MCF7 timecourses

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MCF7 response to Epidermal Growth Factor (EGF) and Heregulin (HRG)

Time course ID: human_MCF7_breast_cancer_cell_line_HRG and human_MCF7_breast_cancer_cell_line_EGF
Sample provider:  Mariko Okada-Hatakeyama and Shigeyuki Magi


Introduction

ErbB receptor family plays a central role in cellular development, proliferation, differentiation and cell death. There are four members, ErbB1/EGFR, ErbB2, ErbB3 and ErbB4 receptors, belong to this family. Dysregulation of the receptors is highly associated with incidence of variety of cancers [1]. Once activated by growth factor binding, the receptor forms homo- and heterodimer and are trans-activated by tyrosine phosphorylation, transmits the phosphorylation signal to downstream kinase such as ERK and Akt, then those kinases activate/phosphorylate transcription factors such as ELK1, CREB, or SRF in nucleus to initiate gene expression.

In human breast cancer MCF-7 cells, the heregulin (HRG; a ErbB3/4 ligand) induces cell differentiation (accumulation of lipid droplets), while epidermal growth factor (EGF; an ErbB1/EGFR ligand) elicits cell proliferation after 5-14 days of the ligand stimulation.

These ligand-stimulated cells show similar time-course profiles in terms of signaling activity and immediate early gene (IEG) mRNA expression (up to 1.5 hours)[2,3]. However, analysis of delayed transcription (up to 72 hours) using qRT-PCR showed the significant expression of transcription factors (c-FOS, FRA-1 and FHL2) specific to HRG but not by EGF [4], suggesting the existence of unknown transcription machinery for cell differentiation during this time-course.

MCF7 Fig1.png
Figure 1: Approximate expression timing of early, mid, delayed response genes.

Samples

The MCF-7 human breast cancer cell line was obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Gibco BRL, Githersburg, MD) supplemented with 10 % fetal bovine serum. Prior to growth hormone treatment, the cells were serum-starved for 16-24 hours, and then EGF (PeproTech House, London, England) or HRG-176-246 (R&D Systems, Inc., Minneapolis, MN) was added. We prepared the EGF or HRG-stimulated time-course samples at 0 (non-treated), 15min, 30min, 45min, 60min, 80min, 100min, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 5hr, 6hr, 7hr and 8hr, to cover the early phase of cell differentiation and to cover early, mid and delayed gene expression. The all cell samples were snap frozen in liquid nitrogen and provided for RNA extraction for CAGE analysis.

MCF-7 cells were incubated in the absence or presence of growth factor in serum-free DMEM. Following cultivation for 14 days, cells were stained with Oil Red O to evaluate differentiation of the cells, which results in lipid droplets accumulation (The figures from Nagashima et al. JBC 2007 [2]).

Quality control

MCF7 Fig3.png
The mRNA expression of c-FOS, FOSL1 and FHL2 was significantly induced for HRG than EGF shown in the past study (The figures from Saeki et al BMC Genomics 2009[4]).

MCF7 Fig4.png
Time-course patterns the CAGE peaks of c-FOS, FOSL1, FHL2 obtained from current analysis was consistent with the mRNA expression data shown in above.

References

[1] Yarden Y1, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2, 127-137, 2001.

[2] Nagashima, et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045-4056, 2007.

[3] Nakakuki, et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141, 884-896, 2010.

[4] Saeki, et al. Ligand-specific sequential regulation of transcription factors for differentiation of MCF-7 cells. BMC Genomics 10, 545, 2009.
























































































Beginning of non-public section

Time-course samples

We prepared the EGF or HRG-stimulated time-course samples at 0 (non-treated), 15min, 30min, 45min, 60min, 80min, 100min, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 5hr, 6hr, 7hr and 8hr. IEGs such as c-FOS and EGR appear at 45-60min, the second wave of transcription factors such as FRA-1 appear at 80-100min followed by FHL2. Transcriptional profile of ERG is mostly transient. There are many reasons suggested for this profile, for example, expression of suppressive transcription factors, siRNA and transient activation of the transcription factors. Basically overall transcriptional profiles should be divided to three categories in this time-scale; fast expression-fast suppression, mid-slow expression-mid-slow suppression, slow expression-no suppression.

Paper Plan

- Several points to discuss.

(1) Similarity and difference in TSS appearance between EGF and HRG-treated cells. Our prelim data showed that expression of TF species are quite common for EGF and HRG, but it became different as time goes. We want to know species of TSS at each time point for both ligands and make statistics of change along with time course.

(2) If there is a significant difference in TSSs for EGF and HRG appeared at some time point, we would like to know what causes this change. By predicting motif and possible binding TF.

(3) Above analysis should be quantitative, because the difference may be small at the early point but such a difference in quantity gradually bigger, and then suddenly make a qualitative difference in TSS.

(4) Also we need to categorize the TSS by time-course patterns as Erik did for QC.

(5) If possible, compare with other time-course data such as adipocyte. HRG-stimulated cells accumulate lipids, so this TSS usage may be very similar to adipocyte.

- figure descriptions; not yet - manuscript drafts - required validation experiments, can they be done in the collaborator's lab? = Partially, yes

Related samples

- already published data that can support for the analysis

[1] Nagashima, et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045-4056, 2007. [2] Nakakuki, et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141, 884-896, 2010. [3] Saeki, et al. Ligand-specific sequential regulation of transcription factors for differentiation of MCF-7 cells. BMC Genomics 10, 545, 2009.

- description of private (non-FANTOM) data that can be considered for the paper In addition to the CAGE data here, we have private time-course dataset of mRNA-seq and microRNA on EGF-and HRG-stimulated cells at 0, 30min, 1hr, 2hr and 6hr.

Quality control - Overall transcriptional profiles should be divided to three categories in this time-scale; fast expression-fast suppression, mid-slow expression-mid-slow suppression, slow expression-no suppression. This trend is most likely presented in the HRG time-course profile which is consistent with our previous work.

Literature references - with links to pubmed

Bioinformatics collaborators - Jess Mar, Kojiro Yano are kindly helping us analysis of the data. - We are also looking for Bioinformatics collaborators to determine difference in transcription factors in EGF(proliferation) and HRG(differentiation)-stimulated cells.


Zenbu configurations and status

ENCODE complementary datasets

Gene expression profiles



MARA based network results

Self-organising maps File:EGF1.pdf

File:HRG.pdf

TSS Switching (Switch Engine)

  • TSS dynamics plots can be found here:Media:mcf7breast_hrg.plots.pdf,Media:mcf7breast_egf1.plots.pdf (it is recommended to download these files instead of viewing in browser as they are large in size)
  • These figures only show those genes for which Switch Engine has detected TSS switching to occur. For now, only the first and last time points are being compared to define a switch.
    • One gene per page, RefSeq and Gene Symbol identifiers on top. Each panel is a separate TSS associated with the gene. TPM expression on y-axis and time on x-axis. Colored points correspond to specific expression, with colors referring to different replicates (key on top). Replicates with a * next to the name are those that have missing data from the currently available release of the DPI clustered and normalized TPM matrix. Such missing data were replaced by imputed values. Black lines are the mean trajectories across replicates. Purple vertical bars correspond to the time points being compared for switch definition. Magnitude of switch is expressed in each panel under TSS identifier with approximate 95% confidence interval in brackets. Note: some confidence intervals may not be accurate or may be missing due to the small sample size. TSS identifiers colored GREEN show an increasing TSS, those colored RED show decreasing TSS, WHITE show no statistically significant change.
    • TSS switch defined as the simultaneous presence of one or more increasing, together with one or more decreasing TSSs per gene. At least one increase and at least one decrease has to be statistically significant (alpha approximately 5%). At least one of the two time points being compared has to be greater than 5 tpm. At least 2 replicates have to agree to call an increasing/decreasing trajectory. TSSs have to be at least 250 base pairs away from one another to call a switch.
  • A summary file can be found here: Media:mcf7breast_hrg.summary.xls,Media:mcf7breast_egf1.summary.xls
    • The file contains the following columns: RefSeq ID, Gene Symbol, TSS ID, X{value}: mean tpm expression at {value} time point, Delta: change in tpm expression from first to last time point, FC: fold change in tpm expression from first to last time point, DeltaSum: net change in tpm expression for this gene (summed across all TSS in this gene), Balance: ratio of increasing to decreasing tpm expression TSSs per gene (e.g. a balance value of 1 means that the total increase in tpm expression in increasing TSSs is equal to the total decrease in tpm expression in decreasing TSSs), Chromosome, Strand, Start position of TSS cluster, Stop position of TSS cluster, DBTSS: distance between TSSs relative to NA value.
  • A list of genes in which TSS switching occurs can be found here: Media:mcf7breast_hrg.genelist.refseq.doc,Media:mcf7breast_egf1.genelist.refseq.doc and Media:mcf7breast_hrg.genelist.symbol.doc,Media:mcf7breast_egf1.genelist.symbol.doc
  • BED file with regions corresponding to TSS clusters involved in TSS switching can be found here: Media:mcf7breast_hrg.switchinglist.bed.xls,Media:mcf7breast_egf1.switchinglist.bed.xls (please remove .xls extension after downloading)



Related samples

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References

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Quality control

Marker gene expression

Media:human_MCF7_breast_cancer_cell_line_EGF1.png

Media:human_MCF7_breast_cancer_cell_line_HRG.png


Short RNA expression

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_MCF7_breast_cancer_cell_line_HRG/miRNA/

Comment from the sample provider

We have identified several marker genes in EGF/HRG-treated MCF-7 of which expression have been analyzed by mRNA-seq, Affymetrix microarray or qRT-PCR. Significant up-regulation of FOS, EGR4, FOSL, DUSP1, MMP1, INHBA, F2RL1, PHLDA1 and FHL2 was confirmed in the HRG-treated MCF-7 in CAGE analysis. Time-course trend is also mostly consistent with our previous data. We could not see a difference in the expression of FABP5, TUBB2A, HBP1, SOCS2, CCNG2, PDCD4, LGI1 in the EGF and HRG-treated cells although we could observe such difference in microarray analysis. In general, we have experienced that the HRG-treated cell shows a robust result, but EGF somewhat shows fluctuation in gene expression. This CAGE data also shows such a property. We think this is a nature of EGF response of MCF-7 cells, because EGF receptor number is low in MCF-7 whereas HRG receptor is 3-5 fold higher (unpublished data analyzed by single molecule imaging), this may affect the gene expression result.

ISMARA analysis results

Response to EGF1

All samples: http://ismara.unibas.ch/timecourses/MCF7EGF1/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/MCF7EGF1-avgd/averaged_report/index.html

Response to HRG

All samples: http://ismara.unibas.ch/timecourses/MCF7HRG/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/MCF7HRG-avgd/averaged_report/index.html

For more information, see ISMARA.

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