br It was interesting to note the change in invasive
It was interesting to note the change in invasive capacity of MCF-7 and MDA-MB-231 Epirubicin upon Ets-1 perturbation. We also found a change in EMT marker expression simultaneously. Previous studies have shown that Ets-1 is not only involved in the development of var-ious tumors but is also associated with tumor invasion (Singh et al., 2002). In mouse mammary tumor (MMT) epithelial cells and in some breast cancer cell lines overexpression of Ets-1 can up regulate MMPs and result in increased cellular invasiveness (Furlan et al., 2008; Park et al., 2008). In concordance with the above documented literature, we observed a significant decrease in the invasion capacity of MCF-7 and MDA-MB-231 breast cancer cells induced by siRNA against the Ets-1 gene. Various studies have established that MMP-9 promotes key events in tumor progression and invasion. Here we found that downregulating Ets-1 results in down regulation of MMP-9, suggesting that Ets-1 pro-motes cellular invasiveness via upregulating MMP-9. r> Tumor progression has its links with EMT (Polyak and Weinberg, 2009) and is characterized by the expression of genes such as Vimentin and N-cadherin (Tiwari et al., 2012) and the activation of certain transcriptional factors such as Snail and Slug (Peinado et al., 2007) which cause loss of cell adhesion to help cells dislodge from primary tumor and invade to other tissues taking help from molecules such as proteases which help in degradation of basement membrane. Thus it was hypothesized to see the effect of Ets-1 knockdown on the expres-sion of above stated EMT markers. Comparing the Ets-1 knock down in MCF-7 and MDA-MB-231, we detected the following EMT molecular markers: Slug, Snail, Vimentin and N-cadherin. It was observed that Ets-1 knockdown resulted in significant decrease in EMT markers in MDA-MB-231 breast cancer cell line; whereas, in contradiction to it MCF-7 showed increase in expression of these markers other than snail. Ets overexpressed proteins in prostrate cells are known to promote an-chorage-independent growth and result in EMT (Hollenhorst et al., Gene 711 (2019) 143952
2010). The involvement of Ets-1 in induction and maintenance of EMT is critical. Reports from chick cranial neural crest have shown that Ets-1 co-operates with slug to induce cell delimination and EMT (Théveneau et al., 2007). In pancreatic cancer cells, Ets-1 overexpression results in upregulation of mesenchymal protein N-cadherin (Li et al., 2015). In epithelial cells knockdown of Ets-1 resulted in reduced expression of Vimentin via RAS/ER signaling pathway (Plotnik et al., 2014). The results are in confirmation with MDA-MB-231 breast cancer cells as these are triple negative/basal like breast cancer cells and show me-senchymal phenotype. But in order to account for the differences in results in MCF-7 cells a number of hypothesis could be postulated such as difference in ER receptor, basal/luminal and P53 status. It is im-portant to note that despite elevated level of Slug, Vimentin and N-cadherin in MCF-7 cells, we noticed a decrease in their invasive po-tential. It implicates the role of other factors being operative in MCF-7 cells, which do not allow EMT markers to exert their role in cell inva-sion. These factors could be working on EMT markers at protein level and/or other network modes. It would be interesting to study this as-pect of cell signaling in MCF-7 cells under Ets-1 influence.
The co-expression of Ets-1 and MMP-9 in our study on breast cancer and from studies on different types of cancers (Sahin et al., 2005) in-dicate a regulatory link between Ets-1 and MMP-9. Bioinformatic ana-lysis also revealed the presence of an Ets-1 binding site in the promoter region of MMP-9. To validate this we performed oligonucleotide pull down and chip assays and could predict that the transcription factor Ets-1 binds directly on MMP-9 gene promoter. Although our results confirmed the direct binding of Ets-1 transcription factor on MMP-9 promoter, further reporter assays need to be performed to confirm the functional role of Ets-1 in MMP-9 transcriptional regulation. In con-cordance with this data other studies have also reported the regulation of MMP-9 by Ets-1 in ovarian, cervical and various other cancers (Ma et al., 2004; Vandooren et al., 2013). Additionally, transcription factors such as AP-1, PEA3, Sp-1 and NF-KB have also been shown to regulate MMP-9 transcriptionally (Yan and Boyd, 2007).
Multiple studies have projected the role of Ets-1 in various cancers but how it functions to regulate the process of carcinogenesis is poorly understood. Our findings clearly provide an insight on the regulation of Ets-1 and its downstream gene MMP-9 in breast cancer. In addition, our study also demonstrates the effect of Ets-1 in the invasion and epithelial to mesenchymal transition in the pathway of breast cancer progression. However, it would be interesting to further dissect the pathways in-volved in Ets-1 dependent and/or MMP-9 in breast cancer carcino-genesis.