肿瘤相关成纤维细胞分泌的WNT2作为食管鳞癌治疗新靶向的研究

Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignances of the digestive tract. Despite advances in diagnosis and multimodality therapies, ESCC continues to carry a grim prognosis. The major reason for this poor survival is the fact that ESCC frequently metastasizes to regional and distant lymph nodes, even at initial diagnosis. Earlier studies from our group found that the tumor fibroblast secreted WNT2 plays a critical role in the malignant progression of esophageal cancer cells via Wnt/β-catenin signaling pathway, supporting the importance of the cancer microenvironment in the development and progression of esophageal cancer. Our preliminary data have demonstrated that conditioned medium (CM) from WNT2 transfected CHO (Chinese Hamster Ovary) cells induces stemness markers (NANOG, SOX2, OCT4 and ALDH1A1) in KYSE30 cells, indicating that stromal signal WNT2 may promote cancer cell metastasis by inducing stem-like cells. Next, we treated the KYSE30 cells cultured in CHO-WNT2-CM with or without anti-WNT2 mouse monoclonal antibody (mAb). The results showed that the anti-WNT2 mouse mAb inhibits the β-catenin nuclear translocation in KYSE30 cells. Importantly, the therapeutic potential of the anti-WNT2 mouse mAb has been demonstrated in a subcutaneous mouse xenograft model showing the paracrine WNT2 bioactivity. In the present research proposal, we extend our studies to investigate therapeutic potential of the microenvironment factor WNT2 in ESCC. Hence, the objectives in the present proposal will be achieved through the following interrelated specific aims: 1) to investigate the biological effects of anti-WNT2 mouse mAb that block secreted WNT2 in ESCC; 2) to investigate the mechanism underlying the biological effects of anti-WNT2 mouse mAb in ESCC; and 3) to investigate the role of anti-WNT2 mouse mAb in mouse xenograft model. These research objectives will be tackled with a multidisciplinary approach combining biochemistry, molecular and cell biology, and animal model. This proposed study will help elucidate the future clinical implications of the WNT2 therapeutic antibody in esophageal cancer.

前期研究发现，肿瘤相关成纤维细胞 (TF)分泌的WNT2蛋白通过激活食管鳞癌细胞Wnt/ β-catenin信号通路以及促进EMT过程的发生，进而参与食管鳞癌的恶性发展进程。预实验结果显示，含有WNT2的条件培养基可诱导食管鳞癌细胞中干细胞标志物表达增强，提示肿瘤微环境因子WNT2与食管肿瘤干细胞的相关性。此外，当鼠源WNT2特异性单克隆抗体中和条件培养基内的WNT2之后，与对照组相比，肿瘤细胞β-catenin的核转移被显著抑制。更重要的是，初步实验结果显示WNT2单抗可有效抑制裸鼠皮下移植瘤的生长，因而成功建立WNT2单抗治疗的体内动物模型。为了进一步了解肿瘤微环境因子WNT2是否可以作为单抗治疗食管癌新的分子靶向，本课题拟采用体外体内实验相结合的方法，详细阐述WNT2单抗通过拮抗旁分泌WNT2对食管鳞癌细胞产生的生物学影响及机制，为发展新型食管癌的治疗方法提供坚实的理论基础。

食管鳞状细胞癌（ESCC）是我国常见的消化系统恶性肿瘤之一。尽管目前在食管癌的诊断和综合治疗方面都取得了相当进展，但其预后仍然不尽人意。导致这种不良预后的主要原因是食管癌容易发生局部和远端的淋巴结转移。我们前期研究发现，食管肿瘤相关成纤维细胞 （CAF）分泌的WNT2蛋白通过激活食管癌上皮细胞的WNT/β-catenin和EMT信号通路促进其侵袭和转移，提示分泌性因子WNT2可成为针对食管肿瘤微环境靶向疗法的理想分子。本课题进一步发现了ESCC细胞中能够与CAF分泌WNT2配体特异性结合的受体FZD7。食管癌组织芯片免疫组化染色显示FZD7蛋白在ESCC肿瘤组织普遍高表达，并与食管癌患者预后不良密切相关。体外细胞实验结果显示WNT2通过FZD7受体发挥其促ESCC细胞转移与干性的功能。为了阻滞WNT2与FZD7之间的相互作用进而抑制ESCC细胞生长与转移，我们研制了具有特异性WNT2蛋白结合能力的鼠源性WNT2单抗，并在有无重组人源性WNT2蛋白（rWNT2）培养的ESCC细胞中加入WNT2单抗（50μg/ml）或加入相同浓度IgG对照处理；采用Wound-healing、Transwell以及干细胞成球等实验分别比较WNT2单抗与对照IgG处理后ESCC细胞的迁移、侵袭及干性特征，发现WNT2单抗能够通过拮抗旁分泌WNT2显著抑制ESCC细胞的侵袭、转移和干性。 机制研究发现WNT2单抗通过抑制WNT/β-catenin和EMT信号通路拮抗WNT2的促ESCC细胞迁移和侵袭能力，并通过抑制p-STAT3降低旁分泌WNT2诱导食管肿瘤干细胞的作用。此外，我们还发现WNT2单抗与STAT3抑制剂在体外对ESCC细胞的迁移和侵袭具有协同抑制作用。裸鼠体内实验结果显示WNT2单抗能够显著抑制食管癌皮下移植瘤和淋巴结转移，同时对裸鼠重要脏器无明显损害。综上所述，食管癌CAFs特异性分泌的WNT2蛋白通过结合ESCC上皮细胞的FZD7受体促进食管鳞癌恶性演进，采用本课题自主研发的WNT2单抗中和拮抗肿瘤微环境中的WNT2蛋白，不仅能够有效阻断ESCC细胞与CAFs的相互作用，从而阻止食管肿瘤进展，并为瞄准恶性实体肿瘤微环境进行高效低毒的食管癌生物治疗提供新思路。