抑制将减少肿瘤血管/淋巴管生成并减少癌细胞凋亡。 这种EphB 4-EFNB 2相互作用定义了一种新的癌症-EC“突触”，其中焦点是串扰， 17，而不仅仅是对EC，从而提供了比目前可用的抗- 18种血管生成疗法。在目标1中，我们将区分靶向EphB 4的房室效应， 19表达癌细胞与表达EFNB 2的EC，通过使用EFNB 2诱导型敲除鼠模型 20在成人EC中选择性缺失EFNB 2。具有shRNA和显性阴性的小鼠HNSCC细胞系 将植入EphB 4在癌细胞上的21（DN）敲低，并将灌注和免疫荧光标记在肿瘤细胞上。 22用于评估对生长和血管生成/淋巴管生成的影响。在目标2和3中，深入审讯 EphB 4-EFNB 2相互作用的潜在信号传导机制将在体内组织以及 使用EphB 4和EFNB 2的shRNA和DN敲低的24种癌症-EC共培养测定。靶向抑制 25 JAK 2-STAT 3-ERK和FasR、BAX和PI 3 K将与遗传和药理学抑制剂一起进行，以评估 26它们在血管发芽和癌细胞凋亡中的作用。将进行邻位连接试验以评价 27细胞内和细胞间的相互作用。最后，将来自体内小鼠模型以及来自临床试验的组织进行了组织切片。 将使用RNAseq、血管生成和血管生成来评估在HNSCC中并入EphB 4-EFNB 2抑制剂的28项试验。 29个凋亡阵列。了解血管系统和血管之间相互作用的机制基础， 30癌细胞将打破几十年来阻碍血管生成领域的障碍。

1 The outcomes for high-risk HPV-negative head and neck squamous cell carcinoma (HNSCC) remain poor 2 despite aggressive therapy. One possible explanation is the development of therapeutic resistance by aberrant 3 regulation of tumor angiogenesis. The receptor tyrosine kinase receptor, EphB4, and its membrane-bound 4 ligand, ephrinB2 (EFNB2), can both signal and have been shown to play pro-tumorigenic and pro-angiogenic 5 roles in numerous malignancies and in early embryonic development. Our data demonstrate strong correlation 6 between elevated gene expression of EphB4 and EFNB2 and survival outcomes in HPV negative HNSCC 7 patients. We show that EphB4 is predominantly expressed on cancer cells and EFNB2 is predominantly 8 present on endothelial cells (EC). Our data also show that targeting HNSCC tumors with a recombinant fusion 9 protein that blocks EphB4-EFNB2 interaction enhances cancer cell apoptosis, reduces 10 angiogenesis/lymphangiogenesis, and decreases tumor growth in patient-derived xenograft models. This, we 11 demonstrate, is associated with an increase in FasR, Bax, Bim, and caspase 3 cleavage apoptotic markers as 12 well as a decrease in VEGFR2/3, JAK2 and Stat3 signaling components. Based on these data, we hypothesize 13 that the interaction between EphB4 and EFNB2 at the cancer-EC junction simultaneously leads to activation of 14 survival and anti-apoptotic pathways for the cancer cell and angiogenic pathways for the EC. Therefore, we 15 propose, its inhibition will decrease tumor vascular/lymph angiogenesis and decreases cancer cell apoptosis. 16 This EphB4-EFNB2 interaction defines a novel cancer-EC “synapse,” where the focus is on the crosstalk and 17 not just on ECs, thus providing an opportunity for greater therapeutic efficacy than the currently available anti- 18 angiogenic therapies. In Aim 1, we will differentiate the compartmental effects of targeting the EphB4- 19 expressing cancer cell versus EFNB2-expressing ECs, by using an EFNB2 inducible knockout murine model 20 with selective deletion of EFNB2 in the adult ECs. Mouse HNSCC cell lines with shRNA and dominant negative 21 (DN) knockdown of EphB4 on cancer cells will be implanted and perfusion and immunofluorescence will be 22 used to assess effects on growth and angiogenesis/lymphangiogenesis. In Aims 2 and 3, in-depth interrogation 23 of signaling mechanisms underlying EphB4-EFNB2 interaction will be tested on in vivo tissue as well as 24 cancer-EC co-culture assays using shRNA and DN knockdowns for EphB4 and EFNB2. Targeted inhibition of 25 JAK2-STAT3-ERK and FasR, BAX, and PI3K will be done with genetic and pharmacologic inhibitors to assess 26 their role in vascular sprouting and cancer cell apoptosis. Proximity ligation assay will be done to evaluate 27 intracellular and intercellular interactions. Finally, tissue from the in vivo mouse model as well as from a clinical 28 trial incorporating an EphB4-EFNB2 inhibitor in HNSCC will be assessed using RNAseq, angiogenic, and 29 apoptotic arrays. Understanding the mechanistic underpinnings of interaction between the vasculature and 30 cancer cell will break down barriers that have stymied the angiogenesis field for decades.