IE1 Sequence Alignment in Several Primary GBM Tissues (S1); HCMV IE is enriched in the SSEA1+ fraction of primary GBM cells and co-localizes with stemness markers in situ (S2); IE siRNA Optimization (S3); HCMV transcripts are enriched in the GSC fraction (S4); HCMV IE modulates Sox2 expression in GBM via miR145 (S5); IE1 overexpression induces Sox2 via miRNA145 inhibition in GSCs (S6); IE attenuation induces a mesenchymal and pro-inflammatory phenotype in HCMV-positive GSC (S7); Immunohistochemical analyses of IE1+/- mouse gliomas (S8); GSC lines used in this study are tumorigenic in vivo (S9); CR208, an IE1 deficient HCMV does not promote GBM stemness to the same extent as Towne parental strain (S10).
ARTICLE ABSTRACTGlioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate-early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with oncogenic properties. Here, we show how HCMV IEs are preferentially expressed in glioma stem–like cells (GSC), where they colocalize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSCs that are endogenously infected with HCMV, attenuating IE expression by an RNAi-based strategy was sufficient to inhibit tumorsphere formation, Sox2 expression, cell-cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSCs elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSCs, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and proinflammatory cytokines, resembling the therapeutically resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miR-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSCs infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared with mock-infected human GSCs. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in GBM cells. Cancer Res; 75(15); 3065–76. ©2015 AACR.