P300/CBP‐associated factor (PCAF)‐mediated acetylation of Fascin at lysine 471 inhibits its actin‐bundling activity and tumor metastasis in esophageal cancer

Abstract

Background Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post‐translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. Methods Immunoprecipitation and glutathione‐S‐transferase pull‐down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP‐associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF‐mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live‐cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. Results Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti‐AcK471‐Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin‐K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin‐K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin‐K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471‐acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin‐bundling activity. Clinically, high levels of AcK471‐Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease‐free survival of ESCC patients. Conclusions Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin‐K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin‐bundling activity.

Fascin lysine 471 acetylation cooperates with serine 39 phosphorylation to inhibit actin‐bundling activity and tumor metastasis in esophageal squamous cell carcinoma

“…Our previous results showed that Lys-471 acetylation increases the mobility of Fascin molecules compared to the WT form [6]. Similarly, rapid mobility was observed in KYSE150 (Figure 1F) and SHEEC (Supplementary Figure S2C) cells expressing Fascin S39D/K471Q .…”

“…potential therapeutic value of mediating Fascin acetylation in tumor metastasis [6]. The phosphorylation of Ser-39 at the Fascin N-terminus and acetylation of Lys-471 at the Fascin C-terminus were inhibited Fasin F-actin bundling activity.…”

“…To assess the clinical significance of the combination of Fascin Ser-39 phosphorylation and Fascin Lys-471 acetylation, we performed immunostaining with phosphorylation-and acetylation-specific antibodies on ESCC samples from the same cohort of patients in our previous work (Figure 1H) [6]. Importantly, Kaplan-Meier survival analysis demonstrated that high levels of both Fascin phosphorylation at the Ser-39 site and acetylation at the Lys-471 site were significantly associated with favorable overall survival (OS; P = 0.006) and disease-free survival (DFS; P = 0.003) compared with the low-level group (Figure 1H).…”

Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma

“…The expression and purification of GST-tagged and His-tagged protein was performed as previously described [60]. Transetta (DE3) Chemically Competent Cells (TransGen Biotech, CD801-03) were transformed with plasmids overnight, Cells were grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) at 37 °C to an OD 600 nm value of 0.7, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Amresco, 0487) and incubated at 16 °C for 16 h. The bacteria were centrifuged and collected, and then lysed with GST lysis buffer (4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 137 mM NaCl, pH 7.3, 0.1% Triton X-100, 1 mM PMSF) and 1 mg/ml lysozyme (Sigma, 62971).…”

Molecular dynamics simulation study on the structures of fascin mutants

“…In the study by Lin et al, monoubiquitinations at K247, K250, K41, K43, K241, K353, K399, K464, and K471 of fascin led to dysfunction of F‐actin binding 24 . Besides ubiquitination modification, for the K471 residue, acetylation can also occur, eliminates F‐actin binding as well (Figure 1A, the magenta sphere) 24,25 . In addition, the mutation of K471 to alanine, making this site unable to be ubiquitinated and acetylation, strongly reduces the ability of fascin to bind to F‐actin 19 …”