The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells

Abstract

BackgroundHuman 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.

Factors in Oncogenesis: Viral Infections in Ovarian Cancer

“…The onset of carcinogenesis begins with the CMV proteins IE1, IE2, pp71, and pUL97 phosphorylating pRB, inhibiting the cell cycle arrest functions of p53 and interfering with the cyclins, Wnt, PI3K/Akt, NF-κB, and STAT3 [153][154][155]. CMV proteins inhibit apoptosis and cell cycle arrest and promotes cell proliferation, angiogenesis, inflammation, and immune evasion [156][157][158][159][160][161]. CMV induces the expression of COX-2, NF-κB, and STAT-3, and the production of proinflammatory cytokines, prostaglandins, and leukotriens [156].…”

“…The interaction of CMV US28 with NF-κB elevates COX2, VEGF, and the migration of macrophages for promoting angiogenesis [158]. CMV strains have been shown to be capable of initiating oncogenesis in human breast epithelial cells by dysregulating p53, pRB, and telomerase [160]. CMV can activate epithelial cells, promote EMT and reverse the process (i.e., mesenchymal to epithelial transition) in tumor cells [160,161].…”

“…CMV strains have been shown to be capable of initiating oncogenesis in human breast epithelial cells by dysregulating p53, pRB, and telomerase [160]. CMV can activate epithelial cells, promote EMT and reverse the process (i.e., mesenchymal to epithelial transition) in tumor cells [160,161]. These CMV-promoted processes may potentially enhance metastatic tumor dissemination through the blood and lymphatic vessels and tumor growth [161].…”

“…CMV may function as an oncomodulator to drive carcinogenesis. It seems to regulate the expression of proteins participating in cell proliferation, cell cycle arrest, cellular metabolism, apoptosis, angiogenesis, inflammation, immune suppression, and EMT plasticity[153][154][155][156][157][158][159][160][161]. It also codes for proteins that function as homologs for human proteins (such as chemokine receptors, Fc receptors, inhibitor of apoptosis).…”

Vaccine Vectors Harnessing the Power of Cytomegaloviruses

“…HCMV infection is associated with some conditions that promote cell transformation, because several HCMV proteins disrupt the cell cycle upon infection [54] or activate the telomerase gene expression [55]. HCMV mediated mutagenesis [56,57], inactivation of tumour suppressors [58], immunoevasion of infected…”