背景:早期转移是导致乳腺癌(BC)预后不良的关键因素。循环肿瘤细胞(CTCs)被认为是肿瘤转移的前体细胞。这最终是BC死亡的主要原因。然而,迄今为止,BC中CTC形成的分子机制尚未得到充分定义。
方法:在早期BC患者(CTC≥5和CTC=0)的原代组织中进行RNA-seq,并在80例未经治疗的BC患者中进行验证研究。在功能研究中使用多种体外和体内模型。荧光素酶报告基因,ChIP-seq,CUT&Tag-seq,和GST下拉,等。用于机械研究。通过CanPatrol™CTC分类系统或LiquidBiospy™微流体芯片对CTC进行计数。ERK1/2抑制剂SCH772984用于体内治疗。
结果:观察到原发性BC组织的高表达FOXD1与BC患者的CTC增加显著相关,尤其是早期BC患者。过表达FOXD1增强了BC细胞的迁移能力,CTC形成和BC转移,通过促进肿瘤细胞的上皮-间质转化。机械上,发现FOXD1通过直接结合RalA启动子诱导RalA表达。然后,RalA与ANXA2和Src形成络合物,促进ANXA2和Src之间的相互作用,从而增加ANXA2的磷酸化(Tyr23)。抑制RalA-GTP形式减弱了ANXA2和Src之间的相互作用。该级联在增强BC细胞转移能力的ERK1/2信号的激活中达到顶峰。此外,用SCH772984(ERK1/2的特异性抑制剂)进行体内治疗用于显著抑制CTC形成和BC转移。
结论:这里,我们报道了FOXD1依赖性RalA-ANXA2-Src复合物,该复合物通过激活BC中的ERK1/2信号促进CTC的形成。FOXD1可作为评估BC转移风险的预后因素。该信号级联是可药用的,并且对于克服来自BC早期阶段的CTC形成是有效的。
BACKGROUND: Early metastasis is a key factor contributing to poor breast cancer (BC) prognosis. Circulating tumor cells (CTCs) are regarded as the precursor cells of metastasis, which are ultimately responsible for the main cause of death in BC. However, to date molecular mechanisms underlying CTC formation in BC have been insufficiently defined.
METHODS: RNA-seq was carried out in primary tissues from early-stage BC patients (with CTCs≥5 and CTCs = 0, respectively) and the validation study was conducted in untreated 80 BC patients. Multiple in vitro and in vivo models were used in functional studies. Luciferase reporter, ChIP-seq, CUT&Tag-seq, and GST-pulldown, etc. were utilized in mechanistic studies. CTCs were counted by the CanPatrol™ CTC classification system or LiquidBiospy™ microfluidic chips. ERK1/2 inhibitor SCH772984 was applied to in vivo treatment.
RESULTS: Highly expressed FOXD1 of primary BC tissues was observed to be significantly associated with increased CTCs in BC patients, particularly in early BC patients. Overexpressing FOXD1 enhanced the migration capability of BC cells, CTC formation and BC metastasis, via facilitating epithelial-mesenchymal transition of tumor cells. Mechanistically, FOXD1 was discovered to induce RalA expression by directly bound to RalA promotor. Then, RalA formed a complex with ANXA2 and Src, promoting the interaction between ANXA2 and Src, thus increasing the phosphorylation (Tyr23) of ANXA2. Inhibiting RalA-GTP form attenuated the interaction between ANXA2 and Src. This cascade culminated in the activation of ERK1/2 signal that enhanced metastatic ability of BC cells. In addition, in vivo treatment with SCH772984, a specific inhibitor of ERK1/2, was used to dramatically inhibit the CTC formation and BC metastasis.
CONCLUSIONS: Here, we report a FOXD1-dependent RalA-ANXA2-Src complex that promotes CTC formation via activating ERK1/2 signal in BC. FOXD1 may serve as a prognostic factor in evaluation of BC metastasis risks. This signaling cascade is druggable and effective for overcoming CTC formation from the early stages of BC.