Regulation of ESR1 expression by promoter methylation and single nucleotide variants in predicted miRNA target sites in the 3' untranslated region

Abstract

Background
The estrogen receptor alpha (ERa), encoded by ESR1, is overexpressed in 75 – 80 % of breast cancers. Treatments targeting ERa are challenged by development of resistance due to loss of ERa expression. It is crucial to increase understanding of regulatory mechanisms of ERa expression and identify alterations in these mechanisms. Modifications of miRNA target sites in ESR1 3’ UTR or methylation of CpG sites in its promoter can alter regulation of ERa.

Specific aims
The project aimed to 1) optimize functional studies of miRNA target sites and 2) develop a method to study ESR1 promoter methylation in a breast cancer cell line sensitive to fulvestrant and a resistant subclone.

Results
Plasmid constructs with ESR1 3’ UTR fragments containing the selected miRNA target sites were cloned and tested in luciferase gene reporter assays in MCF7 and HepG2. None of the miRNA target sites showed strong downregulation of gene expression contrary to a perfect complementary miRNA target with and without mismatch. Genomic DNA from HCC1428 parental and resistant cell line was treated with bisulfite. Two ESR1 promoter region fragments were successfully amplified by PCR and subjected to Sanger sequencing to analyze methylation status.

Conclusion
Functional study of ESR1 miRNA target sites in biological accurate context is complex. Several parameters interplay in regulation of gene expression by miRNA as seen with different site complementarity. Moreover, miRNA have multiple targets. Therefore, reducing the expression level of the plasmid constructs by using a weaker promoter could perhaps prevent overloading of the pool of targets of the miRNA of interest. Regarding analysis of methylation status ESR1 promoter regions, optimization of the amplification of the regions of interest of bisulfite converted DNA is necessary. Analyses of ESR1 promoter regions are currently extended to

Popular Abstract

Regulation of ERa expression, a key to understand breast cancer

Breast cancer is one of the most likely cancers to occur in 1 out of 8 women. Around 8 out 10 diagnosed breast cancers present a singular characteristic: a high level of a protein called estrogen receptor alpha (ERa). This protein regulates expression of genes involved in diverse cellular processes such as cell proliferation and death. Therefore, dysfunction in the regulation of ERa expression can lead to breast cancer formation and progression. Moreover, modifications of its expression pattern have also been associated with development of resistance toward drugs used to treat estrogen-positive breast cancer patients.

Expression of ERa starts by synthesis of an RNA molecule. This RNA molecule is used as a template for synthesis of ERa protein. These steps are regulated by diverse mechanisms. Low level of ERa proteins can be maintained by binding of small RNA molecules, called microRNA, to specific regions of the ERa RNA. Modifications of those specific binding regions have been identified in breast cancer patients. They could impact regulation of ERa by the microRNA and thus result in high level of ERa proteins in breast cancers. These findings raised the question of a potential link between those modifications and high expression of ERa in breast cancers. To answer this question, we first must study those binding regions without modification. Are the non-modified regions initially involved in low expression of ERa ? Therefore, the first aim of this project was to develop a molecular tool to investigate the role of these region in regulation of ERa. No conclusions of a potential role of the tested binding region on regulation of ERa expression could be drawn from the experiments. This project highlighted the complexity of designing a molecular tool to study these binding regions. For further designs, more parameters are likely to be considered to develop a functioning molecular tool.

In breast cancers patients with high level of ERa, the protein is the main therapeutic target. One type of treatment, called Fulvestrant, uses drugs to induce degradation of ERa. However, its performance is challenged by development of resistance. It has been suggested that in response to exposition to fulvestrant, cancer cells adapt to the new conditions by silencing ERa expression via epigenetics. Epigenetic mechanisms are known to regulate the step the step of gene expression transcribing DNA to RNA by modifying the DNA structure in response external factors, such as therapeutics. For instance, the addition of a chemical group in the upper region of the DNA sequence of the ERa gene can prevent synthesis of ERa RNA molecules and ultimately ERa proteins. This could at a later stage lead to resistance to fulvestrant. Is there a difference of chemical group bound to the upper region of the DNA sequence of ERa between breast cancer cells sensitive to fulvestrant and resistant to fulvestrant ? The question can be answered using a sequencing method that allows counting the number of chemical groups bound to the upper region of the DNA sequence of ERa. The first steps of this sequencing method were tested for two portions of the upper region of the DNA sequence of ERa. The optimal conditions of the first steps were successfully identified. These results will be used to create a sequencing library of several portions of the upper region of ERa DNA in multiple breast cancer cell lines. This library could later be used to predict patient response to fulvestrant.

Master’s Degree Project in Molecular Biology 30 credits 2023 Department of Biology, Lund University

Advisor: Persson Helena
Functional Breast Cancer Genomics, department of oncology, faculty of Medicine, Lund University

• author: Binet, Mathilde
• supervisor: Helena Persson
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9135682
• date added to LUP: 2023-08-30 14:58:58
• date last changed: 2023-08-30 14:58:58