b Department of Biochemistry Bundelkhand University Jhansi
b Department of Biochemistry, Bundelkhand University, Jhansi, UP, India
c Dr. B.R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India
d Division of Preventive Oncology, National Institute of Cancer Prevention & Research, Indian Council of Medical Research, Noida, India
Ets-1 is one of the crucial member of transcription factor family which share a unique DNA binding domain. It is predominantly expressed in various tumor subtypes and has shown its association in the regulation of various important genes which include ECM-degrading proteases. Our study aimed to understand the mechanism(s) in the pathogenesis of breast carcinogenesis by Ets-1 transcription factor and its downstream target gene MMP-9. Role of Ets-1 in MCF-7 and MDA-MB-231 breast cancer Rocaglamide A was studied by RNA-interference in combination with pull down and ChIP assays to identify the regulation of MMP-9 in these cell lines. Our results showed that transfection of Ets-1 siRNA in breast cancer cell lines resulted in downregulation of Ets-1 and MMP-9. Ets-1 knock down also showed reduced cell invasion and altered expression of EMT markers. Moreover, we could also predict that MMP-9 gene promoter harbors a binding site for Ets-1 transcription factor may be responsible in direct transactivation of Ets-1 along with EMT markers. Phenotypic changes and molecular alterations that may result in increased aggressiveness/ invasiveness and metastatic nature of cancerous cells may lead to changes in EMT markers. Therefore, these findings may suggest a plausible role of Ets-1 dependent regulation of MMP-9 gene and may have a significant impact on breast carcinogenesis.
Breast cancer is the most common and a leading cause of death in women worldwide (Bray et al., 2018). It is a heterogeneous disease and is classified into several sub-types based on the histological markers or various gene expression profiles (Mehner et al., 2014). Despite several advances in the treatment of breast cancer, invasion and/or metastasis are one of the main causes of increased mortality. During metastasis, the cancer cells evade the matrix adhesion and result in invading sur-rounding tissues or distant organs (Wu et al., 2016). The elucidation of the mechanism involved in invasion and metastasis involves a number of genes and the regulation of the expression of these genes is controlled by transcription factors (Carro et al., 2009).
One such transcription factors is E26 transformation–specific-1 (Ets-1), which has been shown to play a crucial role in carcinogenesis. It is one of the key members of the Ets transcription factor family. This fa-mily has got recognition by its well characterized DNA binding domain
(DBD), which has a helix-turn-helix motif responsible for recognizing specific DNA binding elements called Ets-binding sites that contain GGAA/T consensus sequence as the core motif (Taniguchi et al., 2007; Legrand et al., 2013). The Ets sequence was originally recognized in avian erythroblastosis retrovirus E26 and was called v-ets (viral ets) where it was transformed by viral gag/myb gene fusions. Later, a cel-lular (c) ets-1 was also found; suggesting its derivation from v-ets-1(Watson et al., 1985; Ghysdael et al., 1986). Ets-1 gene is localized on chromosome 11 in humans and acts mainly as transcriptional activator and/or repressor (Dittmer, 2015). Apart from these, Ets-1 shows its involvement in different physiological processes which include differ-entiation, proliferation, migration and apoptosis (de Nigris et al., 2001; Kita et al., 2001; Teruyama et al., 2001; Lulli et al., 2006; Higuchi et al., 2007). Regulation of Ets-1 expression is tightly controlled and its overexpression has been shown to be associated with various invasive pathologies including cancer (Redlich et al., 2001; Dittmer, 2003; Raffetseder et al., 2004). The cellular proliferation and invasion in
Abbreviations: BSA, Bovine serum albumin; ChIP, Chromatin Immunoprecipitation; EMT, Epithelial to mesenchymal transition; Ets-1, E-26 transformation-specific-1; FBS, Fetal Bovine Serum; MMPs, Matrix metalloproteinases; MMP-9, Matrix metalloproteinase-9; PBS, Phosphate Buffer Saline; PI, Protease Inhibitor; SDS PAGE, Sodium dodecayl sulphate polyacrylamide gel electrophoresis; uPA, urokinase Plasminogen Activator; WCL, Whole Cell lysate
E-mail address: [email protected] (S. Hussain).
cancer has been partly explained because of the aberrant expression of Ets-1. This invasiveness occurs because of various protease coding genes which include matrix metalloproteases such as stromelysin-1 and collagenase-1 or urokinase-type plasminogen activator (uPA) controlled by Ets-1. Therefore, in various cancers Ets-1 is currently reflected as a marker for poor prognosis (Davidson et al., 2001; Nakayama et al., 2001; Katayama et al., 2005). In addition, its role in various diseases and sharing of the same DBD in all Ets family members, Ets-1 has un-ique DNA binding affinities that favor its specific biological functions in a controlled manner and to inhibit its own DNA binding it has two inhibitory domains that flank its DBD (Lee et al., 2005).