Shenzhen Key Laboratory of Viral Oncology, The Clinical Innovation & Research Center (CIRC), Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China.
The Third School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong, China.
J Exp Clin Cancer Res. 2022 Oct 13;41(1):301. doi: 10.1186/s13046-022-02504-0.
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.
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.
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.
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.
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（CTC）被认为是转移的前体细胞，是导致 BC 患者死亡的主要原因。然而，目前为止，BC 中 CTC 形成的分子机制还没有得到充分的定义。
对早期 BC 患者（CTC≥5 和 CTC=0 分别）的原发组织进行 RNA-seq 分析，并在 80 例未经治疗的 BC 患者中进行验证研究。在功能研究中使用了多种体外和体内模型。在机制研究中使用了荧光素酶报告基因、染色质免疫沉淀测序（ChIP-seq）、CUT&Tag-seq 和 GST 下拉等实验。通过 CanPatrol™ CTC 分类系统或 LiquidBiospy™ 微流控芯片计数 CTC。将 ERK1/2 抑制剂 SCH772984 应用于体内治疗。
在原发性 BC 组织中，FOXD1 的高表达与 BC 患者中 CTC 的增加显著相关，尤其是在早期 BC 患者中。过表达 FOXD1 通过促进肿瘤细胞的上皮间质转化，增强了 BC 细胞的迁移能力、CTC 的形成和 BC 的转移。机制上，FOXD1 被发现通过直接结合 RalA 启动子来诱导 RalA 的表达。然后，RalA 与 ANXA2 和 Src 形成复合物，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。这一系列反应最终导致 ERK1/2 信号的激活，增强了 BC 细胞的转移能力。此外，体内应用 ERK1/2 的特异性抑制剂 SCH772984 可显著抑制 CTC 的形成和 BC 的转移。
在这里，我们报告了一个 FOXD1 依赖性的 RalA-ANXA2-Src 复合物，它通过激活 ERK1/2 信号促进 BC 中的 CTC 形成。FOXD1 可作为评估 BC 转移风险的预后因素。该信号级联可用于药物干预，从 BC 的早期阶段克服 CTC 的形成。
