Towards an understanding of GPER1 expression and regulation and its significance in human breast cancer', is a presentation of my original research work done at the Department of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, India, under the guidance of Dr. .The entire staff of the Department of Biological Sciences and Bioengineering, IIT Guwahati for efficient and timely assistance whenever required.
Introduction
Aim and scope of the present thesis work
Objectives
Involvement of ERα in estrogen-mediated induction of GPER1 expression in MCF-7 cells expression in MCF-7 cells. Collectively, our results provide clear evidence for the involvement of ERα in the regulation of GPER1 expression in MCF-7 breast cancer cells.
Review of literature
Breast cancer
The relative mRNA levels of GPER1 in the same panel of breast cancer cell lines were assessed by qRT-PCR. In silico analysis of the GPER1 locus in the human genome using Matinspector software (a promoter analysis tool from the Genomatix software package) revealed three potential estrogen response elements (EREs).
GPER1 (G-Protein Coupled Estrogen Receptor 1)
- Localization of GPER1
- Regulation of GPER1 expression
GPER1-mediated signaling
- GPER1 ligands
- GPER1 signaling in normal physiology
- GPER1 signaling crosstalk with other signaling pathways
- GPER1 and cancer
- GPER1 and breast cancer
Analysis of GPER1 locus of the human genome in the UCSC genome browser revealed ERα enrichment in the GPER1 upstream region. Taken together, our results revealed the mechanism of estrogen-mediated regulation of GPER1 in breast cancer.
Materials and methods
Cell culture and treatments
- Cell lines and culture media
- Routine culture and treatment conditions
- Cell counting by dye exclusion method
The monolayer was washed with DPBS before starting the treatments to remove the spent medium components. In experiments involving E2 and/or PPT treatments, cells were preincubated in M2 for 24 h before treatments began to ensure complete removal of residual M1 components.
MTT assay
Cell cycle analysis by flow cytometry
- Sample preparation
- Data acquisition and analysis
The distribution of cells in different phases of the cell cycle was assessed using FCS Express 6 software. Histogram of the FL2 area for the gated population was drawn to visually represent cells in different phases of the cell cycle.
Gene expression analysis
- Primers
- Total RNA isolation and cDNA synthesis
- Routine RT-PCR and quantitative real time RT-PCR
Total RNA was isolated using TRIzol (Invitrogen Corporation, Grand Island, NY, USA), or a similar RNA extraction reagent prepared in house. For quantitative real-time RT-PCR (qRT-PCR) Applied Biosystems® 7500 real-time PCR platform was used.
Generation of polyclonal antibody
- Immunization and immune serum collection
- Affinity purification
- Indirect ELISA
On day two, the peptide column was equilibrated to room temperature and then washed with three RBV coupling buffer. The diluted hyperimmune serum was mixed with the peptide-linked resin and incubated on a shaker for 4 hours at room temperature.
Western blotting
- Total protein isolation and quantification
- SDS-PAGE, transfer and detection
100 μL of pre-immune serum (PIS), hyper-immune serum, or peptide affinity-purified antibody, diluted 1:5000 in PBST containing 1% skim milk, was added to each well and incubated for 2 h at room temperature of the room. Blots were blocked in 1% gelatin in TBST for 1 h at room temperature and then probed with primary antibody diluted in 0.1% gelatin in TBST for 1 h at room temperature.
Immunohistochemistry (IHC)
- IHC staining
- Evaluation of immunostaining pattern
In each series of preparations, normal tissue in the biopsy was taken as an internal control, and a preparation without primary antibodies was taken as a negative control. Normal biopsy tissue was taken as an internal control, and a known positive was taken with each batch of slides, and a slide without primary antibody was taken as a negative control.
Microarray experiments
- RNA isolation, labeling, hybridization and image acquisition
- Data analysis
DNA methylation analyses
- Modified COBRA assay
- Bisulfite sequencing
The sequence of the expected amplicon was retrieved from NCBI and cytosine bases were manually converted to thymine, assuming all CpG sites are methylated. The methylation status of all CpG sites in each of the inserts was represented as a lollipop model.
TCGA data analysis
- Analysis of associations between GPER1 mRNA expression and
- Survival analysis
- Expression-methylation correlation (EMC) analysis using TCGA-BRCA
The GPER1 mRNA expression values of the tumor samples, in each of the above groups, were extracted from the expression table. For a probe-wise analysis of the relationship between GPER1 expression and methylation, two approaches were used.
ChIP-Seq analysis
In the EMC analysis, GPER1 expression data and probe-wise methylation data across tumor samples were analyzed with the Spearman rank correlation test using the statistical package R. This was followed by a pair-wise analysis investigating the difference in mean expression of GPER1 in the hypo- and hyper-methylated groups using a two-sample Welch's t-test.
Statistical analyses
We have also clearly shown that the involvement of dnCpGi in the regulation of GPER1 expression is dispensable. Hyper-methylation of upstream CpG island shore is a potential mechanism of GPER1 silencing in breast cancer cells.
Generation of an anti-GPER1 polyclonal antibody for analysis of
Results
- Polyclonal anti-GPER1 antibody generation and affinity purification
- Immunohistochemical assessment of the peptide-affinity-purified antibody
A western blot of total protein from breast cancer cell lines showed a prominent band of ∼52 kDa (black arrow). Then, the performance of the purified antibody was assessed by Western blotting analysis of the total protein of breast cancer cell lines.
Discussion
The assessment of the importance of GPER1 expression in breast cancer increasingly indicated a connection between GPER1 expression and ERα18, 23, 24. The present thesis focuses on understanding the role of GPER1 expression and regulation in breast cancer. We have successfully demonstrated the expression of three GPER1 transcript variants in breast cancer cell lines.
Taken together, this study provides new insight into the role of GPER1 in breast cancer. Hypermethylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells Gen.
GPER1 expression in human breast tissues: an immunohistochemical
Results
- Results from the analyses of TCGA-BRCA dataset
- Correlation of the GPER1 mRNA expression with that of the breast cancer
- GPER1 expression and prognosis of breast cancer patients
The distributions of GPER1 mRNA expression in molecular subtypes are shown as box plots in Figure 5.3. Analysis of TCGA-BRCA data for association of GPER1 mRNA expression with histopathological parameters revealed that its expression was significantly associated with ERα, PR, HER2 status and molecular subtypes of breast cancer. The distribution of GPER1 mRNA expression values in normal and tumor (A), ERα-positive and -negative (B), PR-positive and -negative (C), and HER2-positive and -negative (D) tumors are shown as frame. plots.
The distribution of GPER1 mRNA expression values in 5 different molecular subtypes of breast cancer are plotted as boxplots. The correlation of GPER1 mRNA expression with that of ERα, PR, HER2 and ERβ was analyzed by Pearson's correlation test.
Discussion
Interestingly, estrogen-mediated induction of GPER1 mRNA variants was abrogated in the absence of ERα (Figure 6.4.A). In this study, we gathered evidence from in vitro and in silico analyzes to demonstrate the involvement of ERα in the regulation of GPER1 expression in breast cancer cells. Taken together, the results of the in vitro cell culture experiments and analysis of the TCGA data strongly suggest a role for DNA methylation in the suppression of GPER1 expression in breast cancer cells and the importance of CpG sites that likely represent upCpGi coastal regions.
In light of the confusion in existing reports, our contribution is important in establishing the role of GPER1 as a tumor suppressor in breast cancer. In the seventh column, Optimal: optimal concentration (> 30 ng/microliter and < 2500 ng/microliter); Suboptimal: Suboptimal concentration (< 30 ng/microliter).
Functional link between GPER1 and ERα in breast cancer
Results
- GPER1 transcript variants
- The involvement of ERα in the estrogen-mediated induction of GPER1
- In silico analyses of ERα binding sites at GPER1 locus
Interestingly, when the relative levels of GPER1 mRNA variants were assessed relative to the 6-hour vehicle-treated group, a time-dependent modulation of mRNA variants was also evident in the vehicle-treated groups (Figure 6.2.B-D). As observed previously, GPER1 mRNA levels in the scrambled siRNA-transfected groups were significantly increased by estrogen (10 nM E2) treatment compared to the corresponding vehicle-treated groups (Figure 6.4.A). Among the predicted sites, only ERE2 was found to be in the positive strand of genomic DNA.
The results were visualized in the UCSC genome browser153 and a snapshot is shown in Figure 6.6. A snapshot of the GPER1 locus in the human genome as seen from the UCSC Genome Browser, along with several tracks for ERα binding sites.
Discussion
Identification of a gene (GPR30) with G protein-coupled receptor superfamily homology associated with estrogen receptor expression in breast cancer. The G protein-coupled receptor GPR30 mediates the upregulation of c-fos by 17beta-estradiol and phytoestrogens in breast cancer cells. G protein-coupled receptor 30 is upregulated by hypoxia-inducible factor-1alpha (HIF-1alpha) in breast cancer cells and cardiomyocytes.
G protein-coupled receptor 30 is critical for progestin-induced growth inhibition in MCF-7 breast cancer cells. Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.
CpG island shore methylation determines the basal GPER1 expression
Results
- GPER1 expression in MCF-7 and MDA-MB-231 cells
- CpG islands in the GPER1 locus
- Differential methylation of the upCpGi
- GPER1 expression inversely correlates with methylation
A qRT-PCR experiment confirmed lower levels of total GPER1 and variant mRNA in MDA-MB-231 cells (Figure 7.1.B). Analysis of 23 inserts from each of the two cell lines revealed that of the 32 CpGs covered by upCpGi, eight CpGs at the 3' end were hypermethylated in MDA-MB-231 cells (Figure 7.3.B). The degree of methylation in the upCpGi residue was similar in both cell lines.
As shown in Figure 7.4, 5-aza treatment caused a significant increase in total GPER1 mRNA and its variants in MDA-MB-231 cells. We also considered a composite methylation score in the upCpGi region as the average beta score of these 12 probes.
Discussion
In vitro studies have demonstrated the importance of GPER1 in progression, development, and drug resistance. Our contributions are significant in terms of revealing the mechanisms of GPER1 expression regulation in breast cancer. The G protein-coupled estrogen receptor (GPER) is expressed in two different subcellular localizations that reflect different tumor properties in breast cancer.
The putative G protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. Study of regulation of steady-state mRNA levels of cystatin A by estrogen in breast cancer cells.
Effect of GPER1 activation on the global transcriptome in
Results
- GPER1 inhibits the E2-induced proliferation of MCF-7 cells
- GPER1 activation arrests MCF-7 cells at G2/M-phase and induces the apoptosis 75
- E2-target genes regulated by GPER1
Effect of G1 stimulation on MCF-7 cells cultured in M1 and M2. MCF-7 cells were seeded in a 96-well plate and cultured for 48 h in M1. GPER1 activation arrests MCF-7 cells in the G2/M-phase and induces apoptosis. G1-treated cells were found to be morphologically different from controls. Unlike the EtOH-treated control group, G1-treated cells were spherical and partially attached to the surface.
The images of the cells were captured at the beginning of treatment (0 h) and after 24 h (24 h). MCF-7 cells were treated with EtOH, 100 nM or 1 µM G1 in M1 medium for 48 hours and the transcription profiles were assessed by microarray.
Discussion
Use of selective estrogen receptor modulators and selective estrogen receptor regulators in breast cancer. Nuclear replacement estrogen receptor GPR30 mediates 17-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts. 17-beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells through activation of extracellular signal-regulated kinase via G protein-coupled receptor 30.
Conclusion
