FOXD1-dependent RalA-ANXA2-Src complex promotes CTC formation in breast cancer.

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.

Conclusion: 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.