Research paperBreast cancer invasion and progression by MMP-9 through Ets-1 transcription factor
Introduction
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 surrounding 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 family 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 cellular (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 differentiation, 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 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 unique 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).
Ets-1 transcription factor regulates the expression of matrix degrading proteinases called matrix metalloproteinase (MMPs). These are also called matrixins and includes a family of zinc-enriched endopeptidases which are initially expressed as inactive pro-enzymes but due to proteolytic processing and/or degradation become active enzymes and impart their important role in extra-cellular matrix-modelling in development, wound healing, inflammation and cancer (Stamenkovic, 2003; Parks et al., 2004). In cancer microenvironment, MMPs play a pivotal role in the initiation, development and progression of cancer via various mechanism(s) (Kessenbrock et al., 2010; Gialeli et al., 2011). One of the key member of MMPs known to play an essential role in cancer is matrix metalloproteinase-9 (MMP-9), also known as gelatinase B has the potential in the degradation of two basic components of basement membrane i.e. denatured collagen and collagen type IV. Its overexpression pays the way for tumor progression and metastasis (Mehner et al., 2014). Strong association in the expression of MMP-9 with aggressive and metastatic breast carcinogenesis has been found to be among the 70 genes in the Rosetta signature for poor prognosis of breast carcinogenesis (van 't Veer et al., 2002). Differential role of MMP-9 has been found in various processes of tumor development which include invasion, induction of angiogenesis and immunomodulation of microenvironment associated with tumor. Further, its substantial role in the creation of pre-metastatic niche that promotes colonization to other organ sites has been established (Kessenbrock et al., 2010). Higher levels of MMP-9 have been associated with increased invasiveness, metastasis and poor prognosis in cervical (Yu et al., 2009), colorectal (Zeng et al., 1996), ovarian (Sillanpaa et al., 2007) and in breast cancer (McGowan and Duffy, 2008). In addition, elevation in the expression level of MMP-9 in serum and urine has shown its association in the metastatic and prognostic analysis of a variety of tumors (Roy et al., 2009).
In view of the above, the present study was designed to investigate the role and regulation of Ets-1 and MMP-9 in the invasion and progression of breast carcinogenesis as MMP-9 is associated with various pathways governed by different genes and transcriptional factors in various cancers (Zhang et al., 2014).
Section snippets
Cell culture
The human breast cancer cell lines MCF-7 and MDA-MB-231 were procured from American Type Culture Collection (ATCC), USA and cultured in Dulbecco’s modified eagle medium low glucose (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) at 370C in 5% CO2.
siRNA transfection followed by semi and quantitative real-time PCR
MCF-7 and MDA-MB-231 breast cancer cells (3 × 105) were plated in 6-well plates and were allowed to adhere for 24 h before transfection experiments. For transfection, 3 μl of Lipofectamine™ 3000 transfection reagent (Invitrogen,
Ets-1 gene expression was repressed efficiently and specifically after the transfection of siRNA
Our previous findings have confirmed that Ets-1 is overexpressed in breast cancer tissue samples and have observed a differential expression pattern between receptor positive and negative status in these breast cancer patients (Nazir et al., 2019). The results prompted us to further explore the potential role and underlying mechanism of Ets-1 in breast cancer cells MCF-7 (ER, PR +ve, Her2 -ve) and MDA-MB-231 (ER, PR and Her2 -ve) and were analyzed by western blotting experiments to check the
Discussion
Breast cancer is the leading cause of mortality among women worldwide (Bray et al., 2018). In the progression of this disease involvement 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 transcription factors show altered expression in the progression of this disease. One
Declaration of Competing Interest
“The authors declare no potential conflicts of interest”.
Acknowledgements
SN acknowledges ICMR for SRF fellowship vide fellowship No. 3/2/2/362/2016-NCD-III. The work was supported by Indian Council of Medical Research (ICMR), New Delhi vide grant No. 5/13/1/TF/NICPR/16/NCD-III.
References (63)
The role of the transcription factor Ets1 in carcinoma
Semin. Cancer Biol.
(2015)- et al.
beta6 integrin induces the expression of metalloproteinase-3 and metalloproteinase-9 in colon cancer cells via ERK-ETS1 pathway
Cancer Lett.
(2014) - et al.
ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3
J. Biol. Chem.
(2012) - et al.
Regulation of in situ to invasive breast carcinoma transition
Cancer Cell
(2008) - et al.
ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression
Biochem. Biophys. Res. Commun.
(2012) - et al.
Matrix metalloproteinases: regulators of the tumor microenvironment
Cell
(2010) - et al.
The structural and dynamic basis of Ets-1 DNA binding autoinhibition
J. Biol. Chem.
(2005) - et al.
Osteopontin promotes the progression of gastric cancer through the NF-kappaB pathway regulated by the MAPK and PI3K
Int. J. Oncol.
(2014) - et al.
Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database
Ann. Oncol.
(2008) - et al.
Expression of the ets-1 proto-oncogene in human colorectal carcinoma
Mod. Pathol.
(2001)
Ets-1 upregulates HER2-induced MMP-1 expression in breast cancer cells
Biochem. Biophys. Res. Commun.
Mesangial cell expression of proto-oncogene Ets-1 during progression of mesangioproliferative glomerulonephritis
Kidney Int.
Prognostic significance of matrix metalloproteinase-9 (MMP-9) in epithelial ovarian cancer
Gynecol. Oncol.
EMT as the ultimate survival mechanism of cancer cells
Semin. Cancer Biol.
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries
CA Cancer J. Clin.
Disseminated tumor cells in bone marrow of gastric cancer patients: correlation with tumor hypoxia and clinical relevance
J Oncol
The transcriptional network for mesenchymal transformation of brain tumours
Nature
Inflammation-related factors predicting prognosis of gastric cancer
World J. Gastroenterol.
Ets-1 messenger RNA expression is a novel marker of poor survival in ovarian carcinoma
Clin. Cancer Res.
The biology of the Ets1 proto-oncogene
Mol. Cancer
Ets-1 triggers and orchestrates the malignant phenotype of mammary cancer cells within their matrix environment
J. Cell. Physiol.
Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein
Proc. Natl. Acad. Sci. U. S. A.
Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting
FEBS J.
Thymomegaly, microsplenia, and defective homeostatic proliferation of peripheral lymphocytes in p51-Ets1 isoform-specific null mice
Mol. Cell. Biol.
The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells
Genes & Cancer
Prostaglandin E2 enhances pancreatic cancer invasiveness through an Ets-1-dependent induction of matrix metalloproteinase-2
Cancer Res.
miR-874 inhibits cell proliferation, migration and invasion through targeting aquaporin-3 in gastric cancer
J. Gastroenterol.
Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with histological grading and growth pattern
Histol. Histopathol.
Expression of dominant-negative form of Ets-1 suppresses fibronectin-stimulated cell adhesion and migration through down-regulation of integrin alpha5 expression in U251 glioma cell line
Cancer Res.
ConTra v3: a tool to identify transcription factor binding sites across species, update 2017
Nucleic Acids Res.
The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage
PLoS One
Cited by (50)
Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells
2023, European Journal of Obstetrics and Gynecology and Reproductive BiologySelf-transfecting GMO-PMO chimera targeting Nanog enable gene silencing in vitro and suppresses tumor growth in 4T1 allografts in mouse
2023, Molecular Therapy Nucleic AcidsE2F3 promotes liver cancer progression under the regulation of circ-PRKAR1B
2021, Molecular Therapy Nucleic AcidsCitation Excerpt :Together, the data from Figure 2 indicated that circ-PRKAR1B could promote liver cancer cell invasion and migration capacities by sponging miR-432-5p. To explore the downstream genes that are involved in the circ-PRKAR1B/miR-432-5p signaling-mediated cell invasion/migration of liver cancer cells, we first selected some genes (MMP9,12,13 E2F3,14 CD44,15 SOX9,16 GSN,17 FOXC2,18 and SMAD419,20) that were reported to play important roles in the progression of many cancers. The data of western blot assay showed E2F3 may be the candidate gene that could be regulated by circ-PRKAR1B, by overexpressing or knocking down circ-PRKAR1B in liver cancer cells (Figure 3A).
Phthalates promote the invasion of hepatocellular carcinoma cells by enhancing the interaction between Pregnane X receptor and E26 transformation specific sequence 1
2021, Pharmacological ResearchCitation Excerpt :It can mediate the transcription of matrix metalloproteinases (MMPs), destroy the extracellular matrix of the primary site of tumor cells, and ultimately cause the invasive growth and metastasis of cells [10–12]. Therefore, the activation of the transcription factor ETS-1 is an essential highly aggressive feature of cancerous cells and is regulated by co-factors/co-regulators [13,14]. Recently, multiple studies have revealed that nuclear receptors, including estrogen receptor (ER)α, androgen receptor (AR), or vitamin D receptor (VDR), could interact with ETS-1 and these activated nuclear receptors induced by their respective ligands could enhance the activation of ETS-1 [15–17].