The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells.
BACKGROUND
Human cytomegalovirus (HCMV) establishes a persistent life-long infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission in humans and we hypothesized that mammary epithelial cells could be one of the main cellular targets of HCMV infection.


METHODS
The infectivity of primary human mammary epithelial cells (HMECs) was assessed following infection with the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infection on expression of p53 and retinoblastoma proteins, telomerase activity and oncogenic pathways (c-Myc, Akt, Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agar assay. CTH cells (CMV Transformed HMECs) were detected in prolonged cultures of infected HMECs. Tumor formation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non coding RNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsies of patients with breast cancer using qualitative and quantitative PCR.


RESULTS
We found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs in vitro. The clinical strain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcripts and proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastoma and p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras, the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted in the development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cells when injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTH cells the presence of a HCMV signature corresponding to a sequence of the long noncoding RNA4.9 (lncRNA4.9) gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSG mice injected with CTH cells and in biopsies of patients with breast cancer using qualitative and quantitative PCR.


CONCLUSIONS
Our data indicate that key molecular pathways involved in oncogenesis are activated in HCMV-DB-infected HMECs that ultimately results in the transformation of HMECs in vitro with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 genomic sequence and give rise to fast growing triple-negative tumors in NSG mice. A similar lncRNA4.9 genomic sequence was detected in tumor biopsies of patients with breast cancer.
['Kumar|Amit|A|', 'Tripathy|Manoj Kumar|MK|', 'Pasquereau|Sébastien|S|', 'Al Moussawi|Fatima|F|', 'Abbas|Wasim|W|', 'Coquard|Laurie|L|', 'Khan|Kashif Aziz|KA|', 'Russo|Laetitia|L|', 'Algros|Marie-Paule|MP|', 'Valmary-Degano|Séverine|S|', 'Adotevi|Olivier|O|', 'Morot-Bizot|Stéphanie|S|', 'Herbein|Georges|G|']
D000818:Animals D001940:Breast D063646:Carcinogenesis D002449:Cell Aggregation D049109:Cell Proliferation D002471:Cell Transformation, Neoplastic D002478:Cells, Cultured D003114:Colony-Forming Units Assay D019938:Cyclin D1 D003587:Cytomegalovirus D003586:Cytomegalovirus Infections D004847:Epithelial Cells D005260:Female D006801:Humans D016688:Mice, Inbred NOD D016513:Mice, SCID D010766:Phosphorylation D010802:Phylogeny D051057:Proto-Oncogene Proteins c-akt D062085:RNA, Long Noncoding D012333:RNA, Messenger D050796:STAT3 Transcription Factor D018874:Spheroids, Cellular D019098:Telomerase D016159:Tumor Suppressor Protein p53 D015854:Up-Regulation D014764:Viral Proteins D014779:Virus Replication
Cytomegalovirus HCMV Oncogenesis HMECs HCMV-DB Transformation CTH cells lncRNA4.9