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

高危HPV阴性的头颈部鳞状细胞癌(HNSCC)的预后仍然很差。 2尽管接受了积极的治疗。一种可能的解释是由异常引起的治疗耐药性的发展。 3肿瘤血管生成的调控。受体酪氨酸激酶受体EphB4及其膜结合 EFNB2是一种配体，它既能发出信号，又能促进肿瘤和血管生成 5在多种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EphB4和EFNB2基因表达升高与预后的关系 7例。我们发现EphB4主要在癌细胞上表达，而EFNB2主要在癌细胞上表达 8表达于内皮细胞(EC)。我们的数据还显示，通过重组融合靶向HNSCC肿瘤 阻断EphB4-EFNB2相互作用的9蛋白促进癌细胞凋亡，减少 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显示，与FasR、bax、bim和caspase 3裂解凋亡标记物的增加有关 12以及VEGFR2/3、JAK2和STAT3信号成分的减少。基于这些数据，我们假设 13 EphB4和EFNB2在肿瘤-EC连接处的相互作用同时导致激活 14癌细胞的存活和抗凋亡途径以及EC的血管生成途径。因此，我们 15提出，它的抑制会减少肿瘤血管/淋巴管的生成，减少癌细胞的凋亡。 16这种EphB4-EFNB2相互作用定义了一种新的癌症-EC“突触”，其中重点是串扰和 17不只是在内皮细胞上，从而提供了一个比目前可用的抗肿瘤药物更有效的治疗机会 18种血管生成疗法。在目标1中，我们将区分靶向EphB4的隔间效应- 19表达癌细胞与表达EFNB2的内皮细胞，使用EFNB2诱导的基因敲除小鼠模型 20在成体内皮细胞中选择性缺失EFNB2。具有shRNA和显性阴性的小鼠HNSCC细胞系 21(DN)敲除癌细胞的EphB4将被植入，并将被灌流和免疫荧光 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和目标3中，深入审讯 23个潜在的EphB4-EFNB2相互作用的信号机制将在体内组织以及 24种肿瘤-EC共培养检测使用shRNA和DN敲除EphB4和EFNB2。靶向抑制 25个JAK2-STAT3-ERK和FasR、Bax和PI3K将用遗传和药物抑制剂进行评估 26它们在血管萌发和癌细胞凋亡中的作用。将进行邻近结扎试验以评估 27细胞内和细胞间的相互作用。最后，来自体内小鼠模型的组织以及来自临床的 28项在HNSCC中加入EphB4-EFNB2抑制剂的试验将使用RNAseq、血管生成和 29个细胞凋亡阵列。了解血管系统和血管系统之间相互作用的机制基础 30癌细胞将打破阻碍血管生成领域数十年的障碍。

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.