S100A14 regulates the invasive potential of oral squamous cell carcinoma derived cell-lines in vitro by modulating expression of matrix metalloproteinases, MMP1 and MMP9

https://doi.org/10.1016/j.ejca.2010.10.012Get rights and content

Abstract

Despite the differential expression of S100A14 (a newly identified S100 member) in various human cancers including oral squamous cell carcinomas (OSCCs), its biological role in tumour invasion has not been characterised. The aim of this study was thus to investigate the possible role of S100A14 in OSCC cell invasion. Using immunohistochemistry in normal (n = 13), dysplastic (n = 10) and OSCC (n = 16) archival tissues, S100A14 protein was found to be down-regulated/lost with concomitant membrane to cytoplasmic translocation in OSCCs, especially in the invading tumour islands. These expression data were corroborated by profiling S100A14 mRNA expression using quantitative RT-PCR (qRT-PCR) in an in vitro human OSCC progression model consisting of cell-lines derived from normal (n = 3), dysplastic (n = 3) and OSCC (n = 8) tissues. Employing in vitro Matrigel invasion assay, we demonstrated that retroviral vector mediated over-expression of S100A14 resulted in significant decrease in the invasive potential of OSCC derived CaLH3 and H357 cell-lines whereas siRNA mediated knockdown resulted in significant increase in the invasive potential of CaLH3 cell-line. Pathway focused PCR array and validation using qRT-PCR revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines. Further, S100A14 over-expression was found to be associated with suppression of MMP9 gelatinolytic activity in CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in 19 cases of OSCCs. Collectively, these data provide the first evidence for a role of S100A14 protein in regulation of OSCC cell invasion by modulating expression of MMP1 and MMP9.

Introduction

Despite ample research and improvement in diagnostic tools and treatment modalities of oral squamous cell carcinoma (OSCC), overall 5 year survival of OSCC patients is poor (<50%) and has remained unchanged for the last three decades.1 OSCC is a highly aggressive pathological condition and frequently metastasizes to the regional lymph nodes. Status of the metastatic involvement of cervical lymph nodes is a major prognostic factor for OSCC patient survival.2, 3 Although the process of tumour cell invasion and metastasis has been suggested to involve a cascade of alterations in cell–cell and cell-extracellular matrix (ECM) interactions and proteolytic degradation of ECM, followed by motility of the cancer cells towards adjacent connective tissue stroma,4, 5 the precise molecular mechanisms remain elusive in OSCC invasion. Hence, a better understanding of the biology of OSCC invasion and identification of key molecules regulating this process are necessary to predict the invasion and metastatic potential of these lesions.

The S100 family is a multifunctional group of EF-hand type calcium binding proteins. Several members of the S100 family have been reported to regulate a number of biological processes like cell growth, cell motility, signal transduction, transcription, cell survival and apoptosis, related to normal development and tumourigenesis.6, 7 Expression of many of the S100 members has been reported to be altered in various human malignancies.8, 9, 10, 11 In addition, several S100 members, namely S100A412, S100A213, S100P14 and S100A1315 have been implicated in tumour invasion and metastasis. Several studies have documented the role of S100 members in regulation of matrix metalloproteinases (MMPs), the key molecules involved in tumour invasion and metastasis.12, 16, 17 MMPs are a large family of zinc-dependent proteolytic enzymes that can degrade virtually any components of ECM, thus allowing migration of cancer cells towards adjacent connective tissue.18, 19 Over-expression of several of the MMPs and their association with tumour progression, invasion and metastasis and poor clinical outcomes has been reported in OSCCs.20, 21

S100A14 protein (previously known as BCMP84, S100A15) is a recently identified member of the S100 family.22, 23 Although differential expression of S100A14 has been reported in different human malignancies including OSCCs,9, 10, 22, 23, 24 biological properties of this molecule are largely unknown. We have recently identified a role for S100A14 in the regulation of cell proliferation by inducing G1-phase cell cycle arrest in OSCC derived cells (Dr. D. Sapkota, University of Bergen). In the present study we investigated the possible role of S100A14 in invasion of OSCCs. Here, we demonstrate that S100A14 plays an important role in the regulation of invasiveness of OSCC derived cell-lines in vitro with concomitant modulation of MMP1 and MMP9 mRNA expressions.

Section snippets

Tissue specimens and immunohistochemistry (IHC)

Immunohistochemical analysis of the S100A14 protein was performed on archival formalin fixed, paraffin embedded tissue specimens of OSCCs (n = 16), oral dysplastic lesions (ODL, n = 10, all mild dysplastic lesions) and normal human oral mucosa (NHOM, n = 13) using Autostainer universal staining system (DAKO-USA, Carpinteria, CA) as described previously.25 All tissue samples, except seven of the NHOM specimens, were collected from the Department of Oral and Maxillofacial Surgery, Khartoum whereas the

S100A14 is strongly expressed at the cell membrane of NHOM epithelial cells, but weakly expressed and with membrane-cytoplasmic switch in the invading OSCC cells

All NHOM tissues showed a strong membranous expression of S100A14 protein confined mainly to the epithelial cells (Fig. 1A). Few scattered cells in the epithelium showed a mixed membranous and cytoplasmic staining as well. None of the epithelial cells showed nuclear staining. All of the ODLs were positive for S100A14 and the expression pattern was similar to that found in NHOM (Fig. 1B). However, the staining intensity was heterogenous across the ODL specimens, with some tissue specimens

Discussion

This study demonstrates that S100A14 regulates the invasive phenotype of oral carcinoma derived CaLH3 and H357 cell-lines and this regulation is associated with the modulation of key genes involved in invasion and metastasis, in particular MMP1 and MMP9. Previously we found down-regulated expression of S100A14 mRNA in OSCC tissues compared to their pair-wised normal controls.9 Assessment of the in vivo specimens in this study further confirmed the down-regulation of S100A14 protein in OSCCs,

Conflict of interest statement

None declared.

Acknowledgements

We are grateful to Dr. Oleg Tsinkalovsky for assistance with the cell sorting and Mrs. Gunnvor Øijordsbakken for excellent technical assistance. This study was supported by the Norwegian State Educational Loan Fund (Quota Programme, DS), Meltzer’s fund (project no: 803172, DS) and NFR (project no: 178601, DEC).

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