Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated
in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.GBM is the most prevalent and the most aggressive primary malignancy of the central nervous system in adults. It is a highly vascularized and infiltrating tumor, rarely cured and prone to recurrence. The median duration of survival after diagnosis is less than 15 months, despite aggressive therapy consisting of surgical resection and concomitant radiotherapy and chemotherapy.
1 Surgical resection of GBMs is typically incomplete, as they are located in the brain and are highly infiltrative. Postoperative radiotherapy and chemotherapy fail to eradicate all remaining GBM cells. Thus, a breakthrough in identifying a new treatment option leading to a cure of this disease is still lacking.GBMs contain a subpopulation of highly tumorigenic cells with unlimited capacity for self-renewal that are commonly resistant to standard therapy. Phenotypically and functionally, these cells resemble neural stem cells and, when implanted in immunodeficient mice, can generate new tumors. As a result, they are referred to as glioma cancer initiating cells or glioma cancer stem cells (GCSCs) (reviewed in Lima
et al.2). Because of their apparent pivotal role in gliomagenesis and tumor recurrence after therapy, GCSCs are a major focus of research whose ultimate goal is to identify more effective therapies for GBM patients.GCSCs were first identified by their surface expression of CD133, based on the findings that these cells grow as neurospheres under nonadherent conditions and that tumors form
in vivo after implantation of only 100 CD133-positive GBM cells but not after implantation of 10
5 CD133-negative GBM cells.
3 The importance of CD133 as a marker of tumor aggressiveness was corroborated by the correlation between CD133 expression in brain tumors and a poor clinical prognosis.
4, 5, 6 However, later studies revealed that CD133-negative cells can give rise to CD133-positive cells
7, 8, 9 and that both CD133-positive and CD133-negative GBM cells can initiate the development of highly aggressive tumors.
10 Moreover, diverse GCSC types – all capable of self-renewal and tumor initiation – coexist within the same GBM.
10 These cells often express markers associated with stem cells such as Sox2, Notch, and Oct-4.
11, 12, 13 This intratumoral heterogeneity and the resulting aggressiveness of GBMs are influenced by the location of the tumor within the brain and by tumor-associated microenvironmental factors (reviewed in Stopschinski
et al.14). While the general validity of CD133 as a major GCSC marker is still debated and its exact function in gliomagenesis remains poorly understood, other GCSC markers have been identified, including Notch1, Oct4, Sox2, and Nestin.
4, 14, 15 The high levels of expression of these markers appear to functionally induce or maintain features that are characteristic of GCSCs.We have focused on developing and testing novel treatments for GBM based on two observations: that 99% of GBMs contain human cytomegalovirus (HCMV) proteins
16, 17 and nucleotide sequences, and that the degree of HCMV protein expression in GBMs is a prognostic factor for patient survival.
18HCMV is a herpesvirus that infects 70–100% of the world''s population. After an active primary infection, usually asymptomatic or subclinical in immunocompetent individuals, the virus establishes latency in the bone marrow and peripheral blood. Latent infections can be reactivated by inflammation. In immunocompromised individuals, primary HCMV infection and reactivation are significantly associated with morbidity and mortality.
19, 20In a clinical trial, we found that the antiviral drug valganciclovir as an add-on to standard therapy led to high survival rates among GBM patients. In a retrospective analysis of patients continuously receiving such therapy for more than 6 months, the 2-year survival rate was 90% and median overall survival was 56.4 months, as compared with 18% and 13.5 months, respectively, in contemporary controls.
17 These results suggest that HCMV has an oncogenic or an oncomodulatory role in GBMs, and highlight the possibility that valganciclovir may eliminate or modulate the behavior of GCSCs that may not be targeted with conventional therapies.In light of these findings, we hypothesized that HCMV infection of GBM cells and the maintenance of a GCSC phenotype could be interrelated events. To test this hypothesis, here we investigated potential co-expression of a GCSC marker with HCMV immediate-early protein in a series of human clinical GBM specimens, and experimentally assessed the ability of HCMV infection to induce a GCSC phenotype in primary human GBM cells.
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