The Molecular Mechanism For TWIST Epigenetic Reppressing Estrogen Receptor-alpha Expression And It’s Clinical Significance In Breast Cancer

objective: To investigate the molecular and functionalregulatory relationship between TWIST, an important transcription factorwhich is correlated to tumor metastasis, and Estrogen Receptor-alpha(ERα), and it’s clinical significance in breast cancer. Methods: First ofall, to establish an inducible and stable expression pcDNA-TWIST cellline; Then, for the purpose of identifing the potential TWIST-bindingsites in human genome, we performed ChIP-Sequencing9analysis inHEK293cell lines that express either Flag-TWIST or Flag control; Inturn, to examine whether the components of NuRD complex are alsorecruited to the TWSIT binding site of the ESR1gene in HEK293cellsexpressing Flag-TWIST, we performed sequential ChIP-re-ChIP assaysand ChIP; Once againe, to identify the important role of TWIST inrepressing ERα expression, we detected the ERα mRNA and protein levelby real time RT-RCR and Western blot analysis in five different type ofbreast cancer cell lines; In addition, to detect whether theTWIST/NuRD-associated region of the ESR1gene has a transcriptionalrepression function, we performed these experiments as follows:1,Luciferase reporter assay was performed;2, we addressed whetherTWIST forced expression would repress endogenous ERα expression in TWIST-negative and ERα-positive breast cancer cells, we used shRNAinterference and cell transfection to knock down or overexpress TWIST;At last, to examine the effect of TWIST on ERα expression in humanbreast tumor and it possible clinical significance, we performedsemi-quantitative RT-PCR and immunohistochemistry (IHC) to measuremRNA and protein in TWIST and ERα in a total of28invasive breastductal carcinomas. Results: An inducible and stable expressionpcDNA-TWIST cell line established successfully. In ChIP-Seq analysis,we identified one TWIST association region within intron7of the humanESR1gene; In ChIP-re-ChIP assays revealed TWIST-associated Mi-2,MTA2and HDAC2were recruited to TWIST binding site of the ESR1gene in Flag-TWIST cells. This indicates that TWIST can efficientlyrecruit the NuRD complex to the TWIST-binding site of the ESR1genein HEK293cells. In highly aggressive ERα-negative cell lines SUM1315,MDA-MB-435and MDA-MB-231, we performed real time RT-PCR andWestern blot analysis and found that high level of TWIST mRNA andprotein were detected in the ERα-negative SUM1315and MDA-MB-435cell lines, and a low level of TWIST protein was also detected in theERα-negative MDA-MB-231cell line. In contrast, TWIST mRNA andprotein were undetectable in T47D and MCF-7cells that express highlevels of ERα. These results indicate that TWIST expression is negativelycorrelated with ERα expression in these cell lines. In luciferase reporter assay, we amplified a DNA fragment containing the six E-Boxes withinthe TWIST-binding region from human ESR1gene, cloned this fragmentinto the pGL3-Pro plasmid with a basal promoter and a luciferase reportercoding sequences. After this pGL3-332ESR1-Pro-Luc reporter plasmidwas co-transfected with a TWIST expression vector or a parent controlvector into HEK293cells, we observed that overexpression of TWISTsignificantly repressed the activity of this reporter. In contrast,overexpression of TWIST did not repress the activity of thepGL3-Pro-Luc parent reporter. These results suggest that theTWIST/NuRD-associated region of the ESR1gene has a transcriptionalrepression function; In shRNA interference and cell transfectionexperiment, to knock down or overexpress TWIST, our analysis revealedthat knockdown of TWIST in MDA-MB-435and4T1cell lines increased2.5-3folds of ERα mRNA expression, in T47D cell line, TWISToverexpression repressed ERα mRNA, and reduced ERα protein level inMCF-7cell line. This indicates that TWIST can repress ERα expressionin breast cancer cell lines. We tested whether Trichostain A (TSA), aHDAC inhibitor, could inhibit TWIST/NuRD-mediated repression of ERexpression. In the TSA-treated4T1cell line，we detected that inhibition ofNuRD HDAC activity can effectively relieve the transcriptionalrepression of the ESR1gene. At last，we collected28invasive breastductal carcinomas, and performed semiquantitative RT-PCR and immunohistochemistry to measure TWIST and ERα mRNA and protein.By analyzing data obtained from these assays we identified17(61%)tumors with high levels of TWIST mRNA and protein and very low orundetectable levels of ERα mRNA and protein,7(25%) tumors with verylow or undetectable levels of TWIST mRNA and protein and high levelsof ERα,2(7%) tumors with high levels of both TWIST and ERα mRNAand pro-teins and2(7%) tumor with very low or undetectable levels ofboth TWIST and ERα mRNA and proteins. These results indicate thatTWIST expression is inversely correlated with ERα expression in most(86%) of the invasive breast carcinomas. Conclusion: We revealed themolecular mechanism for TWIST epigenetic repressing ERα expression.TWIST represses ERα expression by recruiting NuRD complex inTWIST-positive expression breast cancer cell lines. TWIST expressionand ERα expression is negative correlated. The expression or reppressionof ERα in breast cancer cell lines is a reversible process. In those celllines with ERα expression repressed, if we can knockdown TWIST or useTSA, a HADC inhibitor, to recover ERα expression, may allow thesecancers to restore their sensitivity to endocrine therapy in clinic.Consequently, TWIST might be a new target and strategy for breastcancer therapy.