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Molecular cloning and functional studies of tenascin-C isoforms containing the fibronectin-type III repeat additional domain 1 (AD1)

By David Stephen Guttery

Abstract

Tenascin-C is an extracellular matrix glycoprotein expressed at low levels in normal breast tissue and highly expressed in both the stroma and malignant cells of solid tumours. Multiple isoforms of TNC are generated by alternative splicing. The aim of this study was: 1) to investigate the expression of key high molecular weight (MW) TNC isoforms containing domains D, B/D, AD1 and AD2 in normal, benign and malignant breast and relate expression to histopathological features, 2) to investigate the functional significance of AD1 by molecular cloning and 2D invasion assays, 3) to perform differential gene expression analysis using GeneChip arrays and relate to expression of high MW TNC isoforms in MCF-7 cells. \ud AD1 and AD2 were detected in all TNC positive breast cell lines, normal and tumour breast tissues and isolated cells from normal breast tissue, with myoepithelial cells being the major source of AD1. In carcinomas, expression of high MW TNC was significantly associated with younger age (≤ 40 years; p = < 0.05 for all isoforms), negative ER (p = 0.011 for AD1 and 0.032 for AD1/AD2 respectively) and high grade (p = 0.017 and 0.019 respectively). Expression of total TNC, TNC-9/16 and TNC-14/16 was also associated with negative CK14 (p = 0.003 for all), and higher TNC-14/16 expression was associated with lobular carcinomas (p = 0.004). Molecular cloning of AD1 and transfection studies using the TNC-14/AD1/16 isoform significantly increased MCF-7 cell invasion to a level comparable to TNC-9/14/16 (p = < 0.001). Differential gene expression analysis showed that TNC-9/14/16 and TNC-14/AD1/16 significantly increased expression of interferon-inducible transmembrane protein 1 (IFITM1) and profilin-1 (PFN1) in transfected MCF-7 cells. However, quantitative RT-PCR analysis of tissue samples showed significant down-regulation of PFN1 in tumours, compared to normal breast (p = 0.02), which was significantly associated with high TNC-14/16 expression (p = 0.002). \ud In conclusion, high MW TNC isoforms including AD1 have been associated with more aggressive features of breast carcinomas and in-vitro with an invasive phenotype. Moreover, this study also identifies PFN1 as a novel gene target associated with tumours that express high levels of TNC-14/16

Publisher: University of Leicester
Year: 2009
OAI identifier: oai:lra.le.ac.uk:2381/7427

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