Antagonism, Estrogen receptor leads to EMT

E2/ERa signalling, in part through transcriptional activation of luminal/epithelial-related transcription factors, promotes the development of mammary epithelia along a luminal/epithelial lineage. GATA3 and ERa both promote each other (Eeckhoute et al.,2007). In normal breast epithelia, GATA3 is needed for luminal differentiation(Kouros-Mehr et al.,2008) and GATA3 and ERa control many of the same genes (Wilson et al.,2008).  In mice, forcing GATA3 expression in mesenchymal breast cancer cells produces mesenchymal–epithelial transition (MET), a reversible mechanism analogous to EMT, and prevents tumour metastasis (Yan et al.,2010). Another ERa-interacting transcription factor, FOXA1, is essential for luminal lineage in mammary epithelia and stimulates ductal development in mice (Bernardo et al.,2010). FOXA1 enhances ERa gene expression by increasing the accessibility of estrogen-response regions for ERa binding (Nakshatri et al., 2009). In breast cancer cells, on the other hand, E2 appears to increase FOXA1 expression. Importantly, ERa, FOXA1, and GATA3 are all positive breast cancer prognostic factors(Nakshatri et al.,2009).

ERa signalling enhances primary lesion formation (and therefore is mitogenic), but it can control the EMT process (and thus is anti-metastatic) up to a point. Signaling pathways that lead to EMT are antagonised by E2/ERa signalling. TGF-b, for example, has been demonstrated to generate EMT in human mammary epithelial cells, and overexpression of the EMT-inducing protein Snail boosted TGF-b signalling and invasiveness while decreasing adhesion and ERa expression in MCF-7 cells (Taylor et al.,2010). TGF-b has an anti-estrogen impact on MCF-7 cells. Smad2/3 and the Smad-selective E3 ubiquitin ligase Smurf create a ternary complex with ERa, which enhances the proteosomal degradation of Smad proteins, according to Ito et al (Ito et al.,2010).

• Ye et al.  investigated the impact of ERa overexpression in ERa-negative breast cancer cell lines (MDA-MB-468, MDA-MB-231) or ERa knockdown in ERa-positive cell lines (MCF-7, T47D) on Slug and Snail expression and phenotypes in ERa-positive cell lines (MCF-7, T47D)(Ye et al., 2010). Slug is repressed, E-cadherin is increased, and cells develop as adherent colonies with less invasiveness when ERa is forced to get expressed. ERa knockdown, on the other hand, causes an increase in Slug expression, a decrease in E-cadherin, and spindle-shaped invasive cells.

-In luminal-type breast tumours, E2 signalling modulates the activity of ERa and associated cofactors GATA3 and FOXA1 to promote an epithelial phenotype and repress EMT and pro-metastatic progression. In vitro, direct regulatory linkages have been shown, such as increased invasion and beginning of EMT in MCF-7 cells after overexpression of RELB or Snail due to suppression of ERa and E-cadherin, and in vivo results support these findings.

-In breast cancer patients, high levels of ERa, GATA3, and FOXA1 expression are linked to a better prognosis. Invasive basal-like and claudin-low breast cancer subtypes, on the other hand, are associated with high levels of RELB and Snail expression (as well as no/low expression of ERa, GATA3, and FOXA1).

• Wik et al used integrated molecular profiling to examine Endometrial cancer samples from a primary investigation cohort and three independent validation cohorts (Wik et al.,2013). Patient survival was closely linked to ER-a immunohistochemical staining and receptor gene (ESR1) mRNA expression. In the study cohort, ER-a negative was related with activation of genes implicated in Wnt, Sonic Hedgehog, and TGF-b signalling, indicating epithelial–mesenchymal transition (EMT).

-In conclusion, the absence of ER-a in endometrial cancer is linked to EMT and a shorter survival time.

• Bouris et al used specialised shRNA lentiviral particles to create stably transfected MCF-7 cells by knocking down the ER gene (identified as MCF-7/SP10 + cells) and compared them to control cells (MCF-7/c). In MCF-7 cells, ER suppression triggered cellular phenotypic changes as well as significant changes in gene and protein expression of many markers associated with epithelial to mesenchymal transition (EMT). These cells, in particular, showed increased cell proliferation, migration, and invasion(Bouris et al.,2015).

• N1-Guanyl-1,7-Diaminoheptane Sensitizes Estrogen Receptor Negative Breast Cancer Cells to Doxorubicin by Preventing Epithelial-Mesenchymal Transition by Inhibiting Eukaryotic Translation Initiation Factor 5A2 Activation  (Liu et al.,2015)

• Saleh et al. hypothesise that loss of oestrogen receptor function, which causes endocrine resistance in breast cancer, also causes trans-differentiation from an epithelial to a mesenchymal phenotype, which causes enhanced aggressiveness and metastatic tendency(Saleh et al., 2011).

-siRNA-mediated oestrogen receptor silencing in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with cytoskeleton rearrangement and switch from keratin/actin to vimentin, and ability to invade simulated extracellular matrix components.

-Invasion assay of MCF7, E2, pII and MDA231 cells through simulated ECM protein components was carried out.Cells were plated into the upper chambers of the cell invasion plate and incubated for 48 h prior to measurement of the fluorescence intensity of the invading cells in the bottom chambers. MCF7 and E2 cells were considered non- invasive (,20 FU/m), whereas pII and MDA231 cells progressively invaded the BME (100 FU/m).

• Quantitative proteomics demonstrates ER participation in CD146-induced epithelial-mesenchymal transition in breast cancer cells, according to Zheng et al. They used a three-step Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) method to examine whole cell protein profiles of MCF-7 cells that had undergone EMT as CD146 expression increased from moderate to high levels (Zheng et al.,2014).

-In total, 2293 proteins were identified in this investigation, with 103 showing changes in protein abundance that linked with CD146 expression levels, demonstrating substantial morphological and biochemical changes associated with EMT.

-According to the Ingenuity Pathway Analysis (IPA), oestrogen receptor (ER) was the transcription regulator that was most strongly suppressed during CD146-induced EMT. In addition, functional experiments demonstrated that in cells undergoing CD146-induced EMT, ER expression was suppressed, but re-expression of ER eliminated their migratory and invasive characteristics. Finally, we discovered that ER- exerted its effects on CD146-induced EMT via inhibiting Slug, a major EMT transcriptional component.

• By immunohistochemistry, Ye et al found a high direct link between ERa and E-cadherin expression in human breast tumours, showing that ERa signalling may regulate E-cadherin and influence EMT and tumour growth(Ye et al.,2010).They looked at the impacts of ERa signalling in ERa-transfected ERa-negative breast carcinoma cell lines MDA-MB-468 and MDA-MB-231, as well as the effects of ERa knockdown in naturally expressing ERa-positive lines MCF-7 and T47D, to test this theory and the mechanisms behind it.17b-estradiol (E2) decreased slug and increased E-cadherin in ERa-negative lines when ERa was overexpressed.

-In Matrigel, clones that showed the most of these alterations developed in clusters and were less invasive.Slug rose, E-cadherin reduced, cells became spindly, and Matrigel invasion increased when ERa was knocked down in ERa-positive lines. Slug expression was reduced by ERa signalling in two ways: directly, by repression of slug transcription via the formation of a corepressor complex containing ligand-activated ERa, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site oestrogen response elements (EREs); and indirectly, by phosphorylation and inactivation of GSK-3b via phosphoinositide (Akt). Inactivation of GSK-3b suppressed slug expression while increasing E-cadherin. There was a substantial inverse connection between slug and ERa and E-cadherin immunoreactivity in human breast cancer cases. The data suggest that E-cadherin and EMT are regulated by ERa signalling through slug.

No specific Uncertainties and Inconsistencies noted to the best of our knowledge.

- Endogenous ER silencing causes EMT in ER-positive breast cancer cells.

ER-positive MCF-7 cells were infected with ER shRNA lentiviral particles and stable clones were selected with puromycin (optimal dose of 0.8 g/mL) to knockdown ER gene expression (Zheng et al.,2014).

-When the number of cell passages was increased following infection, the expression of ER was gradually knocked down.

-ER gene expression was decreased by roughly 25% four passages after infection compared to control lentiviral particles transfected cells (MCF-7/c cells). The ER gene expression was lowered even more in the following passage (passage 5 post-infection) (by around 50% compared to MCF-7/c cells). In passage 7, a significant reduction in ER gene expression (about 75–80%) was seen, along with a distinct transition of cells from an epithelial to a mesenchymal phenotype.

- When MCF-7 cells reach confluency, they develop as closely packed colonies that produce sheet-like monolayer structures. Stable clones from stage 7 post-infection, on the other hand, grew as more elongated individual cells rather than tight clusters, with a spindle-like shape. For stable clones with a distinct mesenchymal character, a very substantial down-regulation of ER gene expression (above 99 percent) was found from passage 10 and beyond. MCF-7/SP10+ was given to these cells to emphasise the stable transfection (S) and passage 10 or more (P10+). The substantial down-regulation of ERa was confirmed by immunofluorescence and Western blot analysis of the same stable clones (MCF-7/ SP10 + cells).

Downstream key event occurs within hours of the occurrence of the upstream key event.

EMT is inhibited by ERa, and microRNAs either promote or inhibit EMT . These findings raise the question of whether microRNAs have a role in the control of EMT by targeting ERa mRNA. The large (>4000 nt) 30 untranslated region (30-UTR) of human ERa mRNA, as well as results that particular microRNAs are differentially expressed between ERa-positive and ERa-negative breast tumours , suggest the possibility of microRNA-mediated control of human ERa mRNA(Adams et al.,2008).

Pro-metastatic/anti-proliferative (miR-206), pro-metastatic/pro-proliferative (miR-221/222), and anti-proliferative/anti-metastatic (miR-221/223) ERa-targeting microRNAs (miR-130a, miR-145). MiR-17/92 appears to be prometastatic, although it is implicated in several feedback loops, which could make miR-17/92's expression and effects on proliferation extremely reliant on the microenvironment as well as the genetic and epigenetic background.

Accurate identification of micro-RNAs that contribute significantly to a particular pathway (such as EMT) within breast cancers in situ is one hurdle. MicroRNAs have hundreds of potential targets, and in vivo studies will be needed to identify physiologically important targets in the context of breast cancer, as well as to develop effective treatments for breast cancer that involve manipulating microRNA expression levels and identifying off-target effects. (Adams et al.,2007;Zhao et al.,2008;Leva et al.,2010;Stinson et al.,2011;Acunzo et al.,2011;Castellano et al.,2009).