Role of fascin in the proliferation and invasiveness of esophageal carcinoma cells

doi:10.1016/j.bbrc.2005.09.055

Biochemical and Biophysical Research Communications

Volume 337, Issue 1, 11 November 2005, Pages 355-362

https://doi.org/10.1016/j.bbrc.2005.09.055 Get rights and content

Abstract

Fascin, an actin-bundling protein, induces membrane protrusions and increases cell motility in various transformed cells. The overexpression of fascin in esophageal squamous cell carcinoma (ESCC) has been described only recently, but the roles and mechanism still remained unclear. Here, by using RNA interference (RNAi), we have stably silenced the expression of the fascin in EC109 cells, an ESCC cell line. Down-regulation of fascin resulted in a suppression of cell proliferation and as well as a decrease in cell invasiveness. Furthermore, we revealed that fascin might have functions in regulating tumor growth in vivo. The effect of fascin on cell invasiveness correlated with the activation of matrix metalloproteases such as MMP-2 and MMP-9. We examined that fascin down-expression also led to a decrease of c-erbB-2 and β-catenin at the protein level. These results suggested that fascin might play crucial roles in regulating neoplasm progression of ESCC.

Section snippets

Materials and methods

Materials and cell culture. Human ESCC cell line EC109 and uterine cervix cancer cell line HeLa were purchased from the Chinese Academy of Science. They were maintained in 199 medium (Invitrogen) containing 10% fetal calf serum (FCS) in an atmosphere of 5% CO₂ at 37 °C. Monoclonal antibody anti-fascin, anti-PCNA, anti-c-erbB-2 (Dako Corporation), anti-β-catenin (Santa Cruz), anti-β-actin (Sigma), and anti-topoisomerase II (Roche) were used for immunofluorescence and Western blotting. Anti-mouse

Fascin expression in EC109 cells

By using Western blotting, we first examined the level of fascin expression in EC109 cells and HeLa cells. Fascin expression in EC109 cells is almost as high as that in HeLa cells (a putative fascin overexpression tumor cell line), indicating that fascin was also upregulated in EC109 cells (Fig. 1A). Immunofluorescence showed that fascin was localized in the cytoplasm, especially in the membrane protrusions of EC109 cells (Fig. 1B).

Silencing of the fascin gene

To study the function of fascin gene in EC109 cells, pSUPER

Discussion

Fascin is an actin-bundling protein originally found in the extracts of unfertilized sea urchin eggs [2]. Fascins play roles in the organization of two major forms of actin-based structures: dynamic cortical cell protrusions and cytoplasmic microfilament bundles. High-level expression of fascin is observed in many tumor cell lines such as breast carcinoma and esophageal carcinoma, but the roles and mechanism still remained unclear. For a better understanding of the function of fascin, in the

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 39900069, No. 30170428, and No. 30370641); Guangdong Scientific Fund Key Items (No. 37788); Natural Science Foundation of Guangdong Province (No. 010431). We thank Dr. S.J. Fang for the kind help in siRNA design and Professor X.J. Yu for his kind assistance in photomicrography.

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Cited by (90)

AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression
2022, International Journal of Biochemistry and Cell Biology
Fascin is the main actin-bundling protein in filopodia and is highly expressed in metastatic tumor cells. The overexpression of Fascin has been associated with poor clinical prognosis and metastatic progression. Post-translational modifications of Fascin, such as phosphorylation, can affect the proliferation and invasion of tumor cells by regulating the actin-bundling activity of Fascin. However, the phosphorylation sites of Fascin and their corresponding kinases require further exploration. In the current study, we identified novel phosphorylation of Fascin Threonine 403 (Fascin-T403) mediated by AKT serine/threonine kinase 2 (AKT2), which was studied using mass spectrometry data from esophageal cancer tissues (iProX database: IPX0002501000). A molecular dynamics simulation revealed that Fascin-Threonine 403 phosphorylation (Fascin-T403D) had a distinct spatial structure and correlation of amino acid residues, which was different from that of the wild type (Fascin-WT). Low-speed centrifugation assay results showed that Fascin-T403D affected actin cross-linking. To investigate whether Fascin-T403D affected the function of esophageal cancer cells, either Fascin-WT or Fascin-T403D were rescued in Fascin-knockout or siRNA cell lines. We observed that Fascin-T403D could suppress the biological behavior of esophageal cancer cells, including filopodia formation, cell proliferation, and migration. Co-immunoprecipitation (Co-IP) and Duolink in situ proximity ligation assay (PLA) were performed to measure the interaction between Fascin and AKT2. Using in vitro and in vivo kinase assays, we confirmed that AKT2, but not AKT1 or AKT3, is an upstream kinase of Fascin Threonine 403. Taken together, the AKT2-catalyzed phosphorylation of Fascin Threonine 403 suppressed esophageal cancer cell behavior, actin-bundling activity, and filopodia formation.
Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement
2016, Experimental Cell Research
Citation Excerpt :
Fascin is increasingly recognized to play some, even not fully understood, critical functions in cytoskeleton remodeling. Namely, decreased level of fascin correlated with decreased formation of dynamic cell protrusions, decreased proliferation, and reduced invasiveness in cancer cells [35,36]. As expected, we observed a marked decrease in fascin immune staining in INS-treated cells along with the aforementioned sharp decrease in lamellipodia and filopodia (Fig. 8a).
Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.
SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma
2016, Human Pathology
Epigenetic alterations, including DNA methylation and histone modifications, are involved in the regulation of cancer initiation and progression. SET and MYND domain-containing protein 3 (SMYD3), a methyltransferase, plays an important role in transcriptional regulation during human cancer progression. However, SMYD3 expression and its function in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, SMYD3 expression was studied by immunohistochemistry in a tumor tissue microarray from 131 cases of ESCC patients. Statistical analysis showed that overall survival of patients with high SMYD3 expressing in primary tumors was significantly lower than that of patients with low SMYD3-expressing tumors (P = .008, log-rank test). Increased expression of SMYD3 was found to be associated with lymph node metastasis in ESCC (P = .036) and was an independent prognostic factor for poor overall survival (P = .025). RNAi-mediated knockdown of SMYD3 suppressed ESCC cell proliferation, migration, and invasion in vitro and inhibited local tumor invasion in vivo. SMYD3 regulated transcription of EZR and LOXL2 by directly binding to the sequences of the promoter regions of these target genes, as demonstrated by a chromatin immunoprecipitation assay. Immunohistochemical staining of ESCC tissues also confirmed that protein levels of EZR and LOXL2 positively correlated with SMYD3 expression, and the Spearman correlation coefficients (r_s) were 0.78 (n = 81; P < .01) and 0.637 (n = 103; P < .01), respectively. These results indicate that SMYD3 enhances tumorigenicity in ESCC through enhancing transcription of genes involved in proliferation, migration, and invasion.
TGF-β1-induced Fascin1 promotes cell invasion and metastasis of human 786-0 renal carcinoma cells
2016, Acta Histochemica
Citation Excerpt :
According to the follow-up experiment, the cells were divided into six groups including blank control group (also NT group, non-transfected 786-0 cells), negative control group (also si-NC group, 786-0 cells transfected with pGenesil-1-con plasmid), positive group (also si-Fascin1 group, 786-0 cells transfected with pGen-1-FSCN1 plasmid), and three corresponding groups: NT treated with TGF-β1, si-NC treated with TGF-β1 and si-Fascin1 treated with TGF-β1 (Chen, 2014a,b). Based on the method used in the study of Xie et al. (2005), the interference sequence of Fascin1 genes was effectively screened. The most complete coding sequence of Fascin1 gene was found in GenBank by means of bioinformatics methods.
To investigate the effect of transforming growth factor-β1 (TGF-β1) on the expression of Fascin1 protein and its impact on cell invasion and metastasis in human renal carcinoma.
Renal tissue slices of 52 cases when undergoing radical nephrectomy were collected to be the observation group, and the normal renal tissues of 23 cases when undergoing nephrectomy due to trauma were collected to be the control group. The expressions of TGF-β1 and Fascin1 were measured by immunohistochemical staining. Human renal carcinoma 786-0 cell line was selected as the study subject. The cells were divided into six groups including NT (no transfection), si-NC (transfection with pGenesil-1-con) si-Fascin1 (transfection with pGen-1-FSCN1) groups, and three corresponding groups: NT, si-NC and si-Fascin1 groups treated with TGF-β1. RT-qPCR, Western-Blot, Transwell, and flow cytometry method were used in this study.
The expressions of TGF-β1 and Fascin1 in the observation group were significantly higher than those in the control group. The expression of TGF-β1 was positively correlated with that of Fascin1. After 24 and 48 h of treatment with TGF-β1 (10 ng/mL), the invasive and metastatic abilities of the 786-0 cells in the NT and si-NC groups were higher than those before the treatment (P < 0.05). Comparing the three groups before TGF-β1 treatment, the invasive and metastatic ability of 786-0 cells in the si-Fascin1 were significantly lower than those in the NT group and si-NC group (P < 0.05).
TGF-β1 could induce the expressions of 786-0 Fascin1 mRNA and protein and thus improve the invasive and metastatic ability of human 786-0 renal carcinoma cell.
A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation
2024, Open Biology
FSCN1 has a potential indication for the prognosis and regulates the migration of HNSCC
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^☆: Abbreviations: ESCC, esophageal squamous cell carcinoma; RNAi, RNA interference; siRNA, short interfering RNA; FCS, fetal calf serum; TBS, Tris-buffered saline; SEM, scanning electron microscopy; PCNA, proliferating cell nuclear antigen; MMP-2 and MMP-9, matrix metalloproteinases-2 and -9.

¹: These authors contributed equally to this work.

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Role of fascin in the proliferation and invasiveness of esophageal carcinoma cells☆

Abstract

Section snippets

Materials and methods

Fascin expression in EC109 cells

Silencing of the fascin gene

Discussion

Acknowledgments

Curr. Opin. Cell Biol.

Cell

J. Biol. Chem.

Am. J. Pathol.

Cell

Hum. Pathol.

J. Biol. Chem.

Fascins, a family of actin bundling proteins

Cell Motil. Cytoskeleton

Preparation and purification of polymerized actin from sea urchin egg extracts

J. Cell Biol.

Fascin, an echinoid actin-bundling protein, is a homolog of the Drosophila singed gene product

Proc. Natl. Acad. Sci. USA

Redistribution of actin and fascin in sea urchin eggs after fertilization

Cell Motil.

Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin

J. Cell Biol.

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

J. Cell Biol.

Single amino acid mutations in Drosophila fascin disrupt actin bundling function in vivo

Genetics

F-actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

J. Cell Biol.

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones

J. Neurochem.

Fascins, and their roles in cell structure and function

Bioessays

Cell–matrix contact structures

Cell. Mol. Life Sci.