The Mechanism Underlying The Regulation Of Nuclear Receptor ERRα Expression By MicroRNA And The Regulatory Mechanism Of ERRα Controls The Chemokine CCL2Expression In Breast Cancer Cells

The estrogen receptor-related receptor alpha (ERRα) is an orphan member of thenuclear receptor superfamily of transcription factors. It was cloned as a nuclear receptorsharing high level of structural homology with estrogen receptor alpha (ERα). However,there is a difference between ERRα and ERα that no endogenous ligand for ERRα has beenidentified so far. Crystallographic studies have suggested that ERRα can recruitco-regulators to regulate the expression of target genes in a ligand-independent manner. Inanother word, ERRα is a constitutively active orphan nuclear receptor.Recent studies have shown that ERRα plays very important roles in breast cancerinitiation and progression serving as a most important component of breast cancer signalingnetwork. First，breast cancer tissues express a higher level of ERRα, compared to adjacentbenign tissues. The high level ERRα in breast cancer tissues is a prognostic factor of poorclinical outcome. Second, ERRα interferes with some classic breast cancer signalingpathways, such as ERα and HER2signaling pathway, through cross-talk with thesesignaling molecules. Third, a lot of oncogenic signaling pathways that employ thePI3K/AKT as second-signal transducer impinge upon ERRα and its co-activator PGC-1family. Activated ERRα/PGC-1complex can facilitate the progression of breast cancerthrough regulating the expression of a large number of genes implicated in tumor energymetabolism, proliferation, migration and invasion.In summary, ERRα is a signaling molecule widely expressed in various subtypes ofbreast tumor, which independently and/or coordinately modulates the tumor progression.Therefore, finding an effective approach to manipulate the activity or expression of ERRαand elucidating the molecular mechanisms through which it participates in breast cancer progression have profound significance for the deeply understand of ERRα’s role in breastcancer and the therapy invention targeting ERRα. So, we divided our studies into followingtwo parts:At first, we focused on the regulatory mechanism of ERRα gene expression in breastcancer. We elucidated the detail that microRNA regulated the expression of ERRα and itsimpact on the proliferative and migratory capacity of breast cancer cells. We got theseachievements:1. We have identified that miR-137was the microRNA regulating the expression ofERRα employing the bioinformatics search and western blot.2. Using Luciferase-reporter assay combined with site-direct mutation, we have foundtwo functional target sites for miR-137within the ERRα3’-UTR, which located at nt480~486and nt596~602respectively.3. We have checked the expression levels of endogenous miR-137and ERRα innormal breast epithelial cell line and breast cancer cell lines. We found that compared withnormal breast epithelial cell, breast cancer cells expressed higher level of ERRα and lostmiR-137.4. Using Real-time PCR and Western blot, we have demonstrated that miR-137inhibited the expression of endogenous ERRα at both mRNA level and protein level.Moreover, miR-137only took this kind of inhibitory effect on ERRα, rather than on otherERR family members.5. We also have found that miR-137influenced the expression of some ERRα targetgenes and the biological capacities of breast cancer cells though ERRα. In particularly, wehave found that miR-137dramatically inhibited the proliferation of breast cancer cell lineMCF-7, BT-474and SK-BR-3through down-regulating the expression of ERRα. For breastcancer cell line MDA-MB-231, miR-137failed to influence its proliferation butsignificantly impaired its migratory capacity.6. Employing SK-BR-3and MDA-MB-231as cell model, we explored the mechanismunderlying the inhibitory effect of miR-137on cell proliferation and migration respectively.We have demonstrated that miR-137achieved these effects at partly through theinactivation of ERRα-CCNE1and ERRα-WNT11pathway, which are leading course tofacilitate the breast cancer cell proliferation and migration respectively. In the part2of our studies, we also tried to explore the detailed mechanisms throughwhich ERRα participated in the breast cancer malignant progression. The present studiessuggested that Chemokine (C-C motif) ligand2(CCL2)/MCP-1might be a new target geneof ERRα, We have found that:1. The basal level of CCL2generally increased in breast cancer cell lines, comparedwith that of normal breast epithelial cell.2. The change of ERRα expression level in breast cancer cells would dramaticallyinfluence the expression of CCL2.3. Inhibition of the transcriptional regulation activity of ERRα with specific inverseagonist XCT-790significantly impaired the transcription of CCL2gene.4. The luciferase-reporter assay suggested that over-expression of ERRα induced thetranscriptional activity of CCL2promoter. The present data suggested that the promoterregion ranged from-2794bp to-820bp might contain the functional sites mediating theactivation of CCL2promoter induced by ERRα.Taken together, our studies closely focused on ERRα, the novel biomarker of breastcancer, exploring the approach to regulate its expression in breast cancer and themechanism through which it participates in the progression of this disease. We hope ourresearch will provide theoretical foundation of deeply elucidating the relationship betweenERRα and breast cancer and the development of individually therapeutic strategies forERRα positive breast cancer.