To reveal transcriptional changes that underlie glioblastoma, we performed an in-depth analysis of gene expression in malignant stem cells derived from patient tumors in relation to untransformed, karyotypically normal NS cells. These cell types are closely related and it has been hypothesized that gliomas arise by mutations in NS cells or in glial cells that have reacquired stem cell features . We measured gene expression by high-throughput RNA tag sequencing (Tag-seq), a method that features high sensitivity and reproducibility compared to microarrays . qRT-PCR validation further demonstrates that Tag-seq expression values are highly accurate. Other cancer samples and cell lines have recently been profiled with the same method [8, 47] and it should be feasible to directly compare those results to the data presented here.
Through Tag-seq expression profiling of normal and cancer stem cells followed by qRT-PCR validation in a wider panel of 22 cell lines, we identified 29 genes strongly discriminating GNS from NS cells. Some of these genes have previously been implicated in glioma, including four with a role in adhesion and/or migration, CD9, ST6GALNAC5, SYNM and TES [49–52], and two transcriptional regulators, FOXG1 and CEBPB. FOXG1, which has been proposed to act as an oncogene in glioblastoma by suppressing growth-inhibitory effects of transforming growth factor β , showed remarkably strong expression in all 16 GNS cell lines assayed by qRT-PCR. CEBPB was recently identified as a master regulator of a mesenchymal gene expression signature associated with poor prognosis in glioblastoma . Studies in hepatoma and pheochromocytoma cell lines have shown that the transcription factor encoded by CEBPB (C/EBPβ) promotes expression of DDIT3 , another transcriptional regulator that we found to be upregulated in GNS cells. DDIT3 encodes the protein CHOP, which in turn can inhibit C/EBPβ by dimerizing with it and acting as a dominant negative . This interplay between CEBPB and DDIT3 may be relevant for glioma therapy development, as DDIT3 induction in response to a range of compounds sensitizes glioma cells to apoptosis (see, for example, ).
Our results also corroborate a role in glioma for several other genes with limited prior links to the disease. This list includes PLA2G4A, HMGA2, TAGLN and TUSC3, all of which have been implicated in other neoplasias (Additional file 12). PLA2G4A encodes a phospholipase that functions in the production of lipid signaling molecules with mitogenic and pro-inflammatory effects. In a subcutaneous xenograft model of glioblastoma, expression of PLA2G4A by the host mice was required for tumor growth . For HMGA2, a transcriptional regulator downregulated in most GNS cell lines, low or absent protein expression has been observed in glioblastoma compared to low-grade gliomas , and HMGA2 polymorphisms have been associated with survival time in glioblastoma . The set of 29 genes found to generally distinguish GNS from NS cells also includes multiple genes implicated in other neoplasias, but without direct links to glioma (Additional file 12). Of these, the transcriptional regulator LMO4, may be of particular interest, as it is well studied as an oncogene in breast cancer and regulated through the phosphoinositide 3-kinase pathway , which is commonly affected in glioblastoma .
Five of these 29 genes have not been directly implicated in cancer. This list comprises one gene downregulated in GNS cells (PLCH1) and four upregulated (ADD2, LYST, PDE1C and PRSS12). PLCH1 is involved in phosphoinositol signaling , like the frequently mutated phosphoinositide 3-kinase complex . ADD2 encodes a cytoskeletal protein that interacts with FYN, a tyrosine kinase promoting cancer cell migration [61, 62]. For PDE1C, a cyclic nucleotide phosphodiesterase gene, we found higher expression to correlate with shorter survival after surgery. Upregulation of PDE1C has been associated with proliferation in other cell types through hydrolysis of cAMP and cGMP [63, 64]. PRSS12 encodes a protease that can activate tissue plasminogen activator (tPA) , an enzyme that is highly expressed by glioma cells and has been suggested to promote invasion .
By considering expression changes in a pathway context, we identified additional candidate glioblastoma genes, such as the putative cell adhesion gene ITGBL1 , the orphan nuclear receptor NR0B1, which is strongly upregulated in G179 and is known to be upregulated and mediate tumor growth in Ewing's sarcoma , and the genes PARP3 and PARP12, which belong to the poly(ADP-ribose) polymerase (PARP) family of ADP-ribosyl transferase genes involved in DNA repair (Table 4). The upregulation of these PARP genes in GNS cells may have therapeutic relevance, as inhibitors of their homolog PARP1 are in clinical trials for brain tumors .
Transcriptome analysis thus identified multiple genes of known significance in glioma pathology as well as several novel candidate genes and pathways. These results are further corroborated by survival analysis, which revealed a GNS expression signature associated with patient survival time in five independent data sets. This finding is compatible with the notion that gliomas contain a GNS component of relevance for prognosis. Five individual GNS signature genes were significantly associated with survival of glioblastoma patients in both of the two largest data sets: PLS3, HOXD10, TUSC3, PDE1C and the well-studied tumor suppressor PTEN. PLS3 (T-plastin) regulates actin organization and its overexpression in the CV-1 cell line resulted in partial loss of adherence . Elevated PLS3 expression in GNS cells may thus be relevant for the invasive phenotype. The association between transcriptional upregulation of HOXD10 and poor survival is surprising, because HOXD10 protein levels are suppressed by a microRNA (miR-10b) highly expressed in gliomas, and it has been suggested that HOXD10 suppression by miR-10b promotes invasion . Notably, the HOXD10 mRNA upregulation we observe in GNS cells also occurs in glioblastoma tumors, as shown by comparison with grade III astrocytoma (Figure 3b). Similarly, miR-10b is present at higher levels in glioblastoma compared to gliomas of lower grade . It is conceivable that HOXD10 transcriptional upregulation and post-transcriptional suppression is indicative of a regulatory program associated with poor prognosis in glioma.
Tumors from older patients featured an expression pattern more similar to the GNS signature. One of the genes contributing to this trend, TUSC3, is known to be silenced by promoter methylation in glioblastoma, particularly in patients aged over 40 years . Loss or downregulation of TUSC3 has been found in other cancers, such as of the colon, where its promoter becomes increasingly methylated with age in the healthy mucosa . Taken together, these data suggest that transcriptional changes in healthy aging tissue, such as TUSC3 silencing, may contribute to the more severe form of glioma in older patients. Thus, the molecular mechanisms underlying the expression changes described here are likely to be complex and varied. To capture these effects and elucidate their causes, transcriptome analysis of cancer samples will benefit from integration of diverse genomic data, including structural and nucleotide-level genetic alterations, as well as DNA methylation and other chromatin modifications.
To identify expression alterations common to most glioblastoma cases, other studies have profiled tumor resections in relation to non-neoplastic brain tissue [47, 74, 75]. While such comparisons have been revealing, their power is constrained by discrepancies between reference and tumor samples - for instance, the higher neuronal content of normal brain tissue compared to tumors. Gene expression profiling of tumor tissue further suffers from mixed signal due to a stromal cell component and heterogeneous populations of cancer cells, only some of which contribute to tumor progression and maintenance . Part of a recent study bearing a closer relationship to our analysis examined gene expression in another panel of glioma-derived and normal NS cells , but included neurosphere cultures, which often contain a heterogeneous mixture of self-renewing and differentiating cells.
Here, we have circumvented these issues by profiling uniform cultures of primary malignant stem cell lines that can reconstitute the tumor in vivo , in direct comparison to normal counterparts of the same fundamental cell type [4, 5]. While the resulting expression patterns largely agree with those obtained from glioblastoma tissues, there are notable differences. For example, we found the breast cancer oncogene LMO4 (discussed above) to be upregulated in most GNS cell lines, although its average expression in glioblastoma tumors is low relative to normal brain tissue (Figure 3a). Similarly, TAGLN and TES were absent or low in most GNS cell lines, but displayed the opposite trend in glioblastoma tissue compared to normal brain (Figure 3c) or grade III astrocytoma (Figure 3d). Importantly, both TAGLN and TES have been characterized as tumor suppressors in malignancies outside the brain and the latter is often silenced by promoter hypermethylation in glioblastoma [77, 78].