Summary of Study ST002910

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001810. The data can be accessed directly via it's Project DOI: 10.21228/M8RD98 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002910
Study TitleIdentifying the impact of RelA overexpression in triple negative breast cancer cells using mass spectroscopy-based proteomics and metabolomics analysis
Study TypeLC/MS/MS
Study SummaryIntroduction: The incidence of chemotherapeutic resistance among breast cancer patients continues to rise steadily. The expression of RelA, a prominent member of the Rel family, is closely associated with the aggressiveness of triple-negative breast cancer (TNBC) and a poor prognosis for patients. Consequently, it is crucial to deeply investigate the molecular changes that underlie breast cancer progression and chemotherapy resistance associated with RelA overexpression. Materials and Methods: In this study, we performed a comprehensive quantitative analysis of proteomics and metabolomics in triple negative breast cancer (TNBC) cells overexpressing RelA, compared to TNBC cells with basal levels. State-of-the-art Trapped Ion Mobility Spectroscopy, Time-of-Flight Mass Spectrometry (TIMS-TOF-MS) was employed to achieve high-resolution analysis. Results: The results unveiled 27 significantly dysregulated proteins and 21 altered metabolites in MDA-MB-231 RelA cells relative to MDA-MB-231 cells. The upregulated proteins in MDA-MB-231 RelA cells include interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and cytochrome b5, which are involved in tumor migration and progression and regulation of the cellular redox system, respectively. Conversely, the adhesion molecule, integrin alpha-2, was downregulated in MDA-MB-231 RelA cells. In addition, metabolomics analysis revealed significant upregulation in the signal transducer, cyclic AMP, and downregulation in multiple nucleotides such as pyridine, adenine, and thymidine. The integrated analysis of multi-omics data highlighted the highest impacted pathways, including ABC transporters, arginine biosynthesis, purine, pyruvate, pyrimidine, glutathione, and phenylalanine metabolism. Conclusion: This study effectively recognized significantly dysregulated proteins and metabolites in MDA-MB-231 RelA cells, providing valuable insights into potential proteins, metabolites, and signaling pathways that mediate the aggressiveness of TNBC through RelA. Moreover, the multi-omics integrated analysis elucidated RelA role in chemotherapy resistance, tumor progression, migration, and invasion, which would propose potential biomarkers and novel therapeutic targets.
Institute
Sharjah Institute for Medical Research
Last NameFacility
First NameCore
AddressM32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Emailtims-tof@sharjah.ac.ae
Phone+971 6 5057656
Submit Date2023-09-26
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2024-04-05
Release Version1
Core Facility Core Facility
https://dx.doi.org/10.21228/M8RD98
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001810
Project DOI:doi: 10.21228/M8RD98
Project Title:Identifying the impact of RelA overexpression in triple negative breast cancer cells using mass spectroscopy-based metabolomics analysis
Project Type:LC-MS/MS
Project Summary:In this research study, metabolomics alterations associated with the overexpression of RelA in the TNBC cell line, MDA-MB-231, were investigated using liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique. UHPLC-QTOF-MS is a rapid, sensitive, high-resolution mass spectrometry technology that detects and identifies fragment patterns of compounds in complex samples. This analysis would unravel the key metabolites modified due to RelA overexpression, thus highlighting the altered signaling pathways behind the aggressiveness and chemoresistance of TNBC through NF-?B alteration.
Institute:Sharjah Institute for Medical Research
Last Name:Facility
First Name:Core
Address:M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
Email:tims-tof@sharjah.ac.ae
Phone:065057656

Subject:

Subject ID:SU003023
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA316291MDA01-01-4738MDA
SA316292MDA03-02-4743MDA
SA316293MDA03-01-4742MDA
SA316294MDA01-02-4739MDA
SA316295MDA02-01-4740MDA
SA316296MDA02-02-4741MDA
SA316297REL03-02-4737REL
SA316298REL03-01-4736REL
SA316299REL01-01-4732REL
SA316300REL01-02-4733REL
SA316301REL02-01-4734REL
SA316302REL02-02-4735REL
Showing results 1 to 12 of 12

Collection:

Collection ID:CO003016
Collection Summary:Triple negative breast cancer cell line (MDA-MB-231) was purchased from the European Collection of Authenticated Cell Cultures (ECACC). MDA-MB-231 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All incubations were done at 37˚C in a 5% CO2 air atmosphere.
Sample Type:Breast cancer cells

Treatment:

Treatment ID:TR003032
Treatment Summary:P65 plasmid tagged with GFP (Addgene plasmid #23255) was used for transfecting MDA-MB-231 cells. Transfection was performed as described previously [8]. In brief, following the seeding of MDA-MB-231 cells in 6 well plates, cells were transfected with 2.5 μg of RelA/P65 plasmid using lipofectamine 3000 prepared in Opti-MEM I media (Invitrogen Carlsbad, CA, USA). After cells incubated for 6 h, RPMI-1640 culture media was added to replace Opti-MEM I media. Cells were selected using Genticine antibiotic (G418) (InvivoGen, USA, #ant-gn-1).

Sample Preparation:

Sampleprep ID:SP003029
Sampleprep Summary:MDA-MB-231 and MDA-MB-231 RelA cells were cultured in T75 flasks until reaching 70% confluency. Cells were detached using trypsin, and centrifugation was performed to collect the cell pellets. A total of 3×106 cells were collected and stored at -80 ℃ in PBS buffer for further analysis. Cell pellets were prepared in triplicates for metabolomics and proteomics analyses.

Combined analysis:

Analysis ID AN004779
Analysis type MS
Chromatography type Reversed phase
Chromatography system Bruker Elute
Column Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm, 1.8um)
MS Type ESI
MS instrument type QTOF
MS instrument name Bruker timsTOF
Ion Mode POSITIVE
Units AU

Chromatography:

Chromatography ID:CH003611
Chromatography Summary:For metabolomics analysis, a Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 μm beads) was kept at 35 °C. For metabolomics, 10 μL of each sample was injected twice and eluted using a 30-min gradient
Instrument Name:Bruker Elute
Column Name:Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm, 1.8um)
Column Temperature:35
Flow Gradient:a 30-min gradient as follows: 1% ACN was kept for 2 minutes before ramping to 99% ACN for 15 minutes, then holding at 99% ACN for 3 minutes before re-equilibrating to 1% ACN for 10 minutes
Flow Rate:For elution 250 μL/min and 350 μL/min for re-equilibration
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS004525
Analysis ID:AN004779
Instrument Name:Bruker timsTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Using an Apollo II electrospray ionization (ESI) source or an CaptiveSpray ion source, and a timsTOF (Bruker, Bremen, Germany), the MS analysis was carried out. For metabolomics, the nebulizer pressure was adjusted to 2.2 bar, the drying temperature to 220°C, and the gas flow rate to 10 L/min. 4500 V was the capillary voltage, while 500 V was the end plate offset and the scan range for metabolomics was 20-1300 m/z. The instrument was operated in auto-MS/MS mode and the acquisition was performed using the positive mode at 12 Hz. The width of the precursor ion was ±0.5, the cycle time was 0.5 sec., and the threshold was 400 counts per thousand. Active exclusion was excluded after 3 spectra and released after 0.2 min. For MS2 acquisition the data dependent acquisition (DDA) was used, and the collision energy stepping fluctuated between 100 and 250% set at 20 eV. In the first 0.3 minutes of each LC-MS/MS run, sodium formate was injected as an external calibrant during data processing
Ion Mode:POSITIVE
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