Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells

Abstract

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

Human Cytomegalovirus Seropositivity and Viral DNA in Breast Tumors Are Associated with Poor Patient Prognosis

“…Viruses with causative effects in cancer include the Epstein–Barr virus leading to Burkitt’s lymphomas [ 8 , 9 ] and human papillomavirus leading to cervical cancer [ 10 , 11 ]. Some recent studies also show that human cytomegalovirus (HCMV) has oncogenic potential where it directly leads to oncogenesis [ 12 , 13 , 14 ]. Indeed, the virus is found in cancerous tissues, including colorectal [ 15 ], prostate [ 16 ], ovarian [ 17 ] and glioblastoma cancers [ 18 ].…”

“…Increasing evidence also shows that HCMV infection plays a role in breast cancer [ 19 , 20 , 21 ]. This may be oncogenic [ 12 , 13 , 14 ] or oncomodulatory where the infection contributes to cancer progression through the alteration of intracellular signaling pathways [ 22 , 23 ].…”

Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus

“…Basal-like biopsies accounted for the highest percentage of biopsies with high Ki67 mRNA expression level (% of basal-like: 44%) compared to healthy (2%) and luminal biopsies (20%) ( p-value (High Ki67 - Basal : Healthy) = 0.02) ( Figure 7A ). To further study the role of HCMV associated with Ki67 transcript expression, we assessed the presence of HCMV in the biopsies by quantifying IE1 and UL69 genes using qPCR ( 21 , 30 ). In HCMV-positive biopsies, basal-like biopsies showed the highest percentage of biopsies with high levels of Ki67 mRNA (40%) in contrast to the healthy (0%) and luminal (0%) biopsies ( Figure 7B ).…”

“…The study was authorized by the local ethics committees of Besançon University Hospital (Besançon, France) and the French Research Ministry (AC-2015-2496, CNIL n°1173545, NF-S-138 96900 n°F2015). The presence of HCMV, within all the biopsies, was assessed by qPCR using IE1 and UL69 primers as mentioned above and described previously ( 30 ). For Ki67 and GAPDH mRNA quantification, RNA was extracted from the biopsies using E.Z.N.A.…”

Immune Landscape of CMV Infection in Cancer Patients: From “Canonical” Diseases Toward Virus-Elicited Oncomodulation

“…Recent data showed that HCMV could perhaps induce transformation by enhancing the expression of viral genes (for example, UL69 gene). The presence of UL69 gene in CTH cell cultures and UL69 DNA in the majority of breast cancer biopsies indicates a potential significance of UL69 as a target in developing HCMV-vaccine ( 191 ). The usage of HCMV vaccines for the treatment of cancer patients generally and GBM patients in specific might be of high therapeutic value especially that HCMV has been shown to express oncomodulatory functions.…”