Formin homology domains of Daam1 bind to Fascin and collaboratively promote pseudopodia formation and cell migration in breast cancer

Abstract Objectives Cancer cell migration to secondary organs remains an essential cause of death among breast cancer (BrCa) patients. Cell motility mainly relies on actin dynamics. Our previous reports verified that dishevelled‐associated activator of morphogenesis 1 (Daam1) regulates invadopodia extension and BrCa cell motility. However, how Daam1 is involved in actin filament assembly and promotes pseudopodia formation in BrCa cells remains unclear. Materials and methods One hundred human BrCa samples were collected at Women's Hospital of Nanjing Medical University. Immunohistochemistry (IHC) was used to examine Daam1 and Fascin expression. Wound healing and Boyden chamber assays were used to explore cell migration and pseudopodia extension of BrCa cells. Co‐IP/pull down and Western blotting were performed to study the physical interaction between Daam1 and Fascin. Immunofluorescence assays were performed to observe whether Daam1 and Fascin were colocalized and mediated actin filament assembly. Results Fascin was upregulated in BrCa tissues compared with that in paracarcinoma tissues. The downregulation of Fascin caused a decline in pseudopodia formation and cell motility. Moreover, we found that Daam1 interacted with Fascin via formin homology (FH) domains, especially the FH2 domain. Immunofluorescence assays showed that Daam1 and Fascin partially colocalized to actin filaments, and the knockdown of Daam1 or Fascin failed to colocalize to short and curved actin filaments. Conclusions Daam1 specifically binds to Fascin via FH domains and cooperatively facilitates pseudopodia formation and cell migration by promoting actin filament assembly in BrCa.

forces, etc. 2, 3 We have demonstrated that dishevelled-associated activator of morphogenesis 1 (Daam1), a member of formin family proteins, mediates ECM-induced invadopodia extension and cell migration in breast cancer (BrCa). 4 However, the precise role of Daam1 in the assembly of actin filaments and the formation of pseudopodia is still unclear.
Abnormal molecular biological activity of the actin cytoskeleton may strengthen or impair tumour cell motility. 18 A crucial actin filament bundling protein Fascin is involved in tumour cell migration and invasion via modification of the actin cytoskeleton. [19][20][21] High expression of Fascin is shown in ovarian tumours, BrCa, non-small cell lung cancer, colon cancer, prostate cancer, etc, suggesting its oncogenic role in certain cancers mentioned above. [22][23][24][25][26] In mammalian cells, Fascin is localized in filopodia and interacts with Daam1 to promote filopodia formation. 27 Moreover, Daam1 accelerates actin assembly during mouse oocyte meiotic division through the alteration of Fascin expression. 28 However, the underlying mechanism by which Daam1 associates with Fascin to regulate actin filament assembly, pseudopodia information and cell motility, especially in BrCa, is uncertain.
Here, we performed biochemical, immunofluorescent and immunohistochemical assays to reveal the interaction between

| Plasmid constructions and transfections
Human Daam1 cDNA was obtained from our laboratory, and the full-

| Western blotting analysis
Cells cultured in 60 mm dishes were washed with PBS and then lysed with 2 × SDS sample buffer. The lysates were harvested, and abundant protein extracts were separated by 10% SDS-PAGE.

| Coimmunoprecipitation
The cells were lysed with lysis buffer containing 50 mmol/L HEPES,

| In vitro pull-down assay
His-Fascin fusion protein was induced by IPTG for 2-16 hours at 16°C and purified from BL21 bacteria. Next, the fusion protein was eluted three times from Ni-NTA agarose beads by washing buffer

| Immunohistochemistry (IHC)
One hundred BrCa pathological sections were deparaffinized at 55°C for 30 minutes. All sections were washed with xylene twice for 10 minutes each and then rehydrated by sufficient washes in 100%, 90%, 80% and 70% graded ethanol. Next, citrate buffer (pH 6.0; catalog no. P0083, Beyotime) was used to repair antigens at high temperature, and hydrogen peroxidase (0.3%; catalog no. P0100A) was applied to block endogenous peroxidase activity for 5 minutes. In addition, 2% BSA was used to block nonspecific antigens for 1 hour.

MCF-7 cells seeded in 24-well plates and invadopodia adhering to
Boyden chamber membranes were fixed in 4% paraformaldehyde with PBS for 20 minutes. Then, cells and invadopodia were permea- analysed by ZEM software (Zeiss).

| Wound healing assay
MDA-MD-231 cells were cultivated in 96-well cell culture clusters.
When grown to confluence, the cells were transfected with siRNA using Lipofectamine 2000 reagent for 6 hours, then switched to fresh DMEM with 10% FBS and further cultured for 24-48 hours.
Next, the cells were scratched artificially with a plastic pipette tip.
After two rinses with PBS to remove cell residues, the wounded cellular monolayer was permitted to heal for 6 hour in DMEM containing 10% FBS.

| Statistical
All data were analysed using GraphPad Prism 8.0 software.

| Knockdown of Fascin inhibits actin filament assembly and pseudopodia formation
To determine the effect of Fascin on the assembly of actin filaments

| Fascin is highly expressed in BrCa tissues, and downregulated Fascin suppresses the cell migration of BrCa
After understanding the potential mechanism of the inhibition of actin filament assembly owing to the knockdown of Fascin, we further validated whether decreased Fascin mediated cell motility in BrCa. A total of 100 BrCa cases were enrolled, and Fascin expression was examined in clinical samples. These pathological sections were incubated with anti-Fascin antibody via immunohistochemistry (IHC) assay. Fascin was highly expressed in BrCa compared with paracarcinoma tissues (Figure 2A,B). Also, we found that the expression level of Daam1 was associated with the status of lymph-node metastasis of BrCa (P = .040, Table S1). For cell migration assays,

| Daam1 interacts with Fascin and is highly coexpressed in the cytoplasm of BrCa cells
Because of the role of Daam1 in pseudopodia dynamics and the ef- In summary, Daam1 remarkably interacts with Fascin.

| Daam1 binds to Fascin via FH domains
To investigate which domain of Daam1 specifically interacts with

| Daam1 and Fascin recruit each other to promote the assembly of actin filaments
Having verified the physical interaction between Daam1 and Fascin, we studied whether Daam1 and Fascin were colocalized and mediated the assembly of actin filaments. Immunofluorescence assays showed that endogenous Daam1 and Fascin were colocalized with actin filaments (Figure 5A,B). Interestingly, endogenous Daam1 and Fascin were also colocalized in pseudopodia (filopodia; Figure 5C,D).

| D ISCUSS I ON
Cancer cell motility is a central and key part of tumour metastasis.
Although great effort and some research achievements have been made to explore the mechanism of BrCa metastasis, it remains a challenging research topic that needs continuous exploration. 29   that reveals an essential target for BrCa cell metastasis, which may be a promising target for the treatment of BrCa.

ACK N OWLED G M ENTS
This work was supported by grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Yichao Z and LH designed this study. LH wrote the manuscript. LH, YL, XY and Yuerong Z prepared the figures. Yichao Z critically reviewed and edited the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.