HIF-1α上调新型分泌蛋白Canopy2-2促血管生成、诱导EMT促进结直肠癌侵袭转移的分子机制

Metastasis is the main cause of patient death in patients with colorectal carcinoma (CRC). Our recent studies have demonstrated that Canopy2 isoform 1(CNPY2-1) was a secreted angiogenic growth factor. We newly found that Canopy2 isoform 2（CNPY2-2）was a novel secreted protein that significantly induced angiogenesis. CNPY2-2 frequently overexpressed in CRC tissue and peripheral serum, which was positively correlated with the poor prognosis. The Gene Expression Microarray results indicated that CNPY2-2 was associated with epithelial-mesenchymal transition（EMT）. Follow the above project, a series of in vivo and in vitro experiments will be used to further investigate the following issues by combining with CRC clinical samples: 1) to indentify effects of HIF-1a on improving CNPY2-2 expression in CRC cell lines and its associated molecular signaling pathway and mechanisms; 2) to elucidate the molecular mechanisms of CNPY2-2 in promoting CRC cells angiogenesis, EMT, invasion and metastasis through down-regulating p53 pathway; 3) finally, to verify that CNPY2-2 can promote CRC cell invasion and metastasis and find out the relative targets by animal model. The effect of HIF-1a in up-regulating CNPY2-2 expression to promote angiogenesis, invasion and metastasis, as well as its specific molecular mechanisms will be discoved. Our study may provide better experimental basis and novel clues for further revealing the molecular mechanisms of CRC metastasis and potential new targets of anti-cancer treatment.

结直肠癌（CRC）转移是病人最主要的死因。我们前期证实发现CNPY2亚型2（CNPY2-2）是一种新型分泌蛋白、可能促进新生血管形成。最近我们在CRC研究的预实验中发现：CNPY2-2在CRC患者肿瘤组织及外周血中高表达、且与不良预后显著正相关；进一步细胞芯片结果提示CNPY2-2表达与上皮间质转化(EMT)相关。本项目将承前启后，拟在一系列体内外实验中、结合临床检测与分析：1）明确CNPY2-2依赖HIF-1α表达水平的调控机制；2）研究并解析CNPY2-2促CRC血管生成和通过抑制p53通路启动EMT的分子机制；3）在CRC裸鼠肝转移模型中，观察并确证CNPY2-2可促进CRC侵袭转移。深入阐明HIF-1α上调CNPY2-2促血管生成并启动EMT促进CRC侵袭转移的确切分子机制，为揭示CRC转移机理和寻找抗肿瘤治疗新靶点提供实验研究基础和新线索。

本研究《HIF-1α上调新型分泌蛋白Canopy2-2促血管生成、诱导EMT促进结直肠癌侵袭转移的分子机制》（No.81871991）旨在探索结直肠癌相关分泌性蛋白CNPY2参与调控肿瘤相关巨噬细胞向M2型极化及PD-L1水平上调相关机制研究，拟通过系统评价CNPY2血清浓度对直肠癌治疗疗效及预后预测的研究，寻找肿瘤免疫抑制性微环境的形成新的分子靶点。本研究通过体外和体内实验观察了肿瘤相关CNPY2水平与临床不良预后相关，随后深入研究其参与调控肿瘤微环境中TAMs功能表型相关机制研究。结果发现：(1)CNPY2 超表达的的肿瘤细胞，与细胞外间质细胞高表达CNPY2呈现正相关，并且与不良OS及PFS相关。(2) 采用多重免疫荧光组化空间分析提示间质CNPY2表达位置与巨噬细胞重叠，并与相应的抑制性表型密切相关。（3）缺氧诱导肿瘤产生的CNPY2蛋白可促使TAM由M1型转变为M2型，同时PD-L1水平明显上调；另外机制上提示与内质网应激及代谢重编程关系密切。(4)动物实验证敲降CNPY2可以改善CRC肿瘤微环境并协同抗肿瘤作用。（5）此外研究首次确定了RCCI与CLO患者预后之间的关系，并揭示了RCC1在DNA修复和E2F靶标通路中的可能功能。（6）可以通过TROP2表达来对CLO患者进行分层，确定TROP2的表达状态可以帮助制定全面的辅助治疗方案。总的来说，我们的研究与临床治疗密切结合，丰富了 CRC 与 TAMs之间“cross talk”的分子机理，为协同抗肿瘤治疗提供了个体化精准治疗的潜在分子靶点和实验依据。