Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.

Keywords: fascin; migration and invasion; oral squamous cell carcinoma; plectin; prognosis.

Figure 1. Higher expression levels of fascin and plectin in OSCCs

Representative immunohistochemical expression patterns of fascin and plectin in normal oral mucosa and OSCC specimens are shown. Both fascin (A) and plectin (D) expression were limited to the cytoplasm of the basal and suprabasal layers of the normal oral tissue. OSCC tumor cells showed variable distribution and intensity of fascin (B) and plectin (E). Quantification of the positive expression of both fascin (C) and plectin (F) in the ten original pairs of samples used in the LC–MS/MS revealed a significantly higher expression in OSCC cells compared with normal oral mucosa cells *p<0.0001.

Figure 2. Fascin and plectin are overexpressed in OSCCs and OSCC-derived cell lines

Total RNA from fresh samples and cell lines were converted in cDNA and subjected to qPCR. (A) In the fresh samples, the comparison was based in a pool of 11 normal oral tissues, whereas the spontaneously immortalized, but not transformed epithelial cell line HGK was used as reference for the comparison with OSCC-derived cell lines. The amounts of fascin (p<0.01; A) and plectin (p<0.05; C) mRNA were significantly higher in OSCC specimens than in the normal oral mucosa. The mRNA levels of fascin (B) and plectin (D) were also significantly higher in OSCC cell lines compared to HGK cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. Fascin but not plectin is overexpressed in oral dysplasias

(A) Immunodetection of fascin and plectin in representative samples of oral fibrous hyperplasia, characterizing normal oral epithelium, oral dysplasia and OSCCs. The expressions of fascin and plectin were not restricted to the lower layers of the epithelium in the oral dysplasias. (B) As expected, the levels of fascin were significantly higher in OSCCs than in fibrous hyperplasia (p<0.0001). Fascin was also significantly upregulated in the OSCCs when compared with dysplasia samples, independently of the grade (p<0.001). In addition, fascin protein levels were significantly higher in dysplasias than in nontumor tissues (p<0.001). (C) The levels of plectin were significantly higher in OSCCs than in fibrous hyperplasias and dysplasias (p<0.0001), but no differences between fibrous hyperplasia and dysplasias were observed.

Figure 4

Kaplan-Meier cumulative curves for disease-specific and disease-free survivals of patients with OSCC according to expression of fascin (A and B) and plectin (C and D). Patients with high fascin expression corresponded to those with a significantly poorer outcome in disease-specific survival.

Figure 5. Fascin knockdown efficiency in SCC-15 and HSC-3 cells. Cells were transduced with lentivirus expressing shRNA sequences against fascin (shRNA FSCN cells) and control (shRNA Control cells) as outlined in the methods

shRNA FSCN cells showed a marked reduction in both mRNA and protein levels when compared with parental cell and shRNA Control cells.

Figure 6. Downregulation of fascin does not affect viability or proliferation of SCC-15 and HSC-3 cells

Cells were subjected to MTS-cell viability (A and B) and bromodeoxyuridine (BrdU)-labeling cell proliferation (C and D) assays.

Figure 7. Overexpression of fascin is associated with adhesion, migration, invasion and acquisition of EMT properties

(A) Downregulation of fascin significantly induced the adhesive properties of SCC-15 and HSC-3 cells to fibronectin, and of HSC-3 cells on uncoated surfaces. (B) Migration of SCC-15 and HSC-3 cells were significantly decreased by fascin-specific shRNA, as revealed by transwell migration assay. (C) Migration analysis based on scratch wound migration assay showed that fascin-silenced cells (SCC-15 shRNA FSCN and HSC-3 shRNA FSCN) closed the scratch wound significantly more slowly than parental cells (SCC-15 and HSC-3) and shRNA Control cells. (D) Invasion of SCC-15 and HSC-3 cells was significantly inhibited after fascin knockdown. (E) Downregulation of fascin induced significantly the expression of E-cadherin while reducing vimentin expression in SCC-15 cells. For HSC-3 cells, reduction of vimentin expression reached significant levels, while that the induction of E-cadherin did not. *p<0.05, **p<0.01, ***p<0.0001.

Figure 8. Fascin downregulation inhibits the invasion of SCC-15 and HSC-3 cells in the myoma organotypic invasion model

(A) The knockdown of fascin markedly reduced the invasion properties of SCC-15 and HSC-3 cells in the myoma organotypic model when compared with control cells. The invasion depth (B) and the invasion area (C) were significantly reduced for HSC-3 shRNA FSCN cells. *p<0.0001.

Figure 9. The mass of the tumors and the frequency of cervical lymph node metastasis in xenograft tumors formed by HSC-3 shRNA Control and HSC-3 shRNA FSCN cells

(A) In the subcutaneous tumor model, HSC-3 shRNA FSCN tumors were significantly smaller than HSC-3 shRNA Control tumors. (B) In the orthotopic tongue model, no differences in the frequency of cervical lymph node metastasis were observed. *p<0.01.

Figure 10. Inhibition of filopodia formation by fascin knockdown

(A) Actin filaments were labeled with rodhamine phalloidin, and the number of filopodia structures was estimated with the aid of the Image J software. Fascin knockdown significantly reduced the number of filopodia (p<0.0001). (B) Western blot analysis of filopodia-related proteins. Fascin-silenced cells demonstrated reduced expression paxillin in comparison with control cells. No significant effects were observed in FAK and viculin. (C) Confocal analysis revealed the colocalization of fascin and paxillin, and confirmed the decreased expression of paxillin in fascin-silenced cells. (D) HSC-3 shRNA Control and HCS-3 shRNA FSCN were cultured in presence of 50 ng/ml of EGF for 5, 15 and 30 min, and assessed by western blot with antibodies against phospho-fascin, phospho-paxillin and phospho-FAK. Values above bands represent the densitometric analysis. Reduction in phosphorylation of paxillin and FAK occurs upon fascin-silencing in HSC-3 cells. *p<0.0001.

Figure 11. Fascin is target by miR-138

(A) Expression of fascin is inversely correlated with miR-138, but not with miR-145, in OSCC samples. (B) SCC-15 cells were exposed to miR-1 (scramble control) or miR-138 and miR-145 mimics. Levels of fascin mRNA and protein were clearly decreased in miR-138 transfectants, demonstrating that miR-138 regulates fascin mRNA levels. (C) Overexpression of miR-138 significantly inhibited the migration of SCC-15 (p<0.005) and HSC-3 (p<0.01) cells. *p<0.01, **p<0.005.