Abstract
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 cancer cell behaviour. Small interfering RNA (siRNA) 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. The phenotypic alterations caused by TNC knockdown were analysed by Two Dimension (2D) invasion assays and proliferation assays using the mitotic marker Phispho-Histone H3 (pHH3). 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 and TNC-14 p <0.001) and proliferation (total TNC p <0.001 and TNC-AD1 p <0.05). In conclusion, TNC knockdown significantly decreases proliferation and invasion in breast cancer cell lines, confirming its importance in breast cancer progression.
Highlights
Breast cancer is one of the most common cancers in the world with high mortality and morbidity rates, complicated by unique molecular signatures and diverse genetic alterations; each with distinct clinical outcomes [1]
TNC-14/16 and TNC-additional domain 1 (AD1) were expressed in all total TNC positive cell lines, with MDA-MB-231 expressing the highest levels of TNC-14/16 (>1,200,000 molecules per 1 μg RNA), and HBL-100 cells expressed the highest level of TNC-AD1 (>700,000 molecules per 1 μg RNA)
MDA-MB-436 cells showed the highest percentages of high molecular weight TNC (AD1 and 14/16) (>21% total TNC) (Table 3)
Summary
Breast cancer is one of the most common cancers in the world with high mortality and morbidity rates, complicated by unique molecular signatures and diverse genetic alterations; each with distinct clinical outcomes [1]. Alterations in the tumour microenvironment have been shown to play a crucial role in breast cancer progression ([2,3]). Many ECM proteins are altered in breast cancer, one consistent finding was the upregulation of the hexameric protein tenascin-C (TNC) in the tumour stroma ([4,5]). Alternative splicing at the pre-mRNA level of 9 FNIII repeats (exons 10-16, AD1 and AD2), which are included or excluded in a unique manner, allows TNC to show tremendous diversity in isoform expression with as many as 27 different mRNA variants having been identified in the developing mouse brain [7]
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