Human cytomegalovirus : from novel strain, miRNAs to interplay with breast cancer

The prevalence of human cytomegalovirus (HCMV) ranges from 40% to 100% worldwide. A primary HCMV infection results in a lifelong latent or persistent infection. In healthy people, HCMV infection is often asymptomatic, but in immunosuppressed individuals it can cause life- threatening disease, and HCMV congenital infection may result in fetal malformation. Increasing evidence implies that HCMV infection is associated with different malignancies. Several studies have suggested different mechanisms by which HCMV can modulate the tumour and the tumour environment, which could be of relevance in tumour development and progression. However, the role of HCMV in cancer has remained highly controversial. The studies in this thesis investigated the possible role of HCMV in cancer, with a special focus on HCMV encoded-miRNAs. HCMV encodes at least 26 miRNAs, and are expressed during the lytic as well as latent phase of infection. Viral miRNAs are identified to target both host and viral mRNA transcripts and thereby regulate protein expression. They modulate various biological functions of the host cell and the virus life cycle by mediating latency, viral replication, cell cycle control, vesicle trafficking, virus assembly and immune evasion strategies.

In study I (Manuscript), we discovered a novel genetic variant of HCMV lacking intron 2 of the major immediate early (MIE) gene in 48% tumour samples obtained from breast, colon, neuroblastoma, medulloblastoma and glioblastoma patients. In contrast, this variant was less frequently detected in healthy donors, and in patients with HCMV viremia or myocardial infarction. We found, three clinical isolate that contained both the wild type HCMV and the variant HCMV strains; the variant was successfully plaque purified from endothelial cells (HUVEC). Electron microscopy analysis found dense body like particles but no HCMV particles in variant positive HUVEC cells. The variant expressed cytoplasmic IE proteins, which were of multiple sizes. Our results demonstrate a high prevalence of a novel genetic variant of HCMV in cancer patients, which may represent a new virus strain with potential oncogenic properties that warrants further investigation.

In study II (Published in PlosONE 2014), the HCMV microRNA miR-UL112-3p suggested to mediate the latency and immune evasion strategies, also highly expressed in hypertension patients and associated with increased risk of hypertension. In our study, we detected miR- UL112-3p in plasma/serum of 52% (14/27) of Diabetic Mellitus, 25% (5/20) of Glioblastoma patients, in 5% (1/20) of Rheumatoid Arthritis patients and in 10% (2/20) of Healthy Controls. Anti-HCMV IgG was detected in 85%, 65%, 75% of patients and 70% of healthy controls, respectively. Anti-HCMV IgM was found only in one Glioblastoma patient of the 87 examined patients and controls. We didn’t found significant association between serology and miR- Ul112-3p.

In study III (Accepted for publication), HCMV infected cells produce mature virions and defective particles called dense bodies (DBs). In this study, we purified these particles, and found that virions and DBs incorporate viral as well as host encoded RNAs and miRNAs within the particles. Furthermore, we demonstrated that the particle associated miRNAs can be delivered to host cells, are biologically functional, which may affect cellular processes.

In study IV (Published in PLOS One 2013), we found that HCMV proteins IE and LA were abundantly expressed in all breast cancer tissue specimens examined (n=73) and 94% of their paired sentinel lymph node specimens (n=32/34) with metastases. Moreover, we also found HCMV IE and LA proteins in 60% (20/35) sentinel lymph node specimens without metastases. HCMV infections were mostly confined to the neoplastic cells, while some inflammatory cells were also HCMV positive in 79% of lymph nodes with metastases and 60% in metastatic free lymph nodes. We didn’t had enough sample to perform survival analysis, as only six patients died of breast cancer in this cohort. These six patients had high grade HCMV infection. High grade HCMV IE staining are more prevalent, due to the less patients in each group no further statistical correlation analyses with clinical prognostic factors (ER-α, PR and Elston grade) are reported. We conclude that, higher prevalence of HCMV proteins found in lymph nodes with metastasis than lymph nodes with metastasis, and bigger cohort is required to perform the correlation analysis.

Another project by Dr. Afsar Rahbar in our lab, using more number of patients found the high expression of HCMV IE proteins in breast cancer tumours tissues inversely correlation with ER, PR and HER2 levels (manuscript in revision).

In study V (Manuscript) we further studied this phenomenon in vitro; HCMV infection resulted in significant downregulation of mRNA and protein levels of ER-α, PR and Her-2 in breast cancer cell lines. This effect was dependent on viral gene expression. We found potential viral miRNAs which can targets of ER-α (miR-UL22A-3p, miRUL36-3p and miR-US25-2- 3p) and PR (miR-UL22A-3p). Overexpression of each viral miRNAs in a breast cancer cell line resulted in downregulation of mRNA and protein levels of ER-α and PR. Thus, HCMV may promote the establishment of triple negative breast cancer, through viral miRNA induced downregulation of ER-α, PR (and Her-2 by a yet unidentified mechanism). Antiviral therapy could hence be a potential alternative treatment strategy for selected breast cancer patients.

List of scientific papers

I. Wilhelmi V, Chato Taher, Mohammad AA, Assinger A, Khan Z, Pettersson P, Odeberg J, Dzabic M, Xu X, Stragliotto G, Hartman J, Frisell J, Martling A, Varani S, Örvell C, Siesjö P, Kogner P, Peredo I, Landazuri N, Rahbar A, Yaiw KC and Söderberg- Nauclér1 C. Isolation and characterization of novel genetic variant of CMV highly associated with tumors of different origin. [Manuscript]

II. Mohammad AA, Rahbar A, Lui WO, Davoudi B, Catrina A, Stragliotto G, Mellbin L, Hamsten A, Ryden L, Yaiw KC, Soderberg-Naucler C. Detection of circulating hcmv- miR-UL112-3p in patients with glioblastoma, rheumatoid arthritis, diabetes mellitus and healthy controls. PloS one. 2014;9(12):e113740.
https://doi.org/10.1371/journal.pone.0113740

III. Mohammad AA, Costa H, Landázuri N, Lui WO, Hultenby K, Rahbar A, Yaiw KC, and Söderberg-Nauclér C. Human Cytomegalovirus MicroRNAs are carried by Virions and Dense Bodies and are delivered to Target Cells. J Gen Virol. [Accepted]
https://doi.org/10.1099/jgv.0.000736

IV. Taher C, de Boniface J, Mohammad AA, Religa P, Hartman J, Yaiw KC, Frisell J, Rahbar A, Soderberg-Naucler C. High prevalence of human cytomegalovirus proteins and nucleic acids in primary breast cancer and metastatic sentinel lymph nodes. PloS one. 2013;8(2):e56795.
https://doi.org/10.1371/journal.pone.0056795

V. Costa H, Mohammad AA, Lui WO, Joel T, Davoudi B, Bukholm IR, Sauer T, Vetvik K, Jürgen G, Rahbar A, Söderberg-Nauclér C. A potential role for human cytomegalovirus in triple negative breast cancer through its specific mechanisms to downregulate ER-α, PR and HER-2. [Manuscript]