Effects of Alternatively Spliced Tenascin-C Isoforms on Breast Cancer Cell Behaviour

Aims: The tumour microenvironment plays a crucial role in the development of breast cancer. Tenascin-C (TNC), a matricellular protein and its high molecular weight (MW) isoforms have been shown to be over-expressed in the stroma of breast cancers and are associated with poor prognosis. The aim of this study was to investigate the effects of TNC knockdown in TNC expressing invasive breast cancer cell lines on gene expression and cell behaviour. Methods: siRNAs targeting different exons in TNC (24, 14 and 14-AD1) were designed, synthesised and transfected into the highly invasive MDA-MB-231 breast cancer cell line. Down regulation of TNC was confirmed by Western blotting and RT-qPCR. The phenotypic alterations caused by TNC knockdown were analysed by 2D invasion assays and proliferation assays using the mitotic marker (pHH3). cDNA microarray and proteomics were used to analyse the effects of TNC knockdown at the mRNA and protein level. A novel polyclonal antibody was also generated for TNC-AD1 and expression of this and thrombospondin-1 (TSP-1), as a candidate TNC regulated gene was investigated in 36 breast cancer tissues using immunohistochemistry. Results: The siRNA targeted cells showed significant down-regulation of both total TNC (p <0.001) and high MW isoforms (p <0.001) in MDA-MB-231 cells. Moreover, knockdown of total TNC and high MW TNC isoforms significantly decreased both cell invasion (total TNC p <0.001, TNC-14 p <0.001 and TNC-14-AD1 p <0.01) and proliferation (total TNC p <0.001 and TNC-AD1 p <0.05). Microarray analysis following total TNC knockdown revealed significant changes in gene expression: CREBL2, YWHAE, CDC14B and RRAS2 showed down regulation and QKI was specifically up-regulated by knock-down of both total TNC and high MW TNC isoforms. Proteomics and Western blot analysis showed increased levels of thrompospondin-1 (TSP-1) as result of total TNC knockdown as well as high MW TNC isoforms. TNC-AD1 expression in 36 breast cancer tissues was significantly associated with age (>40 years). Conclusion: TNC knockdown significantly decreases proliferation and invasion in breast cancer cell lines, confirming its importance in breast cancer progression.