细胞外基质糖蛋白PCPE-1在骨肉瘤中的功能、机制及临床意义

Osteosarcoma is one of the most malignant bone tumors, and tends to metastasize to lung with poor prognosis. The deeply understanding of mechanism of metastasis is necessary for improving the treatment and prognosis of patients with osteosarcoma. We have recently performed the RNAseq by using osteosarcoma tissues, and found that procollagen C-proteinase enhancer protein (PCPE-1) was markedly up-regulated in human osteosarcoma tissues compared to non-cancerous adjacent tissues. Our preliminary results showed that knockdown or overexpression of PCPE-1 impaired or promoted migration, invasion and the lung metastasis of osteosarcoma cells. The Asn29 N-glycosylation of PCPE-1 positively regulated its protein stability and its secretion, and that this glycosylation may be modulated by ST6GALNAC3. TWIST1 as a key regulator in EMT was able to transcriptionally up-regulate PCPE-1 in osteosarcoma cells. This proposal plans to investigate the following events: To further confirm that PCPE-1 promotes metastasis of osteosarcoma cells in vitro and in vivo; To investigate the function and mechanism of Asn29 N-linked glycosylation modification of PCPE-1 in osteosarcoma cells; To further confirm that TWIST1 transcriptionally up-regulates PCPE-1 in osteosarcoma cells. To explore the clinical significance of TWIST/PCPE-1 signaling axis in patients with osteosarcoma. We believe that this proposal will provide a novel diagnostic biomarker or therapeutic strategy for patients with osteosarcoma

骨肉瘤是一类易转移，预后差的恶性骨肿瘤。深入理解骨肉瘤的转移机制对改善其治疗和预后有重要意义。我们通过骨肉瘤组织转录组测序，鉴定了细胞外基质糖蛋白PCPE-1在癌组织中表达显著高于癌旁组织。预实验显示：敲低/过表达PCPE-1能显著抑制/促进骨肉瘤细胞的迁移、侵袭和转移；PCPE-1的Asn29位点糖基化能够调控其蛋白稳定性、分泌过程，该糖基化可能受糖基转移酶ST6GALNAC3调控；EMT的关键调控因子TWIST1可转录激活PCPE-1表达。本项目拟：细胞水平、动物模型中进一步验证PCPE-1促进骨肉瘤转移的功能；研究PCPE-1的Asn29位点糖基化在骨肉瘤转移中的作用，并阐明其机制；进一步明确骨肉瘤细胞中TWIST1转录激活PCPE-1表达；探讨该TWIST1/PCPE-1通路在骨肉瘤患者中的临床价值。本项目的完成，将为骨肉瘤的诊断治疗提供新的靶标或思路。

骨肉瘤的恶性程度高、无有效药物，90%患者肺转移，急需治疗靶标。我们发现细胞外基质糖蛋白PCPE-1表达受到EMT关键因子TWIST1的转录调控，其高表达与骨肉瘤患者不良预后显著相关，机制上PCPE-1通过加速胞外基质蛋白成熟促进骨肉瘤细胞肺转移，该研究为肉瘤中的类EMT进程的存在提供了理论依据。在该工作的基础上，我们在骨肉瘤中鉴定了一种新的、有功能的exon-intron 融合基因类型 (Rab22a-NeoFs)，其融合蛋白可结合SmgGDS607，促进RhoA的活化，从而促进骨肉瘤的肺转移。我们合成了特异靶向该作用机制的穿膜多肽，可阻断该融合蛋白的功能，为骨肉瘤肺转移的靶向治疗提供了一个新的潜在靶标（Nat Cell Biol, 2020）。进一步研究我们发现该融合蛋白的第七位赖氨酸需被乙酰化才具有功能，其修饰过程受到P300/CBP调控。使用P300/CBP 的小分子化合物抑制剂水杨酸钠，可抑制其乙酰化，从而抑制该融合蛋白的促转移功能（Theranostics, 2020）。同时，我们还发现Rab22a-NeoF1融合蛋白可以通过结合HSP90，并同时携带PYK2一起分泌到外泌体，促进肺转移前微环境的形成及该融合基因阴性的细胞中RhoA活化，从而促进整个骨肉瘤细胞群体的肺转移（Signal Transduct Target Ther, 2021）。近期，我们发现了靶向药物瑞戈非尼和索拉菲尼可以靶向PINK1/Rab22a-NeoF1信号轴阻断其功能，抑制骨肉瘤的肺转移（Adv Sci, 2022）。这一系列研究有望为肺转移的骨肉瘤患者提供新的治疗靶标和策略。