Elevated MED28 expression predicts poor outcome in women with breast cancer
© Yoon et al; licensee BioMed Central Ltd. 2010
Received: 11 September 2009
Accepted: 28 June 2010
Published: 28 June 2010
MED28 (also known as EG-1 and magicin) has been implicated in transcriptional control, signal regulation, and cell proliferation. MED28 has also been associated with tumor progression in in vitro and in vivo models. Here we examined the association of MED28 expression with human breast cancer progression.
Expression of MED28 protein was determined on a population basis using a high-density tissue microarray consisting of 210 breast cancer patients. The association and validation of MED28 expression with histopathological subtypes, clinicopathological variables, and disease outcome was assessed.
MED28 protein expression levels were increased in ductal carcinoma in situ and invasive ductal carcinoma of the breast compared to non-malignant glandular and ductal epithelium. Moreover, MED28 was a predictor of disease outcome in both univariate and multivariate analyses with higher expression predicting a greater risk of disease-related death.
We have demonstrated that MED28 expression is increased in breast cancer. In addition, although the patient size was limited (88 individuals with survival information) MED28 is a novel and strong independent prognostic indicator of survival for breast cancer.
In 2008, an estimated 40,000 women died of breast cancer and over 190,000 women were newly diagnosed with the disease, making breast cancer the second leading cause of cancer death in women . Although treatment based on the molecular characteristics of breast cancer subtypes has helped improve prognosis, much progress needs to be made (reviewed in [2–4]). The characterization of novel markers to augment our understanding of cancer and our ability to predict patient outcomes will greatly improve breast cancer management. A recently identified marker, MED28 (also known as EG-1 or magicin), has been found to be increased in breast cancer and may play a role in the progression of the disease [5–9].
MED28 is a 178 amino acid, ~24 kDa protein that was first identified as being differentially expressed in endothelial cells exposed to conditioned media from tumor cells [5, 6]. Although the exact function of MED28 is unknown, it has been identified as one of approximately 30 subunits within the mammalian Mediator complex, which regulates activation and repression of RNA polymerase II transcribed genes [10–13]. In addition, MED28 has been found to be a binding partner for merlin, a cytoskeleton-related tumor suppressor important in neurofibromatosis 2 development [12, 14].
Clues as to functional consequences of MED28 expression have been found in tumor model systems. The presence of MED28 has been shown to increase cellular proliferation in both cell culture and mouse xenograft models using human breast cancer cells . Inhibition of MED28 expression by either siRNA or anti-MED28 antibody decreases cellular proliferation in vitro and in vivo . Finally, in retrospective studies on human tissue samples, MED28 has been found to be up-regulated in breast, prostate, and colon cancers .
Here we examine the expression levels of MED28 on a population basis using a human breast cancer tissue microarray (TMA). Our findings show that MED28 expression is a significant independent predictor of survival in women with both early and late stage breast cancer.
Characteristics of the Breast TMA
Surgical cases represented on the TMA
A high-density breast TMA was constructed and utilized as previously described [15–17] with appropriate oversight by the UCLA Institutional Review Board. Briefly, the TMA was built using cores from archived formalin-fixed, paraffin-embedded breast tissue samples from 242 cases of patients who underwent surgery at the UCLA Medical Center between 1995 and 2000. Of these 242 cases, 213 women had surgery for suspected breast cancer while 29 women had breast reduction surgery. A "case" is defined as a surgery for which tissue was removed and could be used to construct the TMA. Of the women who had surgery for suspected breast cancer, 134 individuals had their primary surgery at UCLA. An additional 79 women came to the UCLA Medical center for a secondary follow-up surgery.
The spectrum of overall case histologies from the 213 patients who had surgery due to suspected cancer were as follows: 179 cases with invasive breast cancer histology (this was sub-divided into 122 cases which contained both invasive and in situ tumor histologies, and 57 cases which had invasive tumor histology alone); in situ tumor histology alone (22 cases); and individuals who had suspected cancer but who, upon surgery, were found to have ductal hyperplasia, atypical ductal hyperplasia, atypical lobular hyperplasia, or intraductal papilloma (4, 1, 2, and 5 cases respectively). Within the patients with invasive breast cancer (179 cases), 72 cases were associated with metastases. Forty-nine of these patients presented with metastasis at their first surgery (48 lymph node metastases, 1 distant metastasis).
Characteristics of spots on the TMA
At least three samples (cores) of each histology were taken to represent a given histology in each case. In total, the TMA consisted of 2,039 cores of which 924 were readable. Note that unreadable spots primarily included those that contained only stroma or fat or those that had fallen off during processing. The breakdown by core histology was 506 invasive tumors (440 invasive ductal carcinoma (IDC), 66 other breast cancer variants including invasive lobular carcinoma, invasive tubular carcinoma, apocrine carcinoma, mixed invasive ductal and lobular carcinoma, and medullary carcinoma), 98 in situ tumors (92 DCIS + 6 LCIS), 110 metastatic lesions, 14 atypical hyperplasia, 39 ductal hyperplasia, 109 normal matched tissues, 21 benign tissues, and 27 cores from breast reduction cases.
Case inclusion and exclusion criteria for outcomes analyses
The breast TMA was evaluated for MED28 expression using a standard immunohistochemistry protocol as previously described [6, 15–17]. Briefly, 4 μm thick TMA sections were cut, deparaffinized, treated for antigen retrieval with 10 mM sodium citrate, pH 6 (95°C for 20 min), quenched for endogenous peroxidase activity, and blocked with 5% horse serum before incubation for 30 minutes with anti-MED28 primary antibody at a 1:300 dilution. The primary antibody was a polyclonal rabbit anti-human-MED28 antibody produced in the laboratory of Dr. Mai Brooks . The primary antibody was detected by applying a horse anti-rabbit HRP secondary antibody and an avidin-biotin complex (Vector Laboratories, Burlingame, CA) followed by diaminobenzidine. Negative controls included primary incubation with preimmune rabbit serum. Her-2/neu status was determined by immunohistochemistry using the Hercep Test guidelines (DAKO, catalog K5204, Carpinteria, CA).
Semiquantitative evaluation of MED28 staining was performed by a pathologist who tabulated the percentage of glandular cells that exhibited cytoplasmic staining at each intensity, from 0 to 3 (0 being below the level of detection, 1 being weak, 2 being moderate and 3 representing highest expression) as previously described [15–18]. Briefly, an integrated value was used to account for frequency and intensity of staining for each spot. The following formula was used to calculate this integrated value: [3(%x) + 2(%y) + 1(%z)]/100, where x, y, and z represent the percentage of cells staining at intensity 3, 2, and 1, respectively. Survival analyses were analyzed with patient case data. Case data was analyzed by using pooled expression results as previously described [16–19].
Clinico-pathologic characteristics and MED28 expression in individuals with breast cancer
All Invasive Patients
N = 88
N = 66
N = 22
0.946 ± 0.668
Age at Diagnosis
ρ = 0.024b
53 (30 - 89)
53.5 (30 - 89)
52 (36 - 89)
25th to 75th Quartile
45 - 66
45 - 74
45 - 77.2
0.782 ± 0.721
0.918 ± 0.541
1.312 ± 0.708
1.075 ± 0.813
0.598 ± 0.429
1.009 ± 0.698
1.112 ± 0.726
Lymph Node Metastasis
0.833 ± 0.775
1.083 ± 0.612
Tumor Size (cm)
ρ = 0.303b
2.2 (0.1 - 9.0)
2.0 (0.1 - 7.25)
2.5 (0.5 - 9.0)
25th to 75th Quartile
1.18 - 3.00
1.0 - 2.5
1.7 - 4.0
0.812 ± 0.599
1.177 ± 0.734
0.866 ± 0.594
1.271 ± 0.822
0.864 ± 0.562
1.142 ± 0.838
1.181 ± 0.644
0.923 ± 0.661
MED28 levels are elevated in breast cancer
Relatively high levels of MED28 predict a greater likelihood of tumor recurrence
Relatively high levels of MED28 predict a poorer survival outcome
Univariate Cox Model Regression Analysis
95% Confidence Interval
Age at Diagnosis
0.937 - 1.02
Stage (I & II vs. III & IV)
2.16 - 15.7
Grade (I & II vs. III)
0.843 - 6.39
Lymph Node Metastasis
1.4 - 18.1
Tumor Size (cm)
0.926 - 1.52
0.951 - 9.7
0.196 - 1.49
0.209 - 1.51
1.25 - 11.1
1.18 - 3.7
Multivariate Cox proportional hazards analysis
95% Confidence Interval
1.178 - 27.21
Stage (I & II vs. III & IV)
0.821 - 12.23
Grade (I & II vs. III)
0.698 - 4.68
Age at Diagnosis
0.895 - 1.02
0.241 - 2.87
0.399 - 4.54
In this study, we examined the expression of MED28 on a population basis using TMA technology. MED28 levels were increased in DCIS lesions as well as invasive breast cancer compared to morphologically normal breast epithelium. In addition, MED28 was up-regulated in metastatic cells in lymph nodes. These results are in agreement with previous results in which a smaller number of patient samples were examined . Significantly, we further observed that MED28 was a strong predictor of disease outcome with higher levels of MED28 indicating an increased probability of death due to breast cancer in the 88 individuals examined with survival information. These results are consistent with data from in vitro and mouse model systems in which up-regulation of MED28 enhanced cell proliferation and tumor growth . Inhibition of MED28 by antibody or siRNA blocked these effects .
The cellular function of MED28 is currently being elucidated. Although MED28 was initially discovered as a differentially expressed gene in human endothelial cells treated with conditioned media from human cancers , it was also characterized as a binding partner for the actin-associated neurofibromatosis 2 (NF2) tumor suppressor merlin as well as the adaptor protein Grb2 . These and other results are consistent with MED28 functionally linking membrane receptor signaling to cytoskeletal changes. MED28 has further been shown to bind the SH3 domain of src-family members suggesting that one mode of operation is through interaction with kinase signaling molecules Src, Lck, and/or Fyn . Interestingly, over-expression of MED28 in vitro has been shown to activate c-Src and stimulate the c-Src signaling pathway . Src activation can contribute to the malignant phenotype through enhancing processes such as proliferation, invasion, migration, and metastasis (reviewed in [21–24]).
Interestingly, MED28 has been observed both in the cytoplasm and the nucleus as described by us and others [6, 10, 13]. Consistent with this, MED28 is one of the subunits of the highly conserved mammalian mediator complex. This complex functions as a co-activator required for the induction of transcription by RNA polymerase II [25–30]. It has been suggested that MED28 translocates between the nucleus and cytoplasm and therefore may potentially function in transducing membrane-derived signals into gene expression events.
The present study shows that the expression of MED28 is relatively higher in both early and advanced breast cancer lesions. Such elevated levels predict a poorer survival. That MED28 expression was elevated in DCIS lesions compared to normal and was predictive in early as well as late stage patients suggests that alterations in the MED28 signaling axis may be an early indicator of disease progression and a potential therapeutic target. In addition to its potential usefulness as a prognostic factor, MED28 may eventually prove useful for targeted therapy: a recent study showed that inhibition of MED28 resulted in smaller breast tumor xenografts in mice .
ductal hyperplasia, DCIS: ductal carcinoma in situ, ER: estrogen receptor
progesterone receptor, EG-1: endothelial-derived gene-1
We would like to thank Mai N. Brooks for helpful discussion and for anti-MED28 antibody reagents. We would also like to thank Stephanie Hanna and Greg Kanter for excellent technical assistance and Jacob Schatz for stimulating discussion. This work was supported in part by the Early Detection Research Network NCI CA-86366 (LG, DC) and the Jonsson Comprehensive Cancer Center (JCCC) Shared Resource Core Grant at UCLA NIH NCI 2 P30 CA16042-29 (DS).
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 2009, 59 (4): 225-49. 10.3322/caac.20006.View ArticlePubMedGoogle Scholar
- Olopade OI, Grushko TA, Nanda R, Huo D: Advances in breast cancer: pathways to personalized medicine. Clin Cancer Res. 2008, 14 (24): 7988-7999. 10.1158/1078-0432.CCR-08-1211.View ArticlePubMedPubMed CentralGoogle Scholar
- Sotiriou C, Pusztai L: Gene-expression signatures in breast cancer. N Engl J Med. 2009, 360 (8): 790-800. 10.1056/NEJMra0801289.View ArticlePubMedGoogle Scholar
- Dowsett M, Dunbier AK: Emerging biomarkers and new understanding of traditional markers in personalized therapy for breast cancer. Clin Cancer Res. 2008, 14 (24): 8019-8026. 10.1158/1078-0432.CCR-08-0974.View ArticlePubMedGoogle Scholar
- Liu C, Zhang L, Shao ZM, Beatty P, Sartippour M, Lane TF, Barsky SH, Livingston E, Nguyen M: Identification of a novel endothelial-derived gene EG-1. Biochem Biophys Res Commun. 2002, 290 (1): 602-612. 10.1006/bbrc.2001.6119.View ArticlePubMedGoogle Scholar
- Zhang L, Maul RS, Rao J, Apple S, Seligson D, Sartippour M, Rubio R, Brooks MN: Expression pattern of the novel gene EG-1 in cancer. Clin Cancer Res. 2004, 10 (10): 3504-3508. 10.1158/1078-0432.CCR-03-0467.View ArticlePubMedGoogle Scholar
- Lu M, Zhang L, Maul RS, Sartippour MR, Norris A, Whitelegge J, Rao JY, Brooks MN: The novel gene EG-1 stimulates cellular proliferation. Cancer Res. 2005, 65 (14): 6159-6166. 10.1158/0008-5472.CAN-04-4016.View ArticlePubMedGoogle Scholar
- Lu M, Zhang L, Sartippour MR, Norris AJ, Brooks MN: EG-1 interacts with c-Src and activates its signaling pathway. Int J Oncol. 2006, 29 (4): 1013-1018.PubMedGoogle Scholar
- Lu M, Sartippour MR, Zhang L, Norris AJ, Brooks MN: Targeted inhibition of EG-1 blocks breast tumor growth. Cancer Biol Ther. 2007, 6 (6):
- Beyer KS, Beauchamp RL, Lee MF, Gusella JF, Naar AM, Ramesh V: Mediator subunit MED28 (Magicin) is a repressor of smooth muscle cell differentiation. J Biol Chem. 2007, 282 (44): 32152-32157. 10.1074/jbc.M706592200.View ArticlePubMedGoogle Scholar
- Lee MF, Beauchamp RL, Beyer KS, Gusella JF, Ramesh V: Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem Biophys Res Commun. 2006, 348 (3): 826-831. 10.1016/j.bbrc.2006.07.126.View ArticlePubMedGoogle Scholar
- Wiederhold T, Lee MF, James M, Neujahr R, Smith N, Murthy A, Hartwig J, Gusella JF, Ramesh V: Magicin, a novel cytoskeletal protein associates with the NF2 tumor suppressor merlin and Grb2. Oncogene. 2004, 23 (54): 8815-8825. 10.1038/sj.onc.1208110.View ArticlePubMedGoogle Scholar
- Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA, Jin J, Cai Y, Washburn MP, et al: A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol Cell. 2004, 14 (5): 685-691. 10.1016/j.molcel.2004.05.006.View ArticlePubMedGoogle Scholar
- Scoles DR: The merlin interacting proteins reveal multiple targets for NF2 therapy. Biochim Biophys Acta. 2008, 1785 (1): 32-54.PubMedGoogle Scholar
- Shen D, Nooraie F, Elshimali Y, Lonsberry V, He J, Bose S, Chia D, Seligson D, Chang HR, Goodglick L: Decreased expression of annexin A1 is correlated with breast cancer development and progression as determined by a tissue microarray analysis. Hum Pathol. 2006, 37 (12): 1583-1591. 10.1016/j.humpath.2006.06.001.View ArticlePubMedGoogle Scholar
- Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, Wang Q, Chia D, Goodglick L, Kurdistani SK: Global levels of histone modifications predict prognosis in different cancers. Am J Pathol. 2009, 174 (5): 1619-1628. 10.2353/ajpath.2009.080874.View ArticlePubMedPubMed CentralGoogle Scholar
- Mumenthaler S, Yoon N, Li A, Mah V, Chang G, Nooraie F, Elshimali Y, Hanna S, Kim S, Horvath S, et al: Tissue Microarrays: Construction and Utilization For Biomarker Studies. Methods of Cancer Diagnosis, Therapy, and Prognosis. Edited by: Hayat MA. 2008, Springer Netherlands, 1: 217-234. full_text.View ArticleGoogle Scholar
- Mah V, Seligson DB, Li A, Marquez DC, Wistuba II, Elshimali Y, Fishbein MC, Chia D, Pietras RJ, Goodglick L: Aromatase expression predicts survival in women with early-stage non small cell lung cancer. Cancer Res. 2007, 67 (21): 10484-10490. 10.1158/0008-5472.CAN-07-2607.View ArticlePubMedPubMed CentralGoogle Scholar
- Seligson DB, Horvath S, Shi T, Yu H, Tze S, Grunstein M, Kurdistani SK: Global histone modification patterns predict risk of prostate cancer recurrence. Nature. 2005, 435 (7046): 1262-1266. 10.1038/nature03672.View ArticlePubMedGoogle Scholar
- Liu X, Minin V, Huang Y, Seligson DB, Horvath S: Statistical methods for analyzing tissue microarray data. J Biopharm Stat. 2004, 14 (3): 671-685. 10.1081/BIP-200025657.View ArticlePubMedGoogle Scholar
- Kopetz S, Shah AN, Gallick GE: Src continues aging: current and future clinical directions. Clin Cancer Res. 2007, 13 (24): 7232-7236. 10.1158/1078-0432.CCR-07-1902.View ArticlePubMedGoogle Scholar
- Summy JM, Gallick GE: Src family kinases in tumor progression and metastasis. Cancer Metastasis Rev. 2003, 22 (4): 337-358. 10.1023/A:1023772912750.View ArticlePubMedGoogle Scholar
- Martin GS: Fly Src: the Yin and Yang of tumor invasion and tumor suppression. Cancer Cell. 2006, 9 (1): 4-6. 10.1016/j.ccr.2005.12.025.View ArticlePubMedGoogle Scholar
- Resh MD: The ups and downs of SRC regulation: tumor suppression by Cbp. Cancer Cell. 2008, 13 (6): 469-471. 10.1016/j.ccr.2008.05.011.View ArticlePubMedGoogle Scholar
- Gu W, Malik S, Ito M, Yuan CX, Fondell JD, Zhang X, Martinez E, Qin J, Roeder RG: A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol Cell. 1999, 3 (1): 97-108. 10.1016/S1097-2765(00)80178-1.View ArticlePubMedGoogle Scholar
- Ito M, Yuan CX, Malik S, Gu W, Fondell JD, Yamamura S, Fu ZY, Zhang X, Qin J, Roeder RG: Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. Mol Cell. 1999, 3 (3): 361-370. 10.1016/S1097-2765(00)80463-3.View ArticlePubMedGoogle Scholar
- Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP: Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature. 1999, 398 (6730): 824-828. 10.1038/19783.View ArticlePubMedGoogle Scholar
- Naar AM, Beaurang PA, Zhou S, Abraham S, Solomon W, Tjian R: Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature. 1999, 398 (6730): 828-832. 10.1038/19789.View ArticlePubMedGoogle Scholar
- Ryu S, Zhou S, Ladurner AG, Tjian R: The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1. Nature. 1999, 397 (6718): 446-450. 10.1038/17141.View ArticlePubMedGoogle Scholar
- Jiang YW, Veschambre P, Erdjument-Bromage H, Tempst P, Conaway JW, Conaway RC, Kornberg RD: Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc Natl Acad Sci USA. 1998, 95 (15): 8538-8543. 10.1073/pnas.95.15.8538.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/335/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.