Background: The skeletal system is a common site for metastasis from breast cancer. In our prior work, we developed induced tumor-suppressing cells (iTSCs) capable of secreting a set of tumor-suppressing proteins. In this study, we examined the possibility of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes. Methods: The efficacy of ACPs was examined using an MTT-based cell viability assay, a Scratch-based motility assay, an EdU-based proliferation assay, and a transwell invasion assay. To evaluate the mechanism of inhibitory action, a fluorescence resonance energy transfer (FRET)-based GTPase activity assay and a molecular docking analysis were conducted. The efficacy of ACPs was also tested using an ex vivo cancer tissue assay and a bone microenvironment assay. Results: Among the 12 ACP candidates, P18 (TDYMVGSYGPR) demonstrated the most effective anticancer activity. P18 was derived from Arhgdia, a Rho GDP dissociation inhibitor alpha, and exhibited inhibitory effects on the viability, migration, and invasion of breast cancer cells. It also hindered the GTPase activity of RhoA and Cdc42 and downregulated the expression of oncoproteins such as Snail and Src. The inhibitory impact of P18 was additive when it was combined with chemotherapeutic drugs such as Cisplatin and Taxol in both breast cancer cells and patient-derived tissues. P18 had no inhibitory effect on mesenchymal stem cells but suppressed the maturation of RANKL-stimulated osteoclasts and mitigated the bone loss associated with breast cancer. Furthermore, the P18 analog modified by N-terminal acetylation and C-terminal amidation (Ac-P18-NH2) exhibited stronger tumor-suppressor effects. Conclusions: This study introduced a unique methodology for selecting an effective ACP from the iTSC secretome. P18 holds promise for the treatment of breast cancer and the prevention of bone destruction by regulating GTPase signaling.
背景：骨骼系统是乳腺癌常见的转移部位。在我们先前的研究中，我们开发了能够分泌一组肿瘤抑制蛋白的诱导肿瘤抑制细胞（iTSCs）。本研究探讨了从iTSC蛋白质组经胰蛋白酶消化后的蛋白片段中鉴定抗癌肽（ACPs）的可能性。方法：通过基于MTT的细胞活力测定、划痕迁移实验、EdU增殖实验以及Transwell侵袭实验评估ACPs的效能。为探究其抑制机制，进行了基于荧光共振能量转移（FRET）的GTP酶活性测定及分子对接分析。同时，利用离体癌组织实验和骨微环境实验验证了ACPs的效果。结果：在12个候选ACP中，P18（TDYMVGSYGPR）显示出最强的抗癌活性。P18来源于Rho GDP解离抑制因子α（Arhgdia），能有效抑制乳腺癌细胞的活力、迁移和侵袭能力。它还可抑制RhoA和Cdc42的GTP酶活性，并下调Snail和Src等癌蛋白的表达。在乳腺癌细胞及患者来源组织中，P18与顺铂、紫杉醇等化疗药物联用具有协同抑制效应。P18对间充质干细胞无抑制作用，但能抑制RANKL刺激的破骨细胞成熟，并减轻乳腺癌相关的骨质流失。此外，经N端乙酰化和C端酰胺化修饰的P18类似物（Ac-P18-NH2）表现出更强的肿瘤抑制效果。结论：本研究提出了一种从iTSC分泌组中筛选高效ACP的创新方法。P18通过调节GTP酶信号通路，在乳腺癌治疗及骨破坏防治方面具有应用潜力。
肿瘤(癌症)患者之家