br Discussion br Breast cancer is
Breast cancer is the leading cause of mortality among women worldwide (Bray et al., 2018). In the progression of this disease in-volvement of hyper proliferative lesion occurs from in suto localization and results in invasion and metastasis of the disease. The progression of this disease involves invasion and metastasis of the in situ localized hyper proliferative lesion (Hu et al., 2008). Various genes and tran-scription factors show altered expression in the progression of this disease. One such transcription factor is Ets-1 which is reported to be overexpressed in a number of cancers. This overexpression has shown its association with aggressive nature of the invasive tumors and its correlation with Scarf-Bloom-Richardson grading (Katayama et al., 2005) and in poor prognosis of human breast carcinoma (Span et al.,
2002). The association between Ets-1 and MMPs has also been in-dicated in several studies. In addition, Ets-1 has been shown to exhibit the ability to transcriptionally regulate various downstream genes in-cluding the matrix metalloproteinases (MMPs) (Ghosh et al., 2012).
These MMPs are a group of zinc dependent proteases that have shown to play a crucial role in a number of physiological processes which include tissue repaire and/or remodeling, angiogenesis and skeletal morphogenesis during embryonic developmental phases
(Chang et al., 2014; Jiang et al., 2014; Oo et al., 2014; Yang et al., 2014; Zhang et al., 2014). Besides helping in the maintenance of normal functions in a cell, MMPs have also contributed in the development of various cancers. MMP-9 which belongs to this zinc dependent peptidase family has shown its expression in a number of cell types which include smooth muscle cells, keratinocytes, 56390-09-1 of endothelium and macro-phages (Zhang et al., 2013; Bubnovskaya et al., 2014; Jiang et al., 2014; Liu et al., 2014; Ojetti et al., 2014; Zhang et al., 2014). Further, varying levels of this protease have been associated with various types of can-cers. In the in vivo model of transgenic mice increased incidence of tumor occurrence has been associated with the increase in MMP-9 ex-pression where the use of chemical inhibitors against MMP-9 reduced the invasive nature of cancers (Shan et al., 2015). These observations reflect the ability of MMP-9 overexpression and its contribution in the development of tumors, presumably by breaking the extracellular ma-trix resulting in the facilitation of tumor invasion. Involvement of various pathways induced by different genes and transcription factors in various types of cells and diseases has been found in the regulation of MMP-9. Therefore, suggesting the need to understand a specific pathway in a given cell type induced by MMP-9.
A significant co-relation of Ets-1 with breast cancer tumor samples was observed in our recent finding (Nazir et al., 2019). The present study was performed to investigate the regulatory role of Ets-1 tran-scription factor in breast cancer along with its downstream target gene MMP-9 by utilizing RNA interference in breast cancer cell lines MCF-7 and MDA-MB-231. We observed a differential expression of Ets-1 in these cell lines. MCF-7 cell line being less invasive and less aggressive showed lower expression of Ets-1 in comparison to MDA-MB-231 which
belongs to the triple negative breast cancer cell line and is more in-vasive and more aggressive. To check the role of Ets-1 in these cell lines we knocked down the Ets-1 gene by using different concentrations of Ets-1 siRNA in both the MCF-7 and MDA-MB-231 breast cancer cell lines. In case of MDA-MB-231 silencing started at 35 pmole of siRNA and the highest efficiency was achieved at 90 pmole post 48 h of transfection. However, in case of MCF-7 decreased expression of Ets-1 started at 35 pmole of siRNA showed highest effeiciency at 90 pmole but unexpectedly increased at 70 pmole. Though the reason for this phenomenon is not fully understood by us as well, however, there could be several probable reasons such as the cells have a tendency to revert back to the changes introduced to them in specific ways. The MCF-7 cells might be displaying an increase in Ets-1 expression at 70pmole as some off site targets might result in the induction of some different signaling pathways and in turn activating the alternate signaling pathways. Another plausible explanation could be that Ets-1 siRNA may not work efficiently at some higher concentraation in MCF-7 cell line because of overdose/time. Also, as it is siRNA based approach it is likely possible that after transfection the cells may change their behavior or become senescent or resistant resulting in no loss of gene expression. Some biological or experimental variation could be other reason re-sponsible for this type of phenomenon.
On silencing of Ets-1, a corresponding decrease in MMP-9 expres-sion was noted both at the protein and mRNA level as analyzed by using various molecular biology techniques such as western blotting, real time PCR and immunofluorescence. These results suggested direct ef-fect of Ets-1 knockdown on MMP-9 expression. Similar results have also been observed in various other cancers such as pancreas, prostrate, ovary and colon cancer cells (Ito et al., 2004; Ghosh et al., 2012; Kato et al., 2012; Gao et al., 2014). In addition, some other studies have also shown the involvement of various other factors to be associated with MMPs production tunneled by Ets-1 (Ito et al., 2004; Park et al., 2008; Ghosh et al., 2012; Gao et al., 2014). So these studies established the fact that Ets-1 has both direct and indirect role on the expression of MMPs.