Investigation of the effects of downregulation of jumping translocation breakpoint (JTB) protein expression in MCF7 cells for potential use as a biomarker in breast cancer

Abstract

MCF7 is a commonly used luminal type A non-invasive/poor-invasive human breast cancer cell line that does not usually migrate or invade compared with MDA-MB-231 highly metastatic cells, which emphasize an invasive and migratory behavior. Under special conditions, MCF7 cells might acquire invasive features. The aberration in expression and biological functions of the jumping translocation breackpoint (JTB) protein is associated with malignant transformation of cells, based on mitochondrial dysfunction, inhibition of tumor suppressive function of TGF-β, and involvement in cancer cell cycle. To investigate new putative functions of JTB by cellular proteomics, we analyzed the biological processes and pathways that are associated with the JTB protein downregulation. The results demonstrated that MCF7 cell line developed a more "aggressive" phenotype and behavior. Most of the proteins that were overexpressed in this experiment promoted the actin cytoskeleton reorganization that is involved in growth and metastatic dissemination of cancer cells. Some of these proteins are involved in the epithelial-mesenchymal transition (EMT) process (ACTBL2, TUBA4A, MYH14, CSPG5, PKM, UGDH, HSP90AA2, and MIF), in correlation with the energy metabolism reprogramming (PKM, UGDH), stress-response (HSP10, HSP70A1A, HSP90AA2), and immune and inflammatory response (MIF and ERp57-TAPBP). Almost all upregulated proteins in JTB downregulated condition promote viability, motility, proliferation, invasion, survival into a hostile microenvironment, metabolic reprogramming, and escaping of tumor cells from host immune control, leading to a more invasive phenotype for MCF7 cell line. Due to their downregulated condition, four proteins, such as CREBZF, KMT2B, SELENOS and CACNA1I are also involved in maintenance of the invasive phenotype of cancer cells, promoting cell proliferation, migration, invasion and tumorigenesis. Other downregulated proteins, such as MAZ, PLEKHG2, ENO1, TPI2, TOR2A, and CNNM1, may promote suppression of cancer cell growth, invasion, EMT, tumorigenic abilities, interacting with glucose and lipid metabolism, disrupting nuclear envelope stability, or suppressing apoptosis and developing anti-angiogenetic activities. Therefore, the main biological processes and pathways that may increase the tumorigenic potential of the MCF7 cells in JTB downregulated condition are related to the actin cytoskeleton organization, EMT, mitotic cell cycle, glycolysis and fatty acid metabolism, inflammatory response and macrophage activation, chemotaxis and migration, cellular response to stress condition (oxidative stress and hypoxia), transcription control, histone modification and ion transport.

Keywords: Breast cancer; JTB downregulated condition; jumping translocation breakpoint (JTB) protein; proteomics.

Figure 1

Plasmid for downregulation from Creative Biogene.

Figure 2

Workflow for cellular proteomics from 1D-SDS PAGE and in gel-trypsin digestion.

Figure 3

Confocal microscope images showing conformation of stable transfection for control (A) and JTB downregulated condition (B). Left panel is the BF mode, middle panel is the GFP mode and the right panel is a merge between BF and GFP modes.

Figure 4

Downregulation confirmation of hJTB compared to control samples with (A) showing the overexpression at ~45 kDa in upregulated MCF7 cell lysate compared to control using commercially available full length hJTB antibody from Invitrogen; (B) shows GAPDH used as the loading control at 37 kDa.

Figure 5

12% SDS-PAGE gel with 200 µg protein from Control and Down (downregulated hJTB) from MCF7 cell lysate cut into individual gel bands from each lane.