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. 2023 Nov 9;28(22):7501.
doi: 10.3390/molecules28227501.

Two-Dimensional-PAGE Coupled with nLC-MS/MS-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in MCF7 Breast Cancer Cells Transfected for JTB Protein Silencing

Madhuri Jayathirtha  1 , Taniya Jayaweera  1 , Danielle Whitham  1 , Isabelle Sullivan  1 , Brîndușa Alina Petre  1   2   3 , Costel C Darie  1 , Anca-Narcisa Neagu  4
Affiliations

Two-Dimensional-PAGE Coupled with nLC-MS/MS-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in MCF7 Breast Cancer Cells Transfected for JTB Protein Silencing

Madhuri Jayathirtha et al. Molecules. .

Abstract

The identification of new cancer-associated genes/proteins, the characterization of their expression variation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by the rapid and recent advances in bioinformatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team's in vitro studies, we suggest that the human jumping translocation breakpoint (hJTB) protein might be considered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study, we identify DEPs, carcinogenic pathways, and biological processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics applied to a MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs are analyzed using GSEA and KEGG analyses. Almost all DEPs exert pro-tumorigenic effects in the JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that play pro-tumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/β-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α responses, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, tight junctions and cytoskeleton reorganization, the maintenance of stemness, metabolic reprogramming, survival in a hostile environment, and sustain a poor clinical outcome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of the MCF7 BC cell line. The data is available via ProteomeXchange with the identifier PXD046265.

Keywords: JTB protein silencing; MCF7; breast cancer (BC); downregulated JTB interactome; overexpressed JTB interactome; tumorigenic pathways.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The workflow for cellular proteomics followed by 2D-polyacrylamide gel (2D-PAGE) coupled with nLC-MS/MS analysis of the cell lysates.
Figure 2
Figure 2
Images of sh_JTB (left), control_shRNA (middle), control (right) silver stained 2D polyacrylamide gels. Polypeptide spots increased in each compared gels (top vs. bottom) are shown in blue, while spots decreased are outlined in red.
Figure 3
Figure 3
Images of sh_JTB (left), control_shRNA (middle), control (right) silver stained 2D polyacrylamide gels. The circles on each 2D-polyacrylamide gel shows the location of Isoelectric focusing internal standard Tropomyosin of Mw: 33000 and a pI of 5.2.
Figure 4
Figure 4
KEGG pathway analysis of pro-tumorigenic (PT) proteins in downregulated JTB condition; B. Gene ontology (GO) enrichment analysis of proteins in MCF7 BC cell line transfected for JTB downregulation: biological processes (BP) enriched in PT proteins. The analysis was performed using GeneCodis website (https://genecodis.genyo.es/, accessed on 22 October 2023).
Figure 4
Figure 4
KEGG pathway analysis of pro-tumorigenic (PT) proteins in downregulated JTB condition; B. Gene ontology (GO) enrichment analysis of proteins in MCF7 BC cell line transfected for JTB downregulation: biological processes (BP) enriched in PT proteins. The analysis was performed using GeneCodis website (https://genecodis.genyo.es/, accessed on 22 October 2023).
Figure 5
Figure 5
Interaction network of pro-tumorigenic (PT) proteins in MCF7 BC cell line transfected for JTB silencing, by means of STRING on-line database (https://string-db.org/, accessed on 22 October 2023). A total of 27 nodes and 69 edges were mapped in the PPI network with a PPI enrichment p-value of 6.44 × 10−12.
Figure 6
Figure 6
DEPs and their pro-tumorigenic (PT) activity in MCF7 BC cell line transfected for JTB silencing.
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