乳腺癌相关成纤维细胞（CAFs）FOSL2-WNT5A/FAM3C信号促VEGF非依赖性肿瘤血管形成研究

Angiogenesis plays a key role in tumor initiation and development. VEGF-VEGFR signaling has been recognized to control angiogenesis process. However, it has been challenged by its intrinsic refractoriness and resistance of anti-VEGF and VEGFR TKI in clinic, which indicates a VEGF-VEGFR independent angiogenic mechanism. Our previous data showed that the tumor microvessels have a positive relationship with cancer-associated fibroblasts (CAFs) in breast tumor tissues. Axitinib, one of VEGFR inhibitor, cannot completely inhibited the pro-angiogenesis capacity of CAFs, suggesting a VEGF-VEGFR independent angiogenesis in CAFs. The vascular-related transcription factor, FOSL2, and its target WNT5A and FAM3C have been identified in CAFs and preeclampsia by bio-information assay, and experimentally certified that FOSL2 and its targets mediate the angiogenesis via VEGF-independent manner. Here, we try to further verify the pro-angiogenic function of FOSL2 and its targets WNT5A and FAM3C in CAFs. Luciferase and CHIP assay, qRT-PCR, Western blotting, signaling-specific inhibitors will be employed to identify the key signaling to regulate expressions of FOSL2 and its target WNT5A and FAM3C in CAFs. The pro-angiogenesis functions of FOSL2, WNT5A and FAM3C will be tested by tube formation and 3D sprouting assay in co-culture system of HUVEC and supernatants with or without VEGF-neutralized antibody, and specific inhibitor against VEGFR. The activities of VEGF-VEGFR independent downstream signaling pathways will be detected by Western blotting in HUVEC and transplantation tumor tissues from nude mouse. The results may reveal a novel mechanism of breast cancer CAFs in tumor angiogenesis, which will provide a new therapeutic target and guidance for clinical tumor anti-angiogenic therapy.

肿瘤血管形成是肿瘤发生发展的关键，VEGF靶向药物不应性和耐受示肿瘤血管形成的复杂本质。前期发现：癌相关成纤维细胞（CAFs）与乳腺癌血管形成密切，具VEGF非依赖促血管形成能力；CAFs、子痫前期组织存在FOSL2及靶分泌因子WNT5A、FAM3C高表达；实验证实FOSL2促CAFs的血管形成，但不上调VEGF。暗示FOSL2-WNT5A/FAM3C信号轴参与CAFs促VEGF-非依赖血管生成。本项目拟以LUC、CHIP-assay、qRT-PCR、WB等分析CAFs信号活化对FOSL2及FOSL2对WNT5A/FAM3C表达调控；基因过表达、siRNA干扰、去VEGF上清液共培养、特异抑制剂干预等研究FOSL2、WNT5A、FAM3C促HUVEC出芽、成管能力、裸鼠血管形成、VEGF非依赖信号通路活化。研究结果有望揭示乳腺癌CAFs促肿瘤血管形成的新机制，为抗血管肿瘤治疗提供新靶点。

经过4年来努力，课题组全体人员共同努力，圆满完成了本课题的各项研究内容，取得了如下丰硕的成果：1. 乳腺肿瘤微环境基质CAF中特异转录因子FOSL2通过调控WNT5A以VEGF非依赖的方式促进肿瘤血管形成。研究成果揭示estrogen/cAMP/PKA刺激CAFs中FOSL2异常表达，转录调控了Wnt5a的表达，导致分泌因子WNT5A上调与异常分泌，特异激活血管内皮中FZD5/NF-B/ERK信号，导致VEGF非依赖的肿瘤血管形成。WNT5A是一个评估乳腺肿瘤患者预后不良的良好指标。2. 揭示lncRNA SNHG5是乳腺癌CAFs特异表达的ncRNA。LncSNHG5通过调控转录因子ZNF281 mRNA稳定性上调CCL2、CCL5表达激活血管内皮中的P38 MAPK信号，一方面下调与内皮细胞紧密连接相关的ZO-1、Occludin表达，影响血管渗透性；另一方面CCL2、CCL5影响内皮细胞成管、出芽，从而直接参与肿瘤血管形成。该研究成果首次阐明了CAFs通过影响远处组织中与转移前微生境的形成。3. 揭示lncRNA 408（lnc408）促乳腺肿瘤转移及其临床诊断价值。lnc408作为miR-654-5p的海绵样吸附体，通过ceRNA机制抑制miR-654-5p，上调LIMK1表达。LIMK1通过磷酸化p-cofilin影响肌动蛋白F-actin的稳定；同时通过磷酸化CREB调控MMP2、ITGB1、COL1A1表达，发挥了促乳腺肿瘤侵袭转移。.本项目发表高水平SCI收录论文3篇（其中2篇IF>10.0）；正在审稿SCI论文1篇；CSCD核心期刊论文1篇。培养博士研究生2名、硕士研究生2名。