The transcriptome of human mammary epithelial cells infected with the HCMV-DB strain displays oncogenic traits

www.nature.com/scientificreports OPEN Received: 3 April 2018 Accepted: 21 July 2018 Published: xx xx xxxx The transcriptome of human mammary epithelial cells infected with the HCMV-DB strain displays oncogenic traits Fatima Al Moussawi1,2, Amit Kumar 1, Sébastien Pasquereau1, Manoj K. Tripathy Walid Karam2, Mona Diab-Assaf2 & Georges Herbein1,3 1 , Increasing evidence indicates that human cytomegalovirus (HCMV) populations under the influence of host environment, can either be stable or rapidly differentiating, leading to tissue compartment colonization. We isolated previously from a 30-years old pregnant woman, a clinical isolate of HCMV, that we refered to as the HCMV-DB strain (accession number KT959235). The HCMV-DB clinical isolate demonstrated its ability to infect primary macrophages and to upregulate the proto-oncogene Bcl-3. We observed in this study that the genome of HCMV-DB strain is close to the genomes of other primary clinical isolates including the Toledo and the JP strains with the later having been isolated from a glandular tissue, the prostate. Using a phylogenetic analysis to compare the genes involved in virus entry, we observed that the HCMV-DB strain is close to the HCMV strain Merlin, the prototype HCMV strain. HCMV-DB infects human mammary epithelial cells (HMECs) which in turn display a ER−/ PR−/HER2− phenotype, commonly refered to as triple negative. The transcriptome of HCMV-DBinfected HMECs presents the characteristics of a pro-oncogenic cellular environment with upregulated expression of numerous oncogenes, enhanced activation of pro-survival genes, and upregulated markers of cell proliferation, stemcellness and epithelial mesenchymal transition (EMT) that was confirmed by enhanced cellular proliferation and tumorsphere formation in vitro. Taken together our data indicate that some clinical isolates could be well adapted to the mammary tissue environment, as it is the case for the HCMV-DB strain. This could influence the viral fitness, ultimately leading to breast cancer development. Breast cancer, the most common cancer diagnosed among women, exhibits heterogeneous molecular characteristics. Several types of breast cancer have been identified based on the differential gene expression patterns. These groups include among others the normal breast epithelial-like, the luminal epithelial type A and type B, the basal-like and the claudin low groups1. Genetic risk factors and environmental risk factors are some of the etiologic factors involved in breast cancer2. Of all worldwide cancers close to one-fifth could involve infectious agents including viruses, as part of the environmental risk factors3–5. The Betaherpesviridae human cytomegalovirus (HCMV) is know to cause, in the immunocompetent host, an infection that usually range from asymptomatic to mild. Serious complications can however be the result of the infection of immunocompromised hosts6. In opposition to laboratory strains of HCMV, which growth appears to be restricted to fibroblasts only, clinical isolates are able to infect and grow in several types of cells including epithelial cells, endothelial cells, monocytes, macrophages, fibroblasts, stromal cells, hepatocytes, smooth muscle cells, and neural stem/progenitor cells7–10. Recently, a detailed in vivo evolutionary map of HCMV was built by combining population genetics methods and high throughput sequencing. This map provided evidence that viral populations under the influence of host environment can either be stable or rapidly differentiating, leading to tissue compartment colonization11. 1 Department Pathogens & Inflammation-EPILAB, UPRES EA4266, University of Franche-Comté, University of Bourgogne Franche-Comté, F-25030, Besançon, France. 2Université Libanaise, Beyrouth, Lebanon. 3Department of Virology, CHRU Besancon, F-25030, Besançon, France. Fatima Al Moussawi and Amit Kumar contributed equally. Correspondence and requests for materials should be addressed to G.H. (email: ) Scientific RePorTs | (2018) 8:12574 | DOI:10.1038/s41598-018-30109-1 1 www.nature.com/scientificreports/ Figure 1. Comparison of the genomic sequence of HCMV-DB with the genomic sequences of other clinical and laboratory adapted HCMV strains. The upper panel shows the genomic sequence of HCMV-DB as compared to two laboratory adapted strains (AD169 and Towne) and eight low passages clinical isolates (Merlin, Toledo, TR, PH, VR1814, Davis, JP, and TB40/E). The lower panel shows the alignment tree related to the whole viral genome with comparison between HCMV-DB genome and the genome of ten other HCMV strains. Several research groups focusing on inflammatory diseases and on cancer addressed the role played by HCMV in these diseases12–14. Tumor tissues from several cancers, including brain, colon, prostate, liver and breast cancer, have been found positive for HCMV DNA or antigens15–20. In the paradigm of oncomodulation, oncogenesis mechanisms could be amplified by HCMV acting as a cofactor, when infecting the tumor tissue21. In addition, monocytes and macrophages, respectively located in the blood and in the tissues, may act as sites for the establishment of latency, given their role as HCMV cellular reservoirs which are responsible for the dissemination of the virus8,22–24. Finally, in breast carcinomas and glioblastomas, tumor-associated macrophages (TAM) are a marker of poor prognosis, and their development might be influenced by macrophage-tropic HCMV strains25–28. Thus the quest of new HCMV isolates which target monocytes/macrophages and thereby might play a role in oncogenesis has shown increased interest. In the present study, we found that the genomic sequence of the HCMV-DB strain isolated from a cervical swab specimen is close to the sequence of other primary clinical isolates, especially the Toledo strain, originally isolated in the urine of a child presenting a congenital infection by HCMV, and the JP strain, isolated from a glandular tissue, the prostate21,29. Based on the analysis of genes involved in virus entry, our results indicate that the HCMV-DB strain is close to the Merlin strain. Given the scarcity of evidence for a direct role of HCMV in the cellular transformation of epithelial cells, we studied the transcriptome profile of HCMV-DB infected human mammary epithelial cells (HMECs). We observed that the transcriptome of HCMV-DB infected HMECs displays a triple negative ER−/PGR−/HER2− phenotype, presents some oncogenic traits, favors cell cycling and cell proliferation, and modulate angiogenesis and proteolysis. All these phenomena are potentially involved in tumor development. Finally, the infection of HMECs with the HCMV strain DB resulted in enhanced proliferation and tumorsphere formation in vitro. Results Genomic profile of the HCMV-DB strain. We previously isolated a novel HCMV strain, that we characterized and named HCMV-DB8. We compared its genomic sequence to that of ten HCMV strains, including two laboratory a (...truncated)