Epstein–Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation Hai Hu, Man-Li Luo, Christine Desmedt, Sheida Nabavi, Sina Yadegarynia, Alex Hong, Panagiotis A. Konstantinopoulos, Edward Gabrielson, Rebecca Hines-Boykin, German Pihan, Xin Yuan, Christos Sotirious, Dirk P. Dittmer, Joyce D. Fingeroth, Gerburg M. Wulf PII: DOI: Reference:
S2352-3964(16)30209-2 doi: 10.1016/j.ebiom.2016.05.025 EBIOM 618
To appear in:
EBioMedicine
Received date: Revised date: Accepted date:
11 April 2016 16 May 2016 18 May 2016
Please cite this article as: Hu, Hai, Luo, Man-Li, Desmedt, Christine, Nabavi, Sheida, Yadegarynia, Sina, Hong, Alex, Konstantinopoulos, Panagiotis A., Gabrielson, Edward, Hines-Boykin, Rebecca, Pihan, German, Yuan, Xin, Sotirious, Christos, Dittmer, Dirk P., Fingeroth, Joyce D., Wulf, Gerburg M., Epstein–Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation, EBioMedicine (2016), doi: 10.1016/j.ebiom.2016.05.025
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Epstein Barr Virus infection of mammary epithelial cells promotes malignant
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transformation
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Authors: Hai Hu1, Man-Li Luo9, Christine Desmedt2, Sheida Nabavi3, Sina
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Yadegarynia1, Alex Hong8 Panagiotis A. Konstantinopoulos4, Edward Gabrielson5,
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Rebecca Hines-Boykin6, German Pihan10 , Xin Yuan1, Christos Sotirious2, Dirk P.
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Affiliations:
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Dittmer6, Joyce D. Fingeroth7 and Gerburg M. Wulf1*
1 - Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston MA, USA 2 – Institut Jules Bordet - 121 Boulevard de Waterloolaan, Bruxelles 1000 Brussels, Belgium 3 – University of Connecticut, Computer Science and Engineering, 371 Fairfield Way, Storrs CT 06268 4 – Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA 02215
ACCEPTED MANUSCRIPT 5 – Department of Pathology, Johns Hopkins University, 4940 Eastern Ave, Baltimore, MD 21224
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Center, University of North Carolina, Chapel Hill, NC, USA
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6 - Department of Microbiology and Immunology, Lineberger Comprehensive Cancer
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7- Department of Medicine, University of Massachusetts Medical School, 364 Plantation
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Street
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Worcester MA 01605
8-Massachusetts Institute for Technology Department of Biology
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9- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical
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School, 330 Brookline Ave, Boston MA, USA. Present: Breast Tumor Center, Sun YatSen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, P. R. China. 10 – Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston MA, USA
*To whom correspondence should be addressed:
[email protected]
One Sentence Summary: Epstein Barr Virus (EBV) can infect mammary epithelial cells and facilitate cancerous transformation consistent with a ‘hit-and-run’ mechanism.
ACCEPTED MANUSCRIPT Abstract: Whether the human tumor virus, Epstein-Barr virus (EBV), promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here
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we show EBV can infect primary mammary epithelial cells (MECs) that express the
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receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with
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a stem cell phenotype, activates MET signaling and enforces a differentiation block.
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When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors
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was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC). A human
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gene expression signature for MEC infected with EBV, termed EBVness, was
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associated with high grade, estrogen-receptor-negative status, p53 mutation and poor
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survival. In 11/33 EBVness-positive tumors, EBV-DNA was detected by fluorescent in situ hybridization for the viral LMP1 and BXLF2 genes. In an analysis of the TCGA breast cancer data EBVness correlated with presence of the APOBEC mutational signature consistent with past viral infection. We conclude that a contribution of EBV to breast cancer etiology is plausible, through a mechanism in which EBV infection predisposes mammary epithelial cells to malignant transformation, but is no longer required once malignant transformation has occurred.
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Highlights:
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Mammary epithelial cells, but not breast cancer cells, express CD21 and can be
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infected by EBV
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EBV infection of MECs rewires mitogenic signaling with activation of the MET pathway
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and gene expression changes (EBVness)
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EBV infection of MECs lowers the threshold for malignant transformation in xenografts
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Human tumors with ‘EBVness’ are associated with poor prognosis and presence of remnant EBV DNA (but not RNA)
Research in context:
This study shows that Epstein Barr Virus (EBV) infection occurs in breast epithelial cells (MECs), but not breast cancer cells. EBV infection of MECs leads to changes in gene expression, activation of oncogenic signaling via c-MET and lowers the threshold for cancerous transformation. In human breast cancer, these gene expression changes, termed ‘EBVness’, are associated with high grade and the finding of remnant EBV DNA. EBV infection of breast epithelial cells may increase the odds of later breast cancer
ACCEPTED MANUSCRIPT formation in predisposed individuals, which could potentially be avoided by a childhood
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vaccine against EBV.
ACCEPTED MANUSCRIPT Introduction
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The human tumor virus, Epstein Barr Virus (EBV), is causally associated with approximately 200,000 malignancies worldwide annually. The risk of cancer linked to
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EBV infection recently prompted a joint call by the NCI and the NIAID for development
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of an EBV vaccine (Cohen et al., 2011). EBV is typically transmitted early in life as a
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subclinical illness. When delayed until early adulthood symptoms of infectious
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mononucleosis often occur (Evans, 1971). Globally, more than 90% of the adult population has been infected (Evans, 1971, Cohen, 2000). In general, most humans
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tolerate latent EBV infection without adverse effects. However, in certain individuals,
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EBV has been linked to the etiology of cancers, including African Burkitt lymphoma
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(Epstein et al., 1964, Epstein and Barr, 1964); Hodgkin’s disease, nasopharyngeal carcinoma (NPC); gastric adenocarcinoma; and leiomyosarcoma (Niedobitek et al., 2001). Epithelial cancers associated with EBV vary markedly in viral prevalence, from nearly 100% of NPCs to about 10% of gastric carcinomas(Gulley, 2001) and also differ in the patterns of viral genes expressed. As exposure to virus frequently precedes the manifestation of cancer by years, and only a minority of individuals exposed to EBV will develop a related cancer, it is difficult to establish a causal role. An association of EBV infection with breast cancer has been reported from India (Joshi et al., 2009), China (Peng et al., 2014, He et al., 2012), Northern Africa (Fina et al., 2001, Hachana et al., 2011c) and southern Europe (Marrao et al., 2014, Mazouni et al.,
ACCEPTED MANUSCRIPT 2011, Labrecque et al., 1995). However, a potential mechanism of infection has remained unknonwn. Two recent analyses of the RNAseq data in the TCGA dataset did
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not show evidence for actively transcribed virus (Khoury et al., 2013b, Tang et al.,
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2013). These results argued against a role of active EBV infection for the growth of
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established breast cancers, but did not rule out a mechanism of oncogenesis where
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viral infection contributes to transformation but is no longer required once a tumor has established itself. In this report we show that EBV can infect primary human mammary
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epithelial cells (MECs) through CD21 leading to phenotypic changes consistent with
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transformation. These immortalized MECs infected with EBV cooperatively (with
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activated Ras) increase tumor formation in vivo, recapitulating a multistep tumorigenesis
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in an established animal model. Significantly, when a transcriptional profile based on cellular gene expression in EBV+ xenograft tumors was used to interrogate different human breast cancer databases, a subset of high grade breast tumors was identified in which EBV DNA, but not viral RNA, was detected by FISH.
ACCEPTED MANUSCRIPT Methods Cell lines. Immortalized human mammary epithelial cell lines (HMLE and HMEC) were
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provided by Dr. Robert Weinberg, MIT. HMLE was generated from normal human
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primary mammary epithelial that overexpress hTERT (Elenbaas et al., 2001).
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HMEChtert, in short H:MEC, had been generated from primary mammary epithelial
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cells (MECs) that were immortalized with human telomerase (hTERT) and SV40 Large T antigen(Zhao et al., 2003). MCF10A cells were from the ATCC. The EBV producing
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Akata cells were a gift of Dr. Lindsey Hutt-Fletcher, Louisiana State University Health
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Sciences Center, and cultured as described (Kuhn-Hallek et al., 1995)
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Cell Culture, EBV generation, infection and Generation of Cell Lines. MCF-10A
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mammary epithelial cells and HMEC were cultured as described (Debnath et al., 2003). The isolation of primary human mammary epithelial cells (PMECs) was done according to a protocol approved by the Institutional Review Board (IRB) at Beth Israel Deaconess Medical Center. PMECs were isolated from reduction mammoplasty specimens, and cultured in MEGM supplemented with 20 ng/ml EGF, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 1% bovine serum albumin, and 2% calf serum, as described (Burga et al., 2009). Recombinant EBV was isolated from AKATA cells that generate GFP-labeled EBV (GFP-EBV) defective of viral thymidine kinase (TK). Reactivation of virus from AKATA cells was induced by adding Goat anti-human immunoglobulin G at a final concentration
ACCEPTED MANUSCRIPT of 50 μg/ml to the culture medium, as described (Huang et al., 2003). GFP-control cells were generated using retroviral transduction (pBABE-GFP), followed by Neomycin
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selection at 100 μg/ml initially and 50 μg/ml for maintenance. To generate EBV-infected
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MECs, MECs were incubated with GFP-EBV for 2 hours, after which the cells were
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washed. At 72 hrs post infection, selection with Neomycin was started. Care was taken
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to culture the GFP-control MECs exactly like the GFP-EBV cells. For overexpression, vHRasV12D was subcloned into the pBabe retroviral vector
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(Addgene #17756). Production of retroviruses and transduction of MECs was performed
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as described previously (Stewart et al., 2003). Following infection, the cells were
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selected using zeocin (vHRasV12D). Cells were used up to three weeks after selection.
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Fresh cell lines were made for each group of experiments and experiments were performed following at least two separate infections. Flow Cytometry
Anti-CD21-PE antibody (HB5, eBioscience) was used for CD21 detection. Anti-CD24PE and anti CD44-APC antibodies (eBioscience) were used to detect progenitor cells. Anti-CD49f -APC(eBioscience) and anti- EpCAM-FITC (Biolegend) antibody were used for detection of mammary epithelial lineage. Isotype matched antibodies were used as negative controls. Flow cytometry was performed on a LSRII cytometer (BD Biosciences) and data were analyzed with FlowJo.
ACCEPTED MANUSCRIPT Competitive inhibition of EBV infection. HMECs were cultured in 6 well dishes to 40% confluency for the blocking experiments as described in (Fingeroth et al., 1984). Briefly,
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cell were pretreated with HB5 (mouse anti-CD21 antibody) or normal mIgG2a (40μg/ml)
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for 30 min, followed by 3 PBS washes, and then goat F(ab')2 fragments (50 μg/ml) to
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mouse IgG was added and incubated for another 30 min followed by another 3 washes
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with PBS, before EBV were added into the culture medium and incubated for another 4 hours to infect the cells.
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Mammosphere culture. Mammosphere cultures were performed as described (Dontu et
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al., 2003). Single-cell suspension were plated in ultra-low attachment plates (Corning,
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Costar) in DMEM/F-12 HAM medium containing bFGF (20 ng/mL), EGF (20 ng/mL),
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heparin (4 μg/mL) and B-27 supplement (1:50 dilution, invitrogen). The mammospheres were cultured for two weeks and mammospheres with diameter >75 μm were counted. 3D differentiation culture. 10 days mammospheres were resuspended in Matrigel (Gibco) and transferred to culture slides, allowed to solidify and then covered with differentiation medium (MEGM medium (Clonetics) supplemented with 10 ng/ml FGF, 4 ng/ml heparin and 2 μg/ml prolactin) for additional 10–14 days, as described (Mani et al., 2008). Tumorigenesis Assays. Aliquots of indicated numbers of cells were injected subcutaneously into the mammary fat pad of 5-week-old NOD/SCID mice (Jackson Laboratories). Mice were examined at least twice weekly for evidence of tumor growth.
ACCEPTED MANUSCRIPT After tumors were detected, tumor size was measured every two days. All studies involving mice were approved by the Institutional Animal Care and Use Committee at
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Beth Israel Deaconess Medical Center and performed in accordance with the protocol.
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Immunoblotting. Primary monoclonal LMP1 antibody (1:500) (Abcam), Pan-HRas
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antibody (1:1000) (BD), Alpha-Tubulin antibody (1:10000) (Sigma), and polyclonal
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CD21 antibody (1:500) (Abcam), METantibody (1:500) (Santa Cruz), p-METantibody (1:500) (Cell signaling), STAT3 (1:500) (Cell signaling), p-STAT3 (1:500) (Cell signaling)
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antibody were used.
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Immunohistochemistry Immunohistochemical staining for Ki67, p-Met, pSTAT3, K5/6,
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K18, ALDH1, p63, Vimentin was done as described previously(Burga et al., 2009, Burga
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et al., 2011). Immunolabeling was visualized with DAB solution (Vector Laboratories), followed by counterstaining with hematoxylin. phospho-RTK scan. The p-RTK arrays were performed according to the manufacturer’s recommendations (cell signaling Human Phospho-RTKArrayKit, Fluorescent Readout, #7949). The signal was developed using DyLight 680®-linked Streptavidin and then scanned with the Odyssey Infrared Imager (LI-COR). The intensity was read out by Genepix Pro 6.0 (Molecular Devices) and generated the heatmap by MeV4.7 (Saeed et al., 2006). In situ hybridization. EBER in situ hybridization (Fig. 4 C) was performed at the Dana Farber Cancer Center Specialized Histopathology Core Facility. Fish of LMP1 (type 1
ACCEPTED MANUSCRIPT probeset) and glycoprotein gH (BFXL2) (type 6 probeset) in patients’ tumor tissue sections (Fig. 7) were performed following the instructions for the Affymetrix
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QuantiGene View RNA ISH Tissue Assay kit. The LMP1 probe covers bases 70-1115
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of the LMP1 gene. The BfXL2 probe covers bases 450-1358 of the BfXL2 gene. As
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there are no introns in these viral genes, probes can target both EBV DNA and RNA.
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ISH stained tissue sections were scanned at 400 x resolution using a Zeiss LSM501 laser scanning microscope at the Ragon Institute and Tissue FAX software. They were
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scored for LMP1 and BFXL2 by three different physicians (XY, GP and GMW).
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RNAse A and RNAse 1were used to treat controls of Burkitt’s lymphoma before
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hybridization to confirm the probes are RNAse resistant and target to viral DNA.LMP1
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RNA interference and p-METinhibition. The siRNA target sequence to LMP1 mRNA is AAGAGCCUUCUCUGUCCACU (Guasparri et al., 2008). A control scrambled siRNA duplex was obtained from Dharmacon (Scramble II Duplex). Transfection of siRNA was performed using HiPerFect Transfection Reagent (Qiagen). For p-METinhibition, the XL184 (Chemietek) was dissolved in DMSO and introduced into the culture medium at the concentrations indicated in Fig. 5. Microarray Analysis. RNA was extracted using the total RNA isolation mini kit (Agilent). Microarray expression profiles were collected using Affymetrix GeneChip human HT_HG-U133+_PM. Affymetrix CEL files were analyzed with BRB-ArrayTools (Biometrics Research Branch, National Cancer Institute). Array data from our
ACCEPTED MANUSCRIPT experimentally derived tumors can be accessed in the GEO database; (http://www.ncbi.nlm.nih.gov/geo/GSE81068). Four datasets obtained from NCBI’s
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Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) with GEO Series
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accession numbers GSE1456, GSE2990 GSE4922 and GSE2304 were reanalyzed
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together with our raw data. Raw data were processed using Robust Multi-Array (RMA)
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analysis (Irizarry et al., 2003). Signal intensities were normalized, background corrected, and bottom-trimmed at signal intensity of 50. Genes were filtered out if their
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log intensity variation percentile was less than 25% and/or, if they were absent in more
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than 85% of the experiments.
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Hierarchical Clustering. Gene expression data from EBV-infected and GFP-infected
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cells or tumors were used for unsupervised hierarchical clustering with the average linkage method as implemented in the BRB Array Tools Version 3.6. All genes were median-centered across the experiments. Differential expression between the EBVinfected and GFP-labeled cells or tumors were analyzed by BRB-ArrayTools. Statistical significance was set at p ≤ 0.01 at a false discovery rate of 0.15; and at p ≤ 0.001 at a false discovery rate of 0.05. Pathway analysis was performed using the gene set comparison tool of BRB-ArrayTools. We analyzed all pre-defined Biocarta pathways (obtained through the NCI public database) for differential expression between the EBV infected and the GFP labeled cells. The statistical significance for differential expression of each pathway was estimated using the functional class scoring method (Pavlidis et
ACCEPTED MANUSCRIPT al., 2004). In brief, a p-value was computed for each gene in each pathway and then the set of p-values for each pathway was expressed as LS score (mean negative natural
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logarithm of the p-values of the respective single gene univariate test) and the
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Kolmogorov-Smirnov (KS) score (Pavlidis et al., 2004). For each pathway, we tested the
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null hypothesis that the list of differentially expressed genes from each pathway was
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random. N genes (equal to the number of genes of the pathway) were randomly selected from the project gene list, and the LS and KS statistics and their random
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distribution were computed (100,000 random selections). The LS (KS) permutation p-
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value was defined as the proportion of random simulations for which the LS (KS)
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statistic was larger than the LS (KS) statistic computed for the pathway with the original
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gene list. Statistical significance was set at 0.005. Generation of the EBVness signature. To generate the EBVness signature, our data set of EBV infected and GFP labeled cells or tumors was used as the training set. The training was performed with BRB-ArrayTools. The most differentiallly expressed genes between EBV-infected and GFP labeled samples (p
