Understanding the molecular basis of OSCC progression especially invasion and metastasis and identification of potential molecular markers that are contributing to these processes is required to improve the prognosis and the survival of the patients. Invasive and metastatic potential of tumor cells is often associated with reorganization of actin cytoskeleton. Fascin is one such actin bundling protein, found to be up-regulated in several epithelial malignancies including SCC. Its expression has been correlated with aggressiveness of the tumors [12
]. In the present study we have investigated the role of fascin in cell motility, invasion and tumorigenicity using in vitro and xenograft in vivo mouse model. We have further tried to understand the clinical significance of fascin expression in prognostication of OSCC patients using IHC analysis of tumor tissues.
The fascin-overexpressed OSCC cells formed longer and thicker microspikes, develop more filopodia and lamellipodia (Figure ), and showed significant increase in cell motility (Figure ). Several prior studies have shown that fascin overexpression induce lamellipodia and filopodia formation and enhances cell migratory ability in different cell types [35
]. These fascin-overexpressed cells also showed disorganization of cell-cell contacts (Figure ), decrease in E-cadherin and β-catenin levels (Figure ). Previously it has been shown that fascin overexpression led to disorganization of cell-cell contacts and reduction in E-cadherin and β-catenin levels in epithelial cells [37
]. The observed reduction in total β-catenin levels may be due to loss of cell-cell contacts which intern may lead to its degradation at cell surface (Additional file 2
: Figure S1C). Previous reports have also shown reduction or loss of total and/or cell surface levels of β-catenin in OSCC samples and correlated it with aggressiveness of the tumor and poor prognosis [38
]. Importantly, the anchorage-independent growth and xenograft tumor experiments in NOD-SCID mice indicated that fascin also contributes to the development of primary tumors in OSCC (Figure ). To our knowledge this is the first report showing the role of fascin in primary tumor development in OSCC. These results are consistent with the previous reports on colon cancer and oesophageal SCC cells [41
]. Our results further demonstrated significant increase in invasive potential (Figure ) in fascin overexpressed cells. Previously, fascin has been shown to promote invasiveness of the colon, breast and esophageal carcinoma derived cells [22
]. Chen et al. have identified migrastatin analogues which inhibit tumor cell migration, invasion and metastasis by blocking fascin activity using in vitro as well as in vivo model system in breast cancer [45
]. Furthermore, fascin-overexpressed cells also demonstrated increase in MMP-2 activity (Figure ). MMP-2 and MMP-9 are proteolytic enzymes that digest the components of the basement membrane facilitating metastasis of malignant tumors [46
]. Our results are consistent with Xie et al. findings and suggest that, increase in MMP-2 activity in response to fascin upregulation would also contribute to increased invasive ability of the carcinoma cells [42
]. Thus, our results indicate that fascin overexpression leads to increase in cell motility, tumorigenicity and invasive potential of OSCC cells.
Increased cell migration, invasion, tumorigenicity and loss of E-cadherin expression are often observed during Epithelial Mesenchymal Transition (EMT) [47
]. Here fascin overexpressed cells did not show any increase in EMT markers such as snail, slug and vimentin (Figure ; Additional file 2
: Figure S1B). Our results are in agreement with the previous report, in which Vignjevic et al. have shown that fascin mediated tumor cell migration and invasion did not involve EMT in human colon cancer [48
]. These results together suggest that fascin may promote cell migration and invasion of tumor cells through collective cell migration by filopodia formation [49
] in OSCC.
Interactions between cells and their extracellular matrix play an important role in tissue organisation and also influence many aspects of cell behaviour including cell shape and cell migration [50
]. During cell migration, Thrombpspondin-1 induces cross-linking of fascin and F-actin that leads to formation of F-actin based cell protrusions. Fibronectin triggers phosphorylation of fascin at S39, leading to rapid loss of fascin from actin-based structures during initial cell spreading, in a protein kinase Ca (PKCa)-dependent process [53
]. Here, fascin overexpression facilitated cell adhesion to fibronectin, laminin and other ECM molecules in OSCC cells. Previously, it has been shown that fascin may be involved in cell-ECM interaction through integrin [55
]. Previous reports also suggest that fascin regulates focal adhesion disassembly and activation of FAK [56
]. We did not observe any consistent change in α6-integrin, β4-integrin and phosphorylated FAK levels in all the fascin overexpressed clones (Figure ). This suggests that α6β4-integrin-FAK-mediated signalling may not be involved in fascin dependent cell-ECM interaction in OSCC.
Overexpression of fascin is often associated with increased cell proliferation in different types of carcinomas [42
]. Our results have shown increase in cell proliferation in response to fascin overexpression which was accompanied with increased cell proliferation marker PCNA (Figure ). In addition, upregulation of PI3K and MAP kinase pathways was also observed in fascin-overexpressed cells (Figure ). PI3-K and MAP kinase pathways are independently known to regulate cell proliferation and migration in epithelial cells and upregulation of these signalling pathways has been documented in carcinomas including OSCC [58
]. Further, the role of MAPKs in the regulation of MMP expression in carcinoma cells has also been well-studied [61
]. It has been shown that inhibition of p-ERK1/2 may lead to a reduction in the expression of MMP-2 and invasive ability of tumor cells [62
]. These results suggest that fascin possibly regulates MMPs activity and invasiveness through MAPKs pathway. Previously it has been reported that Akt activation is a significant prognostic indicator for OSCC [64
]. Taken together our results suggest that apart from regulating actin polymerization and thereby cell motility, fascin may also activate PI3K and MAP kinase signalling pathways in tumor cells. These events subsequently promote tumor progression.
Next we investigated the clinical significance of fascin expression in prognostication of OSCC patients. Correlation of fascin expression with lymph node metastasis and its presence (Figure ; Table ) in lymph node tissues showing tumor metastasis (Figure ) suggest that fascin may facilitate movement of tumor cells from the primary site to the lymph node. Lymph node metastasis is widely accepted as one of the major prognostic factors in OSCC patients and it's presence is associated with a decrease in overall survival and higher recurrence rates [6
]. High fascin expression was seen in 17% cases where lymph node metastasis was not detected (N0). It will be interesting to follow these cases further to understand whether fascin expression is indicative of "submicroscopic" occult metastasis which is known to have impact on patient survival [65
]. Furthermore, fascin expression was not detected in any of the well differentiated OSCC (Figure ; Table ) and higher fascin expression was mostly found in higher stage tumors (Figure ; Table ). A large number of studies have demonstrated that disease staging has a crucial influence on the outcome [66
]. A number of studies have also shown significant correlation between lower histologic differentiation and poorer prognosis [6
]. We were further able to show correlation between fascin expression and poor patient survival (Figure ). On the other hand fascin non-expression was indicative of disease free survival and decreased chance of recurrence (Figure ). Although, Chen et al. have demonstrated correlation of fascin levels with tumor size, lymph node metastasis and staging, they have not reported correlation study with survival of the patients [27
]. Lee et al. have also shown correlation of fascin expression with nodal metastasis, tumor recurrence and poor patient's survival in 49 OSCC samples [27
]. In this study, we did not observe correlation of fascin expression with tumor size but we could show correlation with differentiation status. Thus apart from confirming the findings of previous studies on larger sample size, we could show its relation with disease free survival and confirm its expression in lymph node metastasis. Thus our results demonstrate prognostic value of fascin expression in OSCC.
Recently, we have shown that knockdown of K8 in an OSCC derived cell line resulted in reduced fascin and β4-integrin levels which correlated with decreased tumorigenicity, migration and invasiveness of these cells [28
]. Therefore, we further analyzed association of fascin in combination with K8 and β4-integrin expression with clinico-pathological parameters of the patients. Fascin and K8 expression together correlated with tumor stage, differentiation status, lymph node metastasis and recurrence while fascin and β4-integrin expression together correlated with differentiation status, lymph node metastasis and per neural tumor extension (Additional file 7
: Table S3). We did not observe correlation between expression of all these proteins (fascin, β4-integrin and K8) together with patient survival (data not shown) possibly because of the smaller sample size. A study with larger sample size is required to highlight the significance of their expression together in prognostication of OSCC.