S100 gene members in oral squamous cell carcinomas (OSCCs) : functional characterization of S100A14 in proliferation and invasion of OSCC derived cells

Abstract

It is generally accepted that carcinogenic substances in the form of tobacco, alcohol or oncogenic viruses, etc cause genetic changes resulting in conversion of normal oral epithelium to a potentially malignant (dysplastic) lesion, and subsequently into the invasive oral squamous cell carcinoma (OSCC). However, the precise molecular mechanism underlying OSCC carcinogenesis remains unclear. Several members of the multifunctional Ca2+ binding S100 proteins have been described in connection with a range of human cancers, including OSCCs. Studies from our group, using high throughput genomic and proteomic methods, have previously identified differential expression of members of the S100 proteins in OSCCs from different populations. S100A14 is a recently identified member of the S100 protein family. Although differential expression of S100A14 has been described in different human cancers, its biological roles in carcinogenesis have not been well characterized. This study aimed (i) to examine the mRNA expression profile of 16 of the S100 gene family members in OSCCs and (ii) to characterize the possible role(s) of S100A14 in proliferation and invasion of OSCC derived cells. We identified significant down-regulation of S100A4, S100A6, S100A8 and S100A14 mRNAs in OSCCs compared to their pair-wised controls. Down-regulation of S100A14 was further validated at the protein level in OSCC archival tissues using immunohistochemistry, and in an in vitro oral cancer progression model both at the mRNA and protein levels. To investigate the functional roles of S100A14, we employed retroviral vector mediated overexpression and siRNA mediated knock-down of the endogenous S100A14 in two OSCC derived cell-lines (CaLH3 and H357). S100A14 over-expression resulted in a significant reduction in CaLH3 cell proliferation due to G1-phase cell cycle arrest, but not apoptosis. This G1- arrest was found to be associated with nuclear accumulation of the tumor suppressor protein p53 and p53-dependent up-regulation of p21. These findings suggest a functional link between S100A14 and cell-cycle regulators p53 and p21 in the regulation of cell cycle in OSCC derived cells and support the idea that S100A14 might function as a tumor suppressor protein working in the p53 pathway. Characterization of the role of S100A14 in tumor invasion showed that overexpression of S100A14 resulted in a significant decrease in the invasive potential of the OSCC derived CaLH3 and H357 cell-lines, whereas siRNA mediated knock-down resulted in a significant increase in the invasive potential of the CaLH3 cell-line in vitro. PCR array and validation using qRT-PCR and gelatin zymography revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines and suppression of MMP9 activity in the CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in OSCC tissue samples. These findings suggest that S100A14 negatively regulates expression and activity of MMP1 and MMP9 and that might be responsible for the S100A14 mediated regulation of tumor cell invasion. In conclusion, findings of this work suggest that differential expression of several of the S100 gene family members is a common genetic alteration in OSCCs. S100A14, similar to other members of the S100 family, is involved in key cellular functions such as cell cycle regulation and tumor cell invasion indicating a tumor suppressor role for S100A14. Uncontrolled cell proliferation and invasion, characteristics of OSCCs, might therefore be related to altered expression of S100A14 frequently observed in these cancers.