HCMV-induced epigenetic alterations : a novel pathway to CNS tumors?

Abstract

Aberrant DNA hypermethylation at CpG islands is a well-established phenomenon involved in transcriptional silencing of tumor suppressor genes. CpG island hypomethylation-mediated reactivation of oncogenes is also documented in several cancer types. DNA methylation and histone modifications catalyzed by DNA methyltransferases (DNMTs), histone deacetylases (HDACs) and histone acetyltransferases (HATs), respectively, result in chromatin structure changes and altered gene regulation. Two categories of drugs, affecting histone acetylation and DNA methylation, are currently considered in epigenetic therapy of cancer. There is some evidence that synergistic effects of HDACi and DNMTi are achieved by their action on common targets, including DNA methyltransferase 1 (DNMT1). During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer including glioblastoma, the most lethal primary brain tumor with poor survival, and medulloblastoma, the most common malignant pediatric brain tumor. HCMV infection in glioblastoma is associated with poor outcome and antiviral treatment, was shown to restrict tumor growth and longer survival in these patients. The aim of this thesis was to investigate events that may play a role in initiation and progression of cancer through epigenetic alteration with a special focus on HCMV infections. The main idea behind this approach is that, if epigenetic alterations and HCMV infection are highly prevalent in cancer, patients might benefit from effects caused by epigenetic and antiviral therapy.

In study I, changes in DNA methylation by the histone deacetylase inhibitor (HDACi), trichostatin A (TSA) was investigated in Hep3B cells. The effect of TSA on DNA methylation was studied at gene specific and global levels. Our results suggest that TSA causes genomic hypomethylation and leads to a decrease of nuclear DNMT1 protein levels by changing the DNA methyltransferases (DNMTs) mobility in the nucleus.

In study II, the impact of human cytomegalovirus infection on the host cell DNA methylation machinery was investigated. The results showed an alteration in the host cell DNA methylation machinery by the re-localization of DNMTs from the nucleus to cytoplasm. The data also suggested that DNA methylation influences cellular susceptibility to HCMV infection.

In study III, we explored the effect of HCMV infection on DNMT1 in medulloblastoma (MB) cells and tumor biopsies as well as human endothelial cells since blood vessels within MB tumors have shown to most often express HCMV proteins, and are important for tumorigenesis. The data demonstrated cytoplasmic localization of DNMT1 in HCMV infected MB and endothelial cells associated with expression of HCMV late gene UL55 (glycoprotein B, gB). Treatment of HCMV infected MB cells by the DNA methylation inhibitor, 5-azacytidine (5AZA), showed significant increased number of cells expressing viral protein. These data suggest that HCMV replication may benefit from inhibition of the host cell nuclear methylation machinery, a fact that may have consequences for using epigenetic drugs in cancer therapy.

In Study IV (Manuscript), the expression and localization of DNA methyltransferase 1 (DNMT1) and various HCMV proteins were assessed in glioblastoma (GBM) cells and patient tissue specimens. Moreover, the effect by 5AZA was analysed in the context of viral replication, proliferation and invasion ability of HCMV infected GBM cells. Our data show that DNMT1 is localized to the extra-nuclear space of GBM cells expressing HCMV-gB proteins and in the cells of blood vessel wall within GBM tumors. In tissue specimens, DNMT1 was shown to be expressed in the nucleus of tumor cells, but localized to the extra-nuclear /cytoplasmic space of cells lining blood vessel walls within the GBM tumors. 5AZA treatment of GBM cells lead to reduced proliferation of uninfected and HCMV infected cells and HCMV infected cells were shown to be vulnerable to 5AZA treatment, leading to a decreased invasion.