Two-Dimensional Polyacrylamide Gel Electrophoresis Coupled with Nanoliquid Chromatography–Tandem Mass Spectrometry-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in the MCF7 Breast Cancer Cell Line Transfected for Jumping Translocation Breakpoint Protein Overexpression

The identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and understand the molecular mechanisms of BC initiation and progression. The jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors, including BC. Thus, the JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial. We previously analyzed the interacting partners of JTBhigh protein extracted from transfected MCF7 BC cell line using SDS-PAGE complemented with in-solution digestion, respectively. The previous results suggested the JTB contributed to the development of a more aggressive phenotype and behavior for the MCF7 BC cell line through synergistic upregulation of epithelial–mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways. In this work, we aim to complement the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography–tandem mass spectrometry (nLC-MS/MS) analysis. We identified six DEPs related to the JTBhigh condition vs. control that emphasize a pro-tumorigenic (PT) role. Twenty-one proteins, which are known to be usually overexpressed in cancer cells, emphasize an anti-tumorigenic (AT) role when low expression occurs. According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process. Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions. Our present and previous results sustain the necessity of the complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by tandem mass spectrometry to avoid their limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged for a better understanding of molecular pathways and neoplastic mechanisms related to the JTB’s role in BC initiation and progression.


Introduction
Currently, the identification of new "key genes" and their association with breast cancer (BC) occurrence is widely used to discover new biomarkers and molecular therapeutic targets to understand the molecular mechanisms of BC progression based on cancer-associated gene/protein expression profiles that could be reliable in clinical studies [1].Jumping translocation breakpoint (JTB), also known as the prostate androgen receptor (PAR), was described as a transmembrane protein gene at 1q21 rearranged in an jumping translocation (JT)-a rare chromosome aberration involved in various types of cancers, including BC [2][3][4].Moreover, recurrent gains in 1q have also been reported in invasive micropapillary carcinomas (IMPC) of the breast [5], a rare type of BC characterized by lymphovascular invasion and inaccurate imaging estimation [6].The JTB gene belongs to the epidermal differentiation complex (EDC) expressed in epithelial cells, following different patterns of cell-type specific expression [3,7].The EDC is a cluster of 62 coding-related genes that span a 2 Mb region, encoding structural and regulatory proteins responsible for epithelial tissue development and repair [8,9].These coding genes are present in four gene families: filaggrin (FLG) and FLG-like, late cornified envelope genes (LCEs), small proline-rich regions (SPRRs), and S100 genes [10].Many of these genes have also been associated with numerous cancers, such as skin, gastric, head and neck, colorectal, lung, ovarian, and renal carcinomas [11].JTB gene amplification has also been associated with many malignancies, such as sarcomas, BC, and other solid tumors [12].
JTB is expressed in normal tissues from different organs and systems: the nervous system, reproductive system, digestive system, respiratory system, endocrine and exocrine glands, and urinary system, as well as in adipose tissue, leukocytes, and spleen [12,13].Thus, JTB was found to be upregulated in many tumor tissues, such as primary BC, compared to normal counterparts, ovary, lung, uterus, colon, rectum, thyroid, prostate, stomach, kidney, and small intestine cancer [13], as well as in leukemia [3] and hepatocellular carcinoma (HCC) [14].In addition, JTB was also upregulated in different cancer cell lines, such as MCF7 and T47D BC cell lines compared to MCF10 normal breast epithelial cell lines or in androgen-sensitive and resistant prostate cancer cell lines compared to normal prostate epithelial cell lines and normal prostatic tissue [12,13].In these malignancies, JTB emphasizes oncogenic activity, leading to the malignant transformation of cells [13].
JTB may influence cell proliferation in vitro, clonogenicity, and in vivo tumorigenicity, being involved in the dysregulation of cell cycle progression, chromosome segregation, spindle formation, and cytokinesis [12].Also, JTB overexpression depresses the membrane potential of mitochondria and cellular growth and conferred resistance to apoptosis induced by TGF-β1, contributing to the tumorigenic process [2].The role of JTB in tumorigenesis is still controversial.Several malignancies from different organs suppress JTB expression, emphasizing a role in the neoplastic transformation of cells [2].Thus, JTB expression detected in the lung, kidney, stomach, and colon decreased significantly in tumor samples compared to normal samples [2].JTB gene expressions have been obtained using isolation and sequencing of JTB cDNA, RNA dot blot, flow cytometry (FC), Northern blot (NB), Western blot (WB), immunoprecipitation (IP), immunofluorescence microscopy (IF), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), apoptosis assays, Aurora A kinase assays, and luciferase reporter gene assays [2,4,12].Our previous results, based on Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by nLC-MS/MS proteomics, suggested that JTB overexpression in MCF7 BC cell line may be associated with mitotic spindle assembly, late estrogen response, epithelial-mesenchymal transition (EMT), and early estrogen response pathways sustained by the deregulation of cytoskeletal organization and biogenesis, mitotic spindle organization, extracellular matrix (ECM) remodeling, cellular response to estrogen, proliferation, migration, metastasis, increased lipid biogenesis, endocrine therapy resistance, tumor microenvironment (TME) acidification, transmembrane transport, glycolytic flux, iron metabolism, oxidative stress (OS), metabolic reprogramming, nucleo-cytosolic mRNA transport, transcriptional activation, chromatin remodeling, modulation of cellular death pathways, and cancer drug resistance as biological processes [15].We also concluded that JTB dysregulation, assessed by in-solution-based proteomics, may promote a more aggressive phenotype and behavior for MCF7 cells by synergistic upregulation of EMT, mitotic spindle organization, and fatty acid metabolism-related pathways [16].
In this work, we aim to investigate the differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB high using 2D-PAGE coupled with nLC-MS/MS proteomics of MCF7 BC cell line in order to complete and complement our previously published results based on SDS-PAGE [15] and in-solution proteomics of MCF7 cells transfected for JTB upregulation [16].

Gene Name Gene Description Role in Biological Processes
Expression in Malignancies and Putative Neoplastic Effects Pathways Neoplastic Behavior

Gene Name Gene Description Role in Biological Processes
Expression in Malignancies and Putative Neoplastic Effects Pathways Neoplastic Behavior

Gene Name Gene Description Role in Biological Processes
Expression in Malignancies and Putative Neoplastic Effects Pathways Neoplastic Behavior
In this experiment, of nine upregulated JTB partners, four proteins may emphasize pro-tumorigenic (PT) functions: retinoblastoma-binding protein 4/histone-binding protein (RBBP4), SET nuclear proto-oncogene/template-activating factor-I β/inhibitor of histone acetyltransferase (SET), actin-binding Rho-activating C-terminal-like (ABRACL) and noncatalytic region of tyrosine kinase (Nck)-associated protein 1 (NCKAP1) (Table 3).These upregulated proteins have been reported as overexpressed in different types of tumors, including BC tissue samples and cell lines, with all of them being involved in the EMT pathway.The EMT process was found to be upregulated in our published analysis conducted in the MCF7 BC cell line transfected for JTB overexpression, using SDS-PAGE [15], as well as in an in-solution digestion [16].RBBP4 and SET are multitasking histone/DNA-binding proteins, which are also involved in chromatin remodeling/organization, DNA replication and repair, transcriptional regulation, histone modification, and cell cycle and promoting cell proliferation, migration, invasion, and anti-apoptosis.ABRACL is known as a regulator of the actin cytoskeleton, cell motility, and cell cycle.Acting as a cytoskeleton/actin dynamics regulator, the non-catalytic region of tyrosine kinase (Nck)-associated protein 1 (NCKAP1) is a part of the WAVE complex that regulates lamellipodia/invadopodia formation and cell mobility, sustaining cell migration.Thus, NCKAP1 promotes EMT activation [31] by HSP90-mediated invasion and metastasis, provoking MMP9 and MMP14 activation [32].Retinoblastoma-binding protein 4 (RBBP4/RbAp48), also known as histone-binding protein RBBP4, is a 48 kDa tumor-specific/oncogenic protein involved in transcription regulatory complexes that control cell cycle gene expression [18], proliferation, migration, invasion, and apoptosis, playing an important role in chromatin metabolism, nucleosome assembly, and histone modification [17,20].RBBP4 was identified as significantly overexpressed in human embryonal and glial brain cancers [18], BC [17], especially in TNBC tissues and cell lines [19], as well as in colon cancer cell lines, promoting malignant progression via increasing activity of the Wnt/β-catenin pathway, in correlation with a high expression of the histone deacetylase 1 (HDAC1) [20].RBBP4 inhibition reduced cell invasion and migration through the regulation of proteins related to the EMT process in colon cancer [20], as well as in TNBC cell lines [19].SET protein, also known as template activating factor-I β (TAF-Iβ), is involved in the cell cycle, migration, apoptosis, transcription, and DNA repair [21].SET was found to be overexpressed in 50-60% of BC tumor samples and BC cell lines [22].In pancreatic cancer (PDAC) progression, the various isoforms of this multifunctional onco-protein sustain EMT by inducing the transcriptional activation of the mesenchymal biomarker N-cadherin, resulting in the promotion of mesenchymal cell characteristics, proliferation, migration, invasion, and colony formation [23].SET overexpression also activated the Rac1/JNK/c-Jun and MEK/ERK signaling pathways that lead to cell migration and proliferation [23].The actin-binding Rho-activating C-terminal-like (ABRACL) protein, which was previously named HSCP280, is a regulator of the actin cytoskeleton, cell motility, and viability [24] through its interaction with cofilin [28].It is highly expressed in several cancers, such as endometrial cancer [26], BC tissues and cells [25], colon cancer cells [28], esophageal carcinoma [27], and gastric cancer [24].ABRACL knockdown suppressed the proliferation, invasion, migration, and EMT of BC cells [25].
Of 23 downregulated JTB partners, two proteins may emphasize PT functions: gelsolin (GSN) and PRDI-BF1 and RIY domain containing 1 (PRDM5/PFM2) (Table 3).GSN is an actin-binding protein downregulated in CC, HCC, GC, cervical cancer, ovarian cancer [64], and BC [65,66].It is known that actin-remodeling proteins have an important role in the regulation of cytoskeletal as well as inflammatory cell responses [129].GSN knockdown leads to EMT in mammary epithelial cells [67].GSN deficiency increases with progression from atypical ductal hyperplasia (ADH) to ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC) [66].Mukherjee et al. (2012) have shown that GSN knockdown in 3T3-L1 adipocyte cells suppressed the expression of lipogenic genes but highly increased that of tumor necrosis factor-α (TNFα) and inflammatory interleukin-6 (IL-6) [130].IL-6 is known to be significantly produced by BC cellsand cancer-associated adipocytes (CAAs), with the potential of inducing proliferation, EMT, stem cell phenotype, angiogenesis, cachexia, and therapeutic resistance in BC cells [131], correlating with clinical disease stage and lymph node metastasis as well as with ER and HER2 expression [132].Moreover, IL-6 is an important growth factor for estrogen receptor-α (ERα)-positive BC [133].Additionally, TNFα is a pro-inflammatory cytokine that was reported as overexpressed in BC, where it correlates with augmented cancer cell proliferation, increased metastasis, higher malignancy grade, and poor prognosis [134].The exposure of the MCF7 BC cell line to a low dose of TNFα enhanced the invasive phenotype by influencing the different genes involved in metastasis [135].PRDM5/PFM2 is a zinc finger protein acting as an epigenetic modifier [113] known as a tumor suppressor; it is frequently silenced/downregulated/inactivated by methylation in multiple carcinoma lines, such as NPC, ESCC [114], GC [115], and HCC and ovarian, cervical, and breast cancers [113,116].PRDM4 knockdown increases cell growth, proliferation, migration, invasion, tumor initiation, and progression [113][114][115], emphasizing an opposite role in melanoma [116].

Anti-Tumorigenic (AT) JTB-Interacting Partners
Two upregulated JTB-related partners, histidine triad nucleotide-binding protein (HINT2) and Armadillo repeats domain of adenomatous polyposis coli (APC), have been detected in this experiment (Table 4).The upregulated APC might emphasize AT functions.It is known as a multi-functional tumor suppressor protein that regulates cell-cell adhesion, cell polarization, migration [43], cell proliferation and differentiation [44], organization of actin and microtubule networks, spindle formation, and chromosome segregation [45].APC was reported as mutated in colon cancer (CC) and liver cancer, where it acts as a negative regulator of canonical Wnt signaling [45].HINT2 is a tumor suppressor protein present in the mitochondrial matrix that sensitizes cells to apoptosis [50] and positively regulates lipid and glucose metabolism as well as mitochondrial respiration [51].HINT2 overexpression induces an anti-EMT gene expression profile in cancer cells, inhibiting cell migration, invasion, and metastasis [52]; induces cell apoptosis; decreases mitochondrial membrane potential; promotes intracellular ROS production; and elevates mitochondrial Ca 2+ levels [53].
Ribosomal proteins (RPs) are involved in tumorigenesis and cell transformation.The knockdown of many RPs, such as RPS3, RPS15A, RPL39, and RPS6, is shown to be linked to a reduction in BC cell growth, proliferation, viability, or metastasis [126].Overexpression is present in BC, including TNBC, especially metastatic TNBC cells, as well as in PCa cell lines [122,124].60S ribosomal protein L7a (RPL7A) blocking may reduce cell migration and invasion [123].Additionally, identified as downregulated in the present JTB-overexpressed condition, 60S ribosomal protein L31 (RPL31) and 40S ribosomal protein S3 (RPS3) may enhance the levels of p53 and p21, decreasing cell growth and cell cycle [125], by promoting ribosomal stress, which impairs ribosomal biogenesis; impedes cell proliferation, invasion, and migration; and increases apoptosis [127].

JTB-Interacting Partners with Controversial Neoplastic Functions
The second mitochondrial-derived activator of caspase (SMAC)/direct IAP-binding protein with low pI (DIABLO) was found to be overexpressed in this experiment.DIABLO is known as a promoter of caspase-dependent apoptosis (HALLMARK_APOPTOSIS) by inhibition of inhibitory apoptotic proteins (IAP) family members.As a promoter of apoptosis, DIABLO is known as an AT protein released from mitochondria into the cytosol in response to the apoptotic stimuli [136].DIABLO has a dysregulated expression in many cancers [137].There are studies that reported DIABLO as overexpressed in GC, CRC, and ovarian cancer and as downregulated in PCa, lung, and soft tissue cancers, as well as in BC tissues, with its expression decreasing with BC progression [47].However, DIABLO was recently found to be involved in non-apoptotic processes that are essential for tumor growth and progression [49], such as phospholipid synthesis associated with tumorigenesis [48].Thus, other published works reported DIABLO overexpression in BC, lung, bladder, cervical, pancreas, prostate, melanoma, glioma [48,49], ER-positive BC cell lines, such as MCF7 that is an ER-positive and PR-positive luminal A BC subtype [138], as well as in ER-positive BC in comparison with ER-negative tissue samples, demonstrating a poor

JTB-Interacting Partners with Controversial Neoplastic Functions
The second mitochondrial-derived activator of caspase (SMAC)/direct IAP-binding protein with low pI (DIABLO) was found to be overexpressed in this experiment.DIABLO is known as a promoter of caspase-dependent apoptosis (HALLMARK_APOPTOSIS) by inhibition of inhibitory apoptotic proteins (IAP) family members.As a promoter of apoptosis, DIABLO is known as an AT protein released from mitochondria into the cytosol in response to the apoptotic stimuli [136].DIABLO has a dysregulated expression in many cancers [137].There are studies that reported DIABLO as overexpressed in GC, CRC, and ovarian cancer and as downregulated in PCa, lung, and soft tissue cancers, as well as in BC tissues, with its expression decreasing with BC progression [47].However, DIABLO was recently found to be involved in non-apoptotic processes that are essential for tumor growth and progression [49], such as phospholipid synthesis associated with tumorigenesis [48].Thus, other published works reported DIABLO overexpression in BC, lung, bladder, cervical, pancreas, prostate, melanoma, glioma [48,49], ER-positive BC cell lines, such as MCF7 that is an ER-positive and PR-positive luminal A BC subtype [138], as well as in ER-positive BC in comparison with ER-negative tissue samples, demonstrating a poor prognosis in BC patients [137].There is evidence that the overexpression of DIABLO increased the mammosphere-forming ability of MCF7 BC cells and activation of the cell survival and proliferation pathways, while it is known that apoptosis itself may induce proliferation and invasion of more aggressive cancer cells to induce tumor growth and expansion [137].The upregulation of DIABLO in the MCF7 BC cell line in the JTB overexpressed condition may be correlated with an increase in aggressiveness and invasion abilities of this BC cell line comparable with the luminal B BC cell lines that show an increased expression of proliferation-related genes and a higher risk of early relapse compared with the luminal A subtype [137,139].Recently, based on proteomics-interactomics-based analysis and a lung cancer mouse model, DIABLO depletion was correlated with the inhibition of tumor cell growth and proliferation, decreased phospholipid levels, activated apoptosis, reversed EMT and altered TME, reduced expression of inflammation-related proteins such as NF-κB and TNF-α, and of the programmed death-ligand 1 (PD-L1), which is associated with immune system suppression [49].The expression of DIABLO inversely correlates with the BC tumor stage, suggesting that this protein may play an important role in BC development [47].
Junction plakoglobin (JUP) and desmoplakin (DSP) are also upregulated in this experiment.DSP is a cell-cell adhesion molecule reported to have an AT activity in non-small-cell lung cancer (NSLC) by inhibition of the Wnt/β-catenin signaling pathway, followed by inhibition of cell proliferation, migration, and invasion and an increase in sensitivity to induced apoptosis [40].Loss of DSP has been involved in BC metastasis [140].JUP, also known as γ-catenin, a member of the Armadillo family of proteins and a paralog of β-catenin, is a component of both desmosomes, in association with DSP, and adherent junctions, along with β-catenin and α-catenin, playing an essential role in the regulation of cell-cell adhesion and participating in cell signaling [141].JUP overexpression was reported in many cancers [34], where it may act as an oncogenic or tumor suppressor protein [36].There are studies focused on various mechanisms by which plakoglobin may inhibit tumorigenesis and metastasis [141].However, other works showed that the aberrant activation of γ-catenin promotes genomic instability and oncogenic effects during tumor progression [34].Also, the overexpression of plakoglobin was able to promote cell survival and metastasis in invasive micropapillary carcinoma of the breast (IMPC) by activation of the PI3K/Akt/Bcl-2 pathway, which may protect the clusters of CTCs from anoikis [35].JUP, as well as DSP, are barrier molecule genes (BMGs) that encode proteins that mediate the mechanical barrier function in normal epithelia, whose overexpression was associated with decreased immune surveillance and shorter patient survival [142].Both JUP and DSP were identified to be upregulated in melanoma cell lines, where they significantly increase tumor burden, VEGF-A, reduced IL-33, and angiogenesis [41], thus emphasizing their PT roles.

Materials and Methods
The MCF7 cell culture, the transfection of hJTB plasmids, and the collection of cell lysates were described previously [15] and briefly described below.

Cell Culture
MCF7 cells were purchased from American Type Culture Collection (HTB-22 ATCC) and grown in RPMI-1640 medium supplemented with 10% FBS, 1% Penicillin Streptomycin, 0.2% Gentamycin, and 0.2% of Amphotericin (growth media) at 37 • C and in 5% CO 2 .The cells were grown until they reached ~70% confluency and transfected with JTB cDNA plasmid for overexpression.

Plasmids for Upregulation
Plasmids were custom-made by Genscript ® .One plasmid with the hJTB gene containing the full coding region of cDNA, ggtaccGCCACCATGCATCATCATCATCATCATCTTGCGGGT-GCCGGGAGGCCTGGCCTCCCCCAGGGCCGCCACCTCTGCTGGTTGCTCTGTGCTTTCAC CTTAAAGCTCTGCCAAGCAGAGGCTCCCGTGCAGGAAGAGAAGCTGTCAGCAAGCAC CTCAAATTTGCCATGCTGGCTGGTGGAAGAGTTTGTGGTAGC AGAAGAGTGCTCTC-CATGCTCTAATTTCCGGGCTAAAACTACCCCTGAGTGTGGTCCCACAGGATATGTAG AGAAAATCACATGCAGCTCATCTAAGAGAAATGAGTTCAAAAGCTGCCGCTCAGCT TTGATGGAACAACGCTTATTTTGGAAGTTCGAAGGGGCTGTCGTGTGTGTGGCCCTGA TCTTCGCTTGTCTTGTCATCATTCGTCAGCGACAATTGGACAGAAAGGCTCTGGAAAA GGTCCGGAAGCAAATCGAGTCCATAGACTACAAAGACGATGACGACAAGTACCCATA CGATGTTCCAGATTACGCTgatatc, corresponding to 146 amino acids of the protein, was used.The hJTB cDNA in sense orientation was inserted into a CMV promoter-based plasmid for JTB overexpression.The plasmid was further customized to have three tags His, HA, and FLAG tags.It also had an eGFP tag to enable confirmation of transfection in MCF7 cells.The second plasmid was an empty vector with an eGFP tag to serve as a control.

Transfection into MCF7 Cells
Lipofectamine™ 3000/DNA and DNA/Plasmid (10 µg/µL) complexes were prepared in Opti-MEM Reduced Serum Media (Invitrogen, Eugene, OR, USA) for each condition and added directly to the cells in culture medium.A total of 2 mg/mL of Neomycin was added after 48 h and incubated at 37 • C. Cells that survived were allowed to reach 80% confluency by replacing the growth media every 48 h with new media containing 2 mg/mL antibiotic.Transfection efficiency was confirmed by visualizing the green fluorescence emitted by the eGFP gene using a confocal microscope.

Western Blot Analysis
Cell lysates from each condition were collected using a lysis buffer containing 20 mM Tris HCl, 150 mM Nacl, 0.2 mM EDTA, 1.1% Triton-X, and protease/phosphatase inhibitors.The lysates were then incubated for 30 min on ice and centrifuged at 14,000 rpm for 20 min at 4 • C. The supernatants were collected, and protein concentration was determined using Bradford assay with bovine serum albumin standards.Lysates containing 20 µg of proteins were run in 14% SDS-polyacrylamide gels and transferred to nitrocellulose membranes.The blots were incubated with blocking buffer containing 5% milk and 0.1% tween-20 overnight at 4 • C with shaking.Primary antibody (JTB Polyclonal Antibody-PA5-52307, Invitrogen) diluted to 1:1000 was added and incubated at 4 • C for 1 h with constant shaking.Secondary antibody (mouse anti-rabbit IgG-HRP sc-2357, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted to 1:2000 ratio was added and incubated for 1 h at room temperature with constant shaking.After each incubation, the blots were washed thrice with TBS-T (1X TBS buffer, containing 0.05% tween-20) for 10 min each with constant shaking.Finally, the enhanced chemiluminescence (CL) substrate (Pierce™ ECL Western Blotting Substrate-32106, Thermo Fisher Scientific, Waltham, MA, USA) was added to the blot, and the blot was analyzed using a CCD imager.For normalization, the blots were treated with Mouse GAPDH monoclonal antibody (51332, cell-signaling technology) and incubated for 1 h, followed by 1 h incubation of goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology) and the addition of ECL substrate.Detection and comparison of the intensity of the bands were carried out using ImageJ software (National Institute of Health, Bethesda, MD, USA).

2D-PAGE and Proteomic Analysis
The entire proteomics-based workflow is shown in Figure 2. We used three biological replicates of control (n = 3) and up_JTB (n = 3).These conditions were analyzed in 2D-PAGE by Kendrick Labs, Inc. (Madison, WI) and nLC-MS/MS, as previously described [15,143].The computer comparisons were carried out for the average of control vs. Up_JTB (n = 3).The dysregulated spots were selected based on the criteria of having a fold increase or decrease of ≥1.7 and p value of ≤0.05.There were 68 dysregulated spots in control vs. up_JTB comparison (Figures S1 and S2 from Supplementary Materials) that were selected for nLC-MS/MS analysis, as previously described [143].The data processing was carried out using ProteinLynx Global Server (PLGS) software, version 2.4, Waters Corp, Millford, MA, USA to convert them to pkl files, and Mascot Daemon software (version 2.4, Matrix Science, Boston, MA, USA) was used to identify the dysregulated proteins.The quantitative analysis of dysregulated spots was conducted using Scaffold software version 4.0 (Proteome Software, Inc, Portland, OR, USA).Gene Set Enrichment Analysis (GSEA) (https://www.gsea-msigdb.org/gsea/index.jsp,accessed on 1 December 2022) was performed to identify the dysregulated pathways as previously described [15].Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/,accessed on 19 September 2023) was used to construct a protein-protein interactions (PPI) network for downregulated DEPs with anti-tumorigenic functions associated with JTB high condition in MCF7 BC cell line.
for nLC-MS/MS analysis, as previously described [143].The data processing was carried out using ProteinLynx Global Server (PLGS) software, version 2.4, Waters Corp, Millford, MA, USA) ) to convert them to pkl files, and Mascot Daemon software (version 2.4, Matrix Science, Boston, MA, USA) ) was used to identify the dysregulated proteins.The quantitative analysis of dysregulated spots was conducted using Scaffold software version 4.0 (Proteome Software, Inc, Portland, OR, USA) ).Gene Set Enrichment Analysis (GSEA) (https://www.gsea-msigdb.org/gsea/index.jsp,accessed on 1 December 2022) was performed to identify the dysregulated pathways as previously described [15].Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/,accessed on 19 September 2023) was used to construct a protein-protein interactions (PPI) network for downregulated DEPs with anti-tumorigenic functions associated with JTB high condition in MCF7 BC cell line.

Data Sharing
Raw data from MassLynx, HTML files from Mascot, and Scaffold files will be provided upon request, according to Clarkson University Material Transfer Agreement.

Statistical Analysis
Data are presented as mean ± S.E.M. Statistical comparisons of three means were made using paired Student's t-test where appropriate p values < 0.05 were considered statistically significant.

Conclusions
Identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and explain the molecular mechanisms of BC initiation and progression based on cancer-associated gene/protein expression profiles that could be reliable in clinical studies.Jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors.JTB protein has

Data Sharing
Raw data from MassLynx, HTML files from Mascot, and Scaffold files will be provided upon request, according to Clarkson University Material Transfer Agreement.

Statistical Analysis
Data are presented as mean ± S.E.M. Statistical comparisons of three means were made using paired Student's t-test where appropriate p values < 0.05 were considered statistically significant.

Conclusions
Identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and explain the molecular mechanisms of BC initiation and progression based on cancer-associated gene/protein expression profiles that could be reliable in clinical studies.Jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors.JTB protein has been reported as overexpressed in some BC cell lines and primary breast tumors compared with their normal tissue samples counterparts, as well as in prostate and liver cancer [3,13].Thus, JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial.We previously analyzed the interacting partners of JTB high protein extracted from transfected MCF7 BC cell line, using SDS-PAGE [15], later complemented by in-solution digestion-based analysis [16].The previous results suggested JTB contribution to the development of a more aggressive phenotype and behavior of MCF7 BC cell line through synergistic upregulation of epithelial-mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways [16].In this work, we complemented the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis.We identified 6 differentially expressed proteins (4 upregulated and 2 downregulated) related to JTB high condition vs. control that emphasize a pro-tumorigenic (PT) role (RBBP4, SET, ABRACL, NCKAP1, GSN, and PRDM5), while 21 proteins, which are known to be usually overexpressed in cancer cells, emphasized an anti-tumorigenic role when low expression occurs as happened in this experiment (UBA1, YWHAZ, TUBB2A, ITGB5, HSPB1, PCBP2, MCCC2, UGDH, TPI1, ATP5F1B, DLSI, FTL, HYOU1, PRDX6, NRDC1, and HNRNPK), as well as three ribosomal proteins (RPL7A, RPL31, and RPS3).Two upregulated proteins, APC and HINT2, also emphasize a tumor-suppressive role.According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process.GSEA was performed for the upregulated JTB condition using H (hallmark gene sets) collection in MSigDB.Analysis of H collection revealed two upregulated pathways, including proteins important for EMT and hypoxia.Two downregulated pathways comprised proteins involved in the mitotic spindle and unfolded protein response (UPR) pathways.Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions, such as decreased cell growth, survival, motility, invasiveness and migration ability, cell proliferation, angiogenesis, colony formation, metabolism, and EMT in addition to an enhancement of apoptosis and other mechanisms of cancer cell death.These results mainly suggest the AT potential of the JTB gene.Moreover, our present and previous results sustain the necessity of complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by MS analysis to avoid their inherent limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged together for a better understanding of molecular pathways and neoplastic mechanisms related to JTB gene or gene product role in BC initiation and progression.

Table 1 .
Significant up-and downregulated pathways in upregulated JTB condition in MCF7 BC cell line, according to GSEA with FDR < 25%.

Table 2 .
DEPs, neoplastic roles, and biological processes expressed in response to JTB upregulation in MCF7 BC cell line.

Table 3 .
Pro-tumorigenic (PT) functions of DEPs in JTB high condition in transfected MCF7 BC cell line.X means that the DEP has the corresponding function.

Table 4 .
Anti-tumorigenic (AT) functions of DEPs in JTB high condition in transfected MCF7 BC cell line.X means that the DEP has the corresponding function.

Table 4 .
Anti-tumorigenic (AT) functions of DEPs in JTB high condition in transfected MCF7 BC cell line.X means that the DEP has the corresponding function.