Elucidating the role of DNA damage and human cytomegalovirus in medulloblastoma and glioblastoma

University dissertation from Stockholm : Karolinska Institutet, Dept of Medicine, Solna

Abstract: The most common primary malignant brain tumor in children is Medulloblastoma, while Glioblastoma is the most common in adults. Treatment for both include some combination of surgery, radiation therapy, and chemotherapy. The evolution of most primary malignant brain tumors is unknown, although varying degree of genomic instability caused by defects in the DNA Damage Response (DDR) is suspected. Lately, even human cytomegalovirus (HCMV) has emerged as a suspected pathogen possibly implicated in malignant tumor evolution. Nevertheless, the causes of the chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies (i.e. radiation and chemotherapy) remain largely unknown. Thus, the main aim of this PhD thesis is to investigate the role of HCMV in the context of DDR in human Medulloblastoma and Glioblastoma. In the 1st study, we turned our attention towards Glioblastoma (GBM). We examined the ability of HCMV to induce a more aggressive cancer stem cell (CSC)-like phenotype in primary GBM cell lines. HCMV infection induced a stem cell phenotype in primary GBM cell lines as determined by changes in the cellular gene expression profile and by the conferred ability of cells to grow as neurospheres in vitro, and this phenotype was prevented by treatment with the anti-viral drug ganciclovir. As CSCs are known to be resistant to chemotherapy, our results imply that HCMV may enhance the malignancy grade of the tumor, and possibly contribute to therapy resistance. In the 2nd study, we found pronounced endogenous DNA damage signaling and constitutive activation of DNA damage checkpoint kinase cascades across our medulloblastoma cohort. The bulk of the specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection. Our results strongly indicate that in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair mechanisms. In the 3rd study, we examined cancer stem cell markers (CD133, CD15, VEGFR2) and HCMV protein expression in human medulloblastoma specimens and medulloblastoma cell lines, at the same time considering also the replication stress and DNA damage response, as cancer stem cells are often more resistant to standard-of-care radiation and chemotherapy treatments. Our immunohistochemistry analysis on clinical material identified widespread expression of the VEGFR2 receptor and CD15, yet more limited expression of CD133 compared to GBM. In addition, assessments of expression of HCMV early and late proteins have been carried out in parallel, along with cell culture experiments with HCMV infection and replication stress responses in medulloblastoma cell lines. Remarkably, we found that unlike the ‘non-stem cell’ medulloblastoma cell lines, the cell line that showed robust stemness phenotype featured a very distinct response to DNA replication stress and HCMV infection, both emerging hallmarks of brain cancers. In the 4th study, we show that HCMV infection induced replication stress (RS) and triggered host DNA damage response (DDR) in permissive and non-permissive human cells. Further, we show that undergoing standard-of-care genotoxic radiochemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV markers after tumor recurrence. We propose a model to explain oncomodulatory effects of HCMV, through RS induction, DDR subversion, cell death inhibition and host-cell’s genome destabilization. Our findings provide fresh insights into HCMV pathobiology and inspiration for future strategies to combine radio-chemotherapy with anti-viral drugs for cancer treatment.

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