Regulation of Med1 protein by overexpression of BAP1 in breast cancer cells

doi:10.21203/rs.3.rs-2987729/v1

Download PDF

Research Article

Regulation of Med1 protein by overexpression of BAP1 in breast cancer cells

https://doi.org/10.21203/rs.3.rs-2987729/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 02 May, 2024

Read the published version in Molecular & Cellular Oncology →

Version 1

posted

You are reading this latest preprint version

Med1, a subunit of the TRAP/mediator complex acts as a transcriptional regulatory mediator, binds to a nuclear receptor and regulated transcription. It is known that elevated Med1 protein expression promotes cancer growth in hormone-dependent breast and prostate cancer, whereas low expression of Med1 protein in melanoma and lung cancer increases cancer metastasis. In this study, Med1 protein expression by deubiquitinating enzymes (DUBs) overexpression was investigated in breast and lung cancer cell lines. Overexpression of BAP1 protein did not affect Med1 mRNA expression in breast cancer and lung cancer cell. However, Med1 protein expression decreased in breast cancer cells while increased in lung cancer cell lines. In addition, Med1 protein expression by overexpression of BAP1 mutant (C91A) in breast and lung cancer cells was not affected. Increased BAP1 expression increased cell growth and metastatic capacity in lung cancer cells. And overexpression of BAP1 in breast cancer cell lines increased the transcriptional regulation of estrogen receptor (ER). In addition, the binding between the Med1 and the BAP1 protein was observed. Therefore, these data suggested that BAP1 was involved in cancer cell growth and metastasis by binding to Med1 protein and regulating Med1 protein expression depending on the cancer cell types.

Med1

BAP1

Breast cancer cells

Protein expression regulation

Deubiquitinating enzyme

The mediator of RNA polymerase II transcription subunit 1 mediator (Med1, also known as TRAP220) plays an important role as a coactivator of a wild range of nuclear receptors including the estrogen receptor (ER), androgen receptor (AR), and thyroid receptor (TR) by targeting and anchoring the complex in transcriptional regulation [1]. Med1 regulates ER-dependent gene transcription by direct interaction with ER [2]. Med1 protein is overexpressed in ~ 50% of all primary breast cancers and breast cancer cell lines [3]. In addition, the knockdown of Med1 impairs the expression of ER-target genes and the estrogen-dependent growth of breast cancer cells [4], but not the expression of genes controlled by other transcriptional activators such as p53 [5]. The overexpression of Med1 in breast [5] and prostate cancers [6] has been described by its function as a hub for nuclear hormone receptors. In contrast, Med1 negatively modulates the expression of metastasis-related genes and is downregulated in melanoma [7] and lung cancer [8]. And Med1 expression considerably decreases during bladder cancer progression from benign urothelium to advanced bladder cancer [9]. Although, Med1 protein expression is related to the development and metastasis of various cancers, the mechanism of regulation of Med1 protein expression is a poorly understood.

Ubiquitylation plays an essential role in eukaryotic cell signaling mechanisms by influence protein degradation by proteasomes, alters cellular location, affect their activity, and promote or prevent protein interactions [10]. The various studies have shown that activation or alterations in ubiquitination processes are associated with the pathogenesis of human carcinoma. Moreover, it has been reported that ubiquitination introduces into malignant tumors through loss of function by inactivating the mutation form, altering of protein expression, or indirectly attenuating of ubiquitination-related proteins [11].

The protease deubiquitinating enzymes (DUBs) is known to play a role in modify proteins by removing ubiquitin or ubiquitin-like molecules or remodeling ub-chains on target proteins, have recently been regarded as important regulators of the ubiquitination-mediated degradation and other functions [12]. BRCA1-associated protein 1 (BAP1) is a nuclear ubiquitin hydrolase and was identified to interaction with the RING finger domain of the tumor suppressor BRCA1 [13]. BAP1 belongs to a DUB with a ubiquitin carboxy-terminal hydrolase (UCH) domain and acts as a transcriptional regulator for mammalian development through cell cycle regulation, chromatin remodeling, DNA damage response, and tumor suppression [14]. The tumor suppressor function of BAP1 required nuclear localization and catalytic activity. Recent studies have clarified the diagnostic significance of BAP1 loss in select tumor types. Dysregulations and mutations, such as lack of deubiquitinating activity (C91A subtraction) or second nuclear localization signal within the BAP1 gene have been identified as drivers in many human cancers, such as uveal melanoma [15], mesothelioma [16], clear-cell renal cell carcinoma [17], leukemia [18], and breast cancer [19].

Therefore, in this study, Med1 protein expression was investigated by various DUBs overexpression in breast cancer cells and cell growth was investigated by wild type and C19A mutant of BAP1, which were identified as Med1 protein regulator in vitro.

Cell culture and Transfection

Breast cancer cell line MCF7 and 293T cells were grown in Dulbecco`s modified Eagle`s medium (DMEM, # 12800-017, Gibco Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco Island, NY, USA) and 1% penicillin-streptomycin. The cells were grown in a 5% CO₂ incubator at 37°C. Full-length human DUBs plasmids were cloned into the pDEST-CMV6 vector with an SRT tag at the N-terminus, and the plasmids are as following: USP3, USP21, USP22, USP25, USP28, USP28, USP35, USP36, USP39, UCH-L1, UCH-L5, and BAP1. To generate a catalytic residue mutation, a site-direct mutagenesis PCR was performed using a site-direct mutagenesis kit (#A14604, Invitrogen) according to the manufacture`s protocol. The plasmid was as followed: USP21 C211A, USP35 C434A, and BAP1C91A. Transfections of DUBs plasmids were performed using transfectamine 2000 reagent (# 11668-019, Invitrogen, Waltham, MA, USA) according to the manufacturer`s protocols.

Western blotting and immunoprecipitation

MCF7 were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 300 mM NaCl and 1% Triton-100) and then incubated for 20 min on ice and centrifuged at 13,000 rpm for 20 min at 4°C. Supernatants were collected and the protein was quantified. Western blotting was performed SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride (PVDF) microporous membranes (# IPVH00010, Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with TBS-T (20 mM Tris-HCl, pH 7.5, 0.05% Tween 20, and 150 mM NaCl) containing 5% skim milk or 5% bovine serum albumin (BSA) for blocking for 1 h and incubated overnight at 4°C with primary antibodies. Primary antibodies used following: anti-Ub (#SC-166553m Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SRT, anti-BAP1 (#SC-28383, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Med1(#SC-8998, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (#SC-8432, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the membranes were incubated for 2 h with the secondary antibody. Blots were detected using an ECL reagent solution (# LF-QC0101, Young-In Frontier Seoul, Korea). The immunoprecipitation (IP) experiment performed to investigate protein interaction. Cell lysates were incubated with an antibody (anti-Med1) 4°C overnight and for 2 h with protein A/G PLUS-Agarose beads (# SC-2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Samples were collected by centrifuged at 13,000 rpm for 10 min at 4°C and washed three times for 5 min each with TBS-T. The samples were boiled in 4X SDS sample buffer for 10 min and analyzed using western blotting.

Luciferase reporter assay

MCF7 cells were transfected with the ERE-Luc reporter luciferase plasmid cloned with pRL-TK-Luc vector and the indicated plasmids (pDEST-CMV6-DUBs). To normalize luciferase activity, Renilla luciferase reporter control vector was co-transfected. At 48 h after transfection, the cells were collected and performed the dual-luciferase reporter assays. Luciferase activity was measured following the manufacture’s protocol (Promega, Madison WI, USA). The firefly luciferase activity was divided by Renilla luciferase activity to figure out the relative luciferase activity. The data represent the average values from three independent experiments.

Quantitative RT-PCR (qPCR)

Total RNA extraction performed according to the manufacture`s protocol of the TRIzol reagent (#10296010, Ambion, ThermoFisher, USA). And cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and oligo-dT primers (Promega, Madison, WI, USA). qPCR was performed in triplicate using the SYBR Green PCR Master Mix (Takara Bio Inc., Bio Inc. Shiga, Japan) and ABI Prism 7500 Real-Time PCR system (Applied Biosystems). The β-actin transcript was used as an internal control for all samples, and the gene-specific mRNA level was normalized to the level of β-actin mRNA. The expression of each mRNA was determined using the 2-^ΔΔCT -threshold cycle method. The following primers were used: Med1: forward 5`-AGTATCATGGGCTCAGCTCC-3’ and reverse 5`-GGTGAGCCCATCATCGACAATTC-3’. BAP1: forward 5`-CCACAAGTCTCAAGAGTCACAG-3` and reverse 5`-CTGCACCATCTGTGTGGTT-3’. uPAR: forward 5`-CGGGCTCCAATGGTTGCCA-3` and reverse 5`-CAGAGTGAGCGTTCGTGAGTG-3’. β-actin: forward 5`-GCTCCTCCTGAGCGCAAG-3` and reverse, 5`-CATCTGCTGGAAGGTGGACA-3`.

In vitro migration and invasion assays

The cell migration assay was performed using multi-chamber well, which was separated from the complete culture medium in the bottom chamber by a polyethylene terephthalate membrane filter (8 µm pore size) (Neuro Probe, Inc., Gaithersburg, MD). The 1 x 10⁵ resuspended cells in serum-free medium were transferred into the upper chamber and complete culture medium was incubated in bottom chamber. FBS was used as a chemoattractant. During incubation for 18 h, cells migrated through pores. Non-migrated cells were carefully removed using a cotton swab, and cells migrated undersurface the membrane filter were fixed with cold methanol for 20 min at room temperature and add the crystal violet to the well for staining the migrated cell. The cells on membrane read density of the stained cells at 595 nm using a microplate reader.

The cell invasion assay was performed using Boyden chambers with Matrigel-pre-coated filter inserts (8 µm pore size) (Costar, Corning, NY). A suspension of 1 x 10⁵ cells in the medium was added to the upper chamber while the lower chamber was fill with medium containing 10% FBS. After 18 h of incubation, the cells that had invaded the membrane were fixed and stained with hematoxylin and eosin (H&E). Stained cells in three randomly chosen fields (100X magnification) were photographed and counted. Data represent the average of three individual experiments, and error bars represent the standard error of the mean (SEM).

Cell proliferation assay

The 1 x 10⁴ cells were seeded each well in 6-well plates and allowed to proliferate for 6 days. The medium was changed every 2 days. After six days, the cells were collected, stained with trypan blue, and counted to measure proliferation. The experiments were repeated three times and performed in triplicates.

The MCF7 cells were seed and treated each 10 ng of EGF (#PHG0311, Invitrogen), E2 (#E4389, Sigma-Aldrich), and TGF-β for 24 h and then harvested. The total cell lysates were immunoblotted with anti-Med1 and anti-BAP1.

Statistical analysis

Densitometric analysis was performed using Image J. The paired t-test and two-way ANOVA were performed by GraphPad Prism version 5 (GraphPad Software, La Jolla, CA USA), p-values of *p < 0.05, **p < 0.01, ***p < 0.001 were deemed significant. All results shown are representative data of at least three independent experiments and are presented as the mean ± standard error of the mean (SEM).

Identification of DUBs regulating the expression of Med1 protein

To determine whether the ubiquitin mechanism is involved in the regulation mechanism of Med1 protein expression, it was performed ubiquitination assay in 293T cells. Ubiquitin of the Med1 protein was observed in the immunoblotting with ubiquitin antibody after immunoprecipitation using the Med1 antibody [Fig. 1A]. Next, Med1 protein expression was investigated by DUBs overexpression using various DUBs plasmids. It was observed that the expression of Med1 was increased by overexpression of DUBs such as ubiquitin specific peptidase 21 (USP21), ubiquitin specific peptidase 22 (USP22), ubiquitin specific peptidase 28 (USP28), and ubiquitin specific peptidase 49 (USP49) compared with the control. However, Med1 protein expression was decreased by ubiquitin-carboxy-terminal hydrolase L1 (UCH-L1) [Fig. 1B]. Abnormal expression of UCHL1 has been implicated in cancer [20, 21]. These results showed that Med1 protein expression was differently regulated by overexpression of various DUBs. And to observe the transcriptional activity of Med1 by overexpression of DUBs, an ERE-luciferase assay was performed. ERE-luciferase activity did not change upon overexpression of the various DUBs. ERE-luciferase activity was elevated, whereas Med1 protein did not affect by ubiquitin specific peptidase 29 (USP29) overexpression. Med1 protein expression was decreased by UCHL1 overexpression, but transcriptional activity was no changed [Fig. 1C].

Regulation of Med1 protein expression and function by BAP1 overexpression

USP21, USP35, and BAP1 differently regulate Med1 protein expression and transcriptional capacity in MCF7 cells. Med1 protein expression highly increased by BAP1 overexpression, whereas did not affect USP21 and USP35 [Fig. 2A]. To confirm these results a mutant DNA plasmid with inhibition of the catalytic activity was used. The expression of Med1 protein was not affected by USP21 C211A and USP35 C434A mutant overexpression [Fig. 2A]. These data suggest that USP21 and USP35 do not regulate Med1 protein expression through catalytic activity. Med1 protein expression was increased by BAP1 wild type overexpression in MCF7 cells but was decreased by BAP1 C91A mutant overexpression [Fig. 2B]. A qPCR result revealed that Med1 mRNA expression was not affected by the overexpression of the BAP1 wild type (WT) or BAP1 C91A mutant [Fig. 2C]. These results demonstrated that Med1 protein regulation by BAP1 was regulated at the protein level not transcriptional level. Next, to verified relation between Med1 and BAP1 protein expression, it was treated with EGF, E2 and TGF -β, which are known to play an important role in breast cancer growth regulation. Med1 protein and mRNA (data not shown) were increased by EGF treatment, but not increased by E2 and TGF-β. On the other hand, BAP1 was significantly increased by E2 and TGF treatment [Fig. 2D]. These results addressed that the regulation of Med1 protein expression by BAP1 is regulated at the protein level and is regulated by different mechanisms from growth factors.

Med1 is associated with BAP1

To investigate the interaction between Med1 and BAP1 protein, co-immunoprecipitation was performed in the BAP1 overexpressing cell line, BAP1 wild type and BAP1 C91A mutant were overexpressed, and IP was performed using an anti-Med1 antibody in BAP1 or BAP1 C91A overexpressed cells. Immunoblotting was performed using anti-SRT and anti-BAP1 antibodies. Med1 was observed to bind to BAP1. However, the BAP1 C91A mutant was observed to bind weakly compared with the BAP1 control [Fig. 3A]. These results demonstrates that BAP1 is associated with Med1, but not the BAP1 catalytic mutant.

BAP1 overexpression regulates cell growth and cancer metastasis in vitro.

Med1 regulates the transcription of various genes and plays a role in cell growth and cancer metastasis. Therefore, to investigates whether overexpression of BAP1 affects proliferation and, metastasis by Med1 protein expression, cell proliferation, and invasion assays were performed in MCF7 breast cancer cells. There was no difference in cell growth in the BAP1 overexpressing cells compared with the control in the MCF7 cell. However, cell growth was reduced in the BAP1 C91A mutant overexpressing cells [Fig. 3B]. In a previous report, BAP1 increased cell growth whereas abnormal activity of BAP1 inhibited cell growth in several cancers. It was observed that overexpression of BAP1 wild type slightly decreased invasion ability, but increased cell migration. And overexpression of BAP 1 C91A mutants was observed to increase metastatic ability and mRNA level of the urokinase-type plasminogen receptor (uPAR) gene, a metastasis-related gene, was significantly increased by overexpression of the BAP1 mutant (Fig. 3D). Many studies have reported that inhibition of Med1 expression increased cancer metastasis and associated with uPAR gene expression. Therefore, these results demonstrate that cell growth is regulated by overexpression of BAP1, and metastatic potential can be modulated by aberrant activation of BAP1 or Med1 protein expression.

Med1 (also known as Trap220 or DRIP205) is a subunit of the mediator complex. Recent studies have suggested that Med1 protein expression play a role in tumorigenesis and cancer metastasis. The deficiency of the Med1 protein functions as a tumor suppressor gene and inhibits invasion and metastasis in lung carcinoma [8] and melanoma cells [22]. Loss of Med1 significantly decreases the proliferation of prostate cancer cells and breast cancer [23]. In contrast, a deficiency of Med1 protects hepatocytes from chemical carcinogen-induced hepatocarcinogenesis [24] and osteoblastoma [25, 26]. Some kinases such as AKT, ERK and DNA-PK mediate the phosphorylation of Med1 and enhance Med1 association transcriptional regulation [6, 27, 27]. And overexpression of Med1 abrogated the effects of miR-1 on cell proliferation [28]. However, there have been few studies on the mechanism of Med1 protein expression regulation. In this study, it was investigated that overexpression of various DUBs regulates the expression of the Med1 protein. Some DUBs such as USP21 and USP35 wild type increased the Med1 protein expression, but it did not regulate the Med1 protein expression by overexpression of catalytic mutant forms (USP21 C211A, and USP35 C434A). These results explain the UPS21 and UPS35 affect Med1 protein expression by indirect regulation mechanism and the underlying mechanism is unclear. However, Med1 protein was increased by the overexpression of BAP1 and decreased by BAP1 C91A overexpression. Various studies have reported that BAP1 acts as a tumor suppressor by binding BRCA1 in breast cancer [13] and BAP1 cancer syndrome, in which metastatic breast cancer develops with loss of BAP1 protein expression [29]. Overexpression of BAP1 in breast cancer is involved in the transcriptional regulation of ER by stabilizing the Med1 protein. It also affected the growth and metastasis of cancer cells. Location of BAP1 and Med1 are in the nucleus, and they modulated transcriptional activity. In addition, an examination of the interaction of Med1 and BAP1 proteins, showed that Med1 and BAP1 proteins are binding, and are involved in the development and metastasis of cancer [Fig. 3]. Although the binding of Med1 and BAP1 involved in the cancer growth or metastasis, mechanisms are still unclear. However, in the future, the results of this study will contribute to various research fields, such as the mechanism of regulating the expression of target genes by the binding of Med1 and BAP1 and the growth and metastasis of cancer by Med1 and BAP1. In addition, it may contribute to various studies, such as what role Med1 protein expression by post-translational phosphorylation of Med1, which forms or maintains an active chromatin structure, plays a role in binding to BAP1 and ubiquitination. And as a further study, it can investigate whether Med1 phosphorylation affect the binding ability of BAP1 protein and the role in cancer cell growth and metastasis capacity. Thus, Med1 and BAP1 can serve as a biomarker for cancer development or metastasis, and targeting agent serves as a potential gene therapeutic target in various cancers.

Funding

This work was supported by the Dong-A Univdersity research fund.

Acknowledgement

I thank Kyungkyu Kim (Sungkyunkkwan Uuniversity) for providing deubiquitinating enzyme DNA plasmids (pDEST-CMV16 SRT tag vector).

Mitsuhiro Ito, Robert G, Roeder. The TRAP/SMCC/Mediator complex and thyroid hormone receptor function. Trends in endocrinology and metabolism. 2001;12:127–34.
Zhang X, Krutchinsky A, Fukuda A, Chen W, Yamaura S, Brian T. Chait and Robert G. Roeder.Med1/Trap220 exists predominantly in a TRAP/Mediator subpopulation Enriched in RNA polymerase II and is required for ER-mediated transcription. Mol Cell. 2005;19:89–100.
Yijun Zhu C, Qi S, Jain MM, Le Beau R, Espinosa G, Brandon Atkins MA, Lazar AV, Yeldandi M, Sambasiva Rao, Janarda K. Reddy. Amplification and overexpression of peroxisome proliferator activated receptor binding protein (PBP/PPARBP) gene in breast cancer. Proc Natl Acad Sci USA. 1999;96:10848–53.
Marissa Leonard and Xiaoting Xhang, Zhejian J. Estrogen receptor coactivator mediator subunit 1 (MED1) as a tissue-specific therapeutic target in breast cancer. Univ Sci B. 2019;20:381–90.
Frade R, Balbo M, Barel M. RB18A regulates p53-dependent apoptosis. Oncogene. 2002;31:861–6.
Feng Jin S, Irshad W, Yu M, Belakavadi M, Chekmareva MM, Ittmann C, Abate-Shen, Joseph D, Fondell. ERK and AKT signaling drive MED1 overexpression in prostate cancer in associate with elevated proliferation and tumorigenicity. Mol Cancer Res. 2013;11:736–47.
Ndong JDeLCroix, Jean D, Rousselet N, Frad R. Down-regulation of the expression of RB18A/MED1, a cofactor of transcription, triggers strong tumorigenic phenotype of human melanoma cells. Int J Cancer. 2009;124:2597–606.
Kim H-J, Roh MS, Son CH, Kin AJ, Jee HJ, Song N, Kim M, Seo S-Y. Young Hyun Yoo, Jeanho Yun. Loss of Med1/Trap220 promotes the invasion and metastasis of human non-small-cell lung cancer cells by modulating the expression of metastasis-related genes. Cancer Lett. 2012;28:195–202.
Niklas Klumper I, Syring W, Vogel D, Schmidt SC, Muller J, Ellinger D, Adler. Johannes Bragelmann, and Sven Perner. Mediator complex subunit Med1 protein expression is decreased during bladder cancer progression. Front Med (Lausanne) 2017 4, 30F.
Kirby N. Swatek & David Komander. Ubiqutin modifications. Cell Res. 2016;26:399–422.
Tianshui Sun Z, Liu Q. The role of ubiquitination and deubiquitylation in cancer metabolism. Mol Cancer. 2020;19:pp146.
He M, Zhou Z, Zou AAShahH, Tao J, Chen Q. Yong Wan. The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics. Cell & Bioscience. 2016;6:62.
David E, Jensen M, Proctor ST, Marquis HP, Gardner, Seung I, Ha, Lewis A, Chodosh, Alexander M, Ishov N, Tommerup H, Vissing Y, Sekido J, Minna A, Borodovsky DC, Schultz, Keith D, Wilkinson, Gerd G, Maul N, Barlev SL, Berger GC, Prendergast, Frank J, Rauscher. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene. 1998;16:1097–112.
Nazar Mashtalir S, Daou H, Barbour, Nadine N, Sen J, Gagnon IH, Martel, Haider H, Dar. Marc Therrien, El Bachir. Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol Cell. 2014;54:392–406.
William Harbour Michael J, Onkenelishad D, Robersonshenghui Duanli Caolori DO, Laurin Councilkatie AWorleym. Matatallcynthia Helms, Anne M. Bowcock. Frequent mutation of BAP1 in metastasizing Uveal Melanomas. Science. 2010;330:1410–3.
Joseph R, Cheung M, Pei J, Below JE, Tan Y, Sementino E, Cox NJ, Dogan U, Pass HI, Trusa S. Mary Hesdorffer, Masaki Nasu, Amy Powers, Zeyana Rivera, Sabahattin Comertpay, Mika Tanji, Giovanni Gaudino, Haining Yang, Michele Carbone. Germline BAP1 mutations predispose to malignant mesotheliom. Nat Genet. 2011;28:1022–5.
Celis SPena-LlopisSVaga-Rubin-de, Liao A, Leng N, Pavia-Jimenez A, Wang S, Yamasaki T, Zhrebker L, Sivanad S, Spence P, Kinch L, Hambuch T, Jain S, Lotan Y, Margulis V, Sagalowsky AI, Pia Banerli SUmmerour, Wareef Kabbani SW, Wendy Wong N, Grishin M, Laurent X-J, Xie CD. Haudenschild, Mark T. Ross David R. Bentley, Payal Kapur, and James Brugarolas. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012, 44, 751–759.
Lu Wang NW, Birch Z, Zhao CM, Nestler A, Kazmer A, Shilati A, Blake. Patrick Alexander Ozark, Emily Jane Rendleman, Didi Zha, Caila Ann Ryan, Marc Alard Jonathan Morgan, Ali Shilatifard. Epigenetic targeted therapy of stabilized BAP1 in ASXL1 gain-of-function mutated leukemia. Nat Cancer. 2021;2:515–26.
Qin J, Zhou Z, Chen W, Wang C, Zhang H, Ge G, Shao M, You D, Fan Z, Xia H, Liu R. Ceshi Chen. BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. Nat Commun. 2015;6:8471.
Zhihua Liu RK, Meray TN, Grammatopoulos RA, Fredenburg MR, Cookson Y, Liu T Logan and, Peter T. Lansbury. Membrane-associated farnesylated UCH-L1 promotes α-synuclein neurotoxicity and is a therapeutic target for Parkinson`s disease. Proc Natl Acad Sci USA. 2009;106:4635–40.
Yichin Liu, Hilal A, Lashuel S, Choi X, Xing A, Case J, Ni L-A, Yeh GD, Cuny, Ross L, Stein, Peter T, Lansbury. Discovery of inhibitors that elucidate the role of UCH-Li activity in the H1299 lung cancer cell line. Chem Biol. 2013;10:837–46.
Gade P, Singh AK, Roy SK, Reddy SP, Kalvakolanu DV. Down-regulation of the transcriptional mediator subunit Med1 contributes to the loss of expression of metastasis-associated dapk1 in human cancers and cancer cells. Int J Cancer. 2009;125:1566–74.
Chen Z, Zhang C, Wu D, Chen H, Rorick A, Zhasng X, Wang Q. Phospho-Med1-enhanced UBE2C locus looping drives castration-resistant prostate. cancer growth EMBO. 2011;30:2405–19.
Rasool RU, Natesan R, Deng Qu, Aras S, Lal P, Effron SS, Mitchell-Velasquez E, Posimo JM, Carskadon S, Baca SC, Mark M, Pomerantz J, Siddiqui LE, Schwartz, Daniel J, Lee. Nallasivam Palanisamy, Goutham Narla, Robert B Den, Mattew L Freedman, Donita C Brady, Irfan A Asangani. CDK7 inhibition suppresses castration-resistant prostate cancer through Med1 inactivation. Cancer Discov. 2019;9:1538–55.
Madesh Belakavadi, Pradeep K, Pandey. Ravi vijayvargia, Joseph D Fondell. Med1 phosphorylation promotes its association with mediator: implications for nuclear receptor signaling. Mol Cell Biol. 2008;28:3932–42.
Med1 subunit. of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis. Gene Expr. 2014;16:63–75.
Schiano C, Rienzo M, Casamassimi A, Napoli C. Gene expression profile of the whole mediator complex in human osteosarcoma and normal osteoblasts. Med Oncol. 2013;30:7392013.
Jiang C, Chen H, Shao L. Qiaojie Wang.MicroRNA-1 functions as a potential tumor suppressor in osteosarcoma by targeting Med1 and Med3. Oncol Rep. 2014;32:1249–56.
Ana Bltnik D, Ribnikar B, Reric. Petra Skerl, Gasper Klancar, Mateja Krajc. BAP1-defiicient breast cancer in a patient with BAP1 cancer syndrome. Case Report 2022, 29, 921–927.

Download PDF

Journal Publication

published 02 May, 2024

Read the published version in Molecular & Cellular Oncology →

Version 1

posted

You are reading this latest preprint version

Regulation of Med1 protein by overexpression of BAP1 in breast cancer cells

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture and Transfection

Western blotting and immunoprecipitation

Luciferase reporter assay

Quantitative RT-PCR (qPCR)

Cell proliferation assay

Statistical analysis

Results

Identification of DUBs regulating the expression of Med1 protein

Regulation of Med1 protein expression and function by BAP1 overexpression

Med1 is associated with BAP1

Discussion

Declarations

Funding

Acknowledgement

References

Status:

Journal Publication

Version 1