Many under- and several overexpressed genes are observed within the region of the 1p/19q co-deletion
We considered all oligodendrogliomas with 1p/19q co-deletion and compared their gene expression profiles to normal brain references to identify differentially expressed genes. We observed 3,068 (23.8%) under- and 3204 (24.9% of genes) overexpressed genes in oligodendrogliomas (q-value < 0.05, Additional file 4: Table S3). Only few strongly underexpressed tumor suppressors (log-ratio <−2: ANO3 and CDH1), but several strongly overexpressed oncogenes (log-ratio > 2: MYC, EGFR, PDGFRA, PIK3CA, PRRX1, ASCC3, ZNF117, CRISPLD1, CSMD3, ALDH1L2, MDGA2, TSHR and H3F3A) were among these genes.
Considering chromosomal locations, we found that 524 underexpressed genes (45.2% of genes on 1p/19q) and interestingly also 130 overexpressed genes (11.2% of genes on 1p/19q) were located within the region of the 1p/19q co-deletion. We observed strong underexpression for 74 of the 524 underexpressed genes on 1p/19q (log-ratio <−2). The ten most strongly underexpressed genes on 1p/19q were LC17A7, PRKCG, RIMS3, KIAA1324, AK5, SLC6A17, CD22, HPCA, MAG and CHD5. We also observed strong overexpression for 10 of the 130 overexpressed genes on 1p/19q (log-ratio > 2: SAMD11, SLC35E2, HES5, GRHL3, RCC1, SPOCD1, HFM1, DLL3, IL4I1 and CACNG6). Interestingly, DLL3 and HES5 are part of the Notch signaling pathway involved in oligodendrocyte specification [56] restricting cell proliferation and tumor growth in glioma mouse models [22].
We further analyzed all differentially expressed genes in the context of known cancer-relevant signaling pathways (Fig. 2). We observed that especially the Notch and Hedgehog signaling were strongly enriched for overexpressed genes, whereas MAPK signaling was enriched for underexpressed genes (Fig. 2a). In addition, also ErbB signaling and the Adherens junction pathway tended to show an enrichment of underexpressed genes. Considering metabolic pathways, we found that the oxidative phosphorylation pathway was enriched for underexpressed genes (Fig. 2b). Also the pyrimidine, purine and pentose phosphate pathway tended to show some enrichment of differentially expressed genes.
Transcriptional regulatory networks predict tumor gene expression levels
To provide the basis for the impact quantification of gene copy number mutations on cancer-relevant pathways, we used regNet [64] to learn genome-wide transcriptional regulatory networks based on gene copy number and expression data of 178 histologically classified oligodendrogliomas with and without 1p/19q co-deletion. We repeated the genome-wide network inference ten times utilizing different training and test data sets (see “Materials and methods” section for details). The resulting networks had on average 67,900 ± 1080 directed links between regulators and target genes (Additional file 3: Figure S3). More than three quarters of these links were activator links (78%) and the others were inhibitor links.
Next, we integrated the outgoing links of each gene across the ten networks to derive a connectivity score that accounts for the co-occurrence of links (see “Materials and methods” for details). This score is higher for genes with more stable outgoing links across n of networks than for genes with less co-occurring links. We utilized these scores and found that tumor suppressor genes, oncogenes, essential genes and signaling pathway genes had significantly greater connectivity scores than genes that were not included in these categories (Wilcoxon rank sum tests: P=0.035 for tumor suppressors, P=0.028 for oncogenes, P=5.39·10−9 for essential genes, P=0.01 for signaling pathway genes). The ten genes with the greatest connectivity score were (GARS, CCDC85B, NDUFA1, SPRED2, BIRC6, MRPL45, EDA2R, HMGCS1, SLC17A7, RAB40B; Additional file 3: Figure S4). CCDC85B is a known downstream target of p53 signaling with reported function as tumor suppressor [26]. Also SPRED2 is a known tumor suppressor that induces autophagy [32]. SLC17A7 has been observed as a tumor suppressor in a glioblastoma stem cell line [44]. BIRC6 can inhibit apoptosis in glioblastoma cell lines [11]. RAB40B is a member of the RAS oncogene family potentially involved in the remodeling of the extracellular matrix during invasion of breast cancer [27]. Other genes like GARS, NDUFA1, HMGCS1, and SLC17A7 have known functions in cellular metabolism. This clearly indicates that major regulators in our networks are known to have important cancer-relevant functions.
We further tested the capability of each network to predict the expression level of each of the 12,285 genes in independent oligodendroglioma (59 randomly selected tumors left out from network learning) and closely related oligoastrocytoma (118 samples including 34 tumors with and 84 tumors without 1p/19q co-deletion) test sets that were not considered for network inference. To realize this, we computed correlations between originally measured gene expression levels and corresponding network-based predicted gene expression levels across all tumor samples in each test set for each of the ten networks to analyze the prediction quality. Corresponding median gene-specific correlations integrating the prediction results of the ten networks are summarized in Fig. 3 (see Additional file 3: Figure S5 for individual networks). Overall, the vast majority of genes showed strong positive correlations between measured and predicted expression values with a median correlation of 0.75 for the oligodendroglioma test sets and a median correlation of 0.73 for the oligoastrocytoma test set. We also compared these results to predictions of gene expression levels that were obtained from random networks of same complexity as the originally learned networks (degree-preserving network permutations). We found that our networks made significantly better predictions of originally measured gene expression levels than corresponding random networks (Fig. 3, Wilcoxon rank sum test: P<2.2·10−16 for each of both test sets).
Genes directly affected by the 1p/19q co-deletion strongly impact on cancer-relevant signaling pathways
We utilized the learned networks to determine impacts of differentially expressed genes located within the region of the 1p/19q co-deletion on known cancer-relevant signaling pathway genes (see Fig. 1 for an illustration). To realize this, we considered the 654 differentially expressed genes observed within the 1p/19q region (524 under- and 130 overexpressed genes with q-values < 0.05, Additional file 4: Table S3) and applied regNet [64] to compute impacts of these genes on the expression of signaling pathway genes using network propagation. We did this independently for each network and computed corresponding impacts for each gene pair under random networks. We further integrated the scores of the ten networks and determined all differentially expressed 1p/19q-genes with significantly greater impacts on the expression of known cancer signaling pathway genes than under random networks (paired Wilcoxon rank sum tests, q-value < 0.05, see “Materials and methods” section for details). Predicted high-impact genes are shown in Fig. 4a and provided in Additional file 5: Table S4. We performed in-depth literature searches and analyzed gene annotations [62] to characterize cellular functions and known cancer-relevant impacts of these genes.
The gene with the greatest impact on signaling pathway genes was ELTD1 located on the 1p arm. ELTD1 encodes for a G-protein coupled receptor. The deletion of one copy of the 1p arm in oligodendrogliomas did not lead to a reduced expression of ELTD1. We found ELTD1 significantly overexpressed in oliogodendrogliomas compared to normal brain tissue. ELTD1 has been identified to represent a key player of tumor angiogenesis [50]. ELTD1 has also been functionally validated as oncogene in glioblastomas [72, 86]. The microRNA-139-5p has been reported to act as a tumor suppressor inhibiting ELTD1 expression in glioblastoma cell lines [15].
The only detected high-impact gene located on the 19q arm with strong impact on signaling pathway genes was KLK6. KLK6 encodes for a serine-protease and was strongly underexpressed in oligodendrogliomas compared to normal brain tissue. High expression levels of KLK6 have been associated with poor prognosis of intracranial tumors [18] and resistance of glioblastomas to cytotoxic agents [19]. KLK6 has recently been found to be involved in the control of metastasis formation in colon cancer [66].
PTAFR located on the 1p arm encodes for a G-protein coupled receptor involved in the regulation of cell proliferation and angiogenesis. PTAFR was strongly underexpressed in oligodendrogliomas compared to normal brain. PTAFR is a putative oncogene and has been reported to play a role in different types of cancer including the activation of PI3K-Akt signaling in esophageal cancer [12] or the support of prostate cancer development via ERK1/ERK2 signaling [30].
ZBTB17 located on the 1p arm was underexpressed in oligodendrogliomas compared to normal brain tissue. ZBTB17 encodes a transcriptional regulator interacting with MYC-genes. Reduced expression of ZBTB17 due to heterozygous loss of 1p36 has been reported to increase the aggressiveness of neuroblastomas [25]. This suggests that ZBTB17 is a putative tumor suppressor gene.
CAP1 located on the 1p arm was underexpressed in oligodendrogliomas compared to normal brain tissue. CAP1 is involved in the cyclic AMP pathway and interacts with the actin cytoskeleton influencing cell adhesion [82]. CAP1 expression has been reported to be positively correlated with proliferation, migration, invasion, and WHO grade of gliomas [3, 21].
So far, no roles in cancer have been reported for the two overexpressed high-impact genes THAP3 and ZMYM1 located on the 1p arm. Both genes are likely to encode transcription factors. THAP3 is involved in the regulation of cell proliferation [51]. ZMYM1 could be involved in the regulation of the cytoskeletal organization and cell morphology.
Further, we used our network propagation algorithm to predict potential regulatory downstream effects of high-impact genes on individual cancer-relevant signaling pathways (Additional file 3: Figure S6a). Especially the overexpression of ELTD1 and the underexpression of PTAFR in oligodendrogliomas tend to influence the expression of several signaling pathways suggesting complex regulatory dependencies that support or counteract oligodendroglioma growth. Specific impacts of individual high-impact genes are summarized in Additional file 3: Texts S1.
In summary, depending on the expression states in combination with reported roles in cancer, genes like ELTD1, ZBTB17, or THAP3 are likely to support oligodendroglioma growth, whereas other genes like KLK6, PTAFR, or CAP1 may restrict the speed of tumor growth. This might contribute to the overall better prognosis of oligodendroglioma patients in comparison to patients with other gliomas [69].
Genes directly affected by the 1p/19q co-deletion strongly impact on metabolic pathways
Similar to the analysis of signaling pathways, we used network propagation to identify those differentially expressed genes within the region of the 1p/19q co-deletion that had strong impacts on the expression of metabolic pathway genes. We predicted 14 high-impact genes widespread across the 1p/19q region (q-value < 0.05, Fig. 4b, Additional file 6: Table S5). All genes were underexpressed in oligodendrogliomas compared to normal brain, except for overexpressed DPH5. Two genes with strong impact on signaling pathways (KLK6, PTAFR) were also among these high-impact genes. In contrast to our previous impact quantification for signaling pathways that revealed only one high-impact gene on the 19q arm (Fig. 4a), we now found six genes (MAG, COX6B1, EIF3K, SEPW1, SLC17A7, KLK6) with strong impact on the expression of metabolic pathway genes on 19q (Fig. 4b). We again performed in-depth gene annotation analyses and literature searches to summarize known functions and roles in cancer.
SLC17A7, the gene with the greatest impact on the expression of metabolic pathways, has been observed as tumor suppressor in a glioblastoma stem cell line [44]. SLC17A7 is located on the 19q arm, encodes for a vesicle-bound, sodium-dependent phosphate transporter expressed in neuron-rich regions, and was strongly underexpressed in oligodendrogliomas compared to normal brain.
SDHB is located on the 1p arm, encodes for the succinate dehydrogenase complex subunit B, and was underexpressed in oligodendrogliomas in comparison to normal brain. Germline mutations of SDHB have been reported for patients with head and neck paraganglioma [5] and phaeochromocytomas [7]. Succinate accumulated in SDHB-mutated cells inhibits alpha-ketoglutarate-dependent enzymes leading to the activation of hypoxia induced genes and hypermethylation of DNA and histones in paraganglioma [4, 40]. Similarly, a knockdown of SDHB in mouse ovarian cancer cells enhanced cell proliferation and induced hypermethylation of histones promoting an epithelial-to-mesenchymal transition [2]. All these findings suggest that reduced expression of SDHB in oligodendrogliomas may support and possibly enhance the epigenetic reprogramming via the same pathomechanism induced by a heterozygous IDH-mutation that is found in each oligodendroglioma [13, 48].
PARK7 located on the 1p arm was underexpressed in oligodendrogliomas compared to normal brain tissue. PARK7 encodes for a peptidase that protects cells against oxidative stress. Downregulation of PARK7 has been associated with a reduction of cell proliferation, migration, and invasion of glioma cell lines [31]. Downregulation of PARK7 in clear renal cell carcinoma cells increased cisplatin-induced apoptosis [73]. PARK7 has been reported as oncogene in different cancers activating PI3K-Akt, MAPK, and mTOR signaling to protect cells against hypoxic stress [77].
HBXIP located on the 1p arm was underexpressed in oligodendrogliomas compared to normal brain tissue. HBXIP functions as a cofactor of survivin in the suppression of apoptosis [49]. HBXIP has been reported to promote the proliferation and migration of breast cancer cells [45]. Conversely, suppression of HBXIP has been found to reduce cell proliferation, migration and invasion of bladder carcinomas [42]. This suggests that the underexpression of HBXIP could counteract oligodendroglioma growth.
SEPW1 is located on 19q, encodes for a selenoprotein that functions as an glutathione antioxidant, and was underexpressed in oligodendrogliomas compared to normal brain. SEPW1 has been mapped to a putative tumor suppressor region on the 19q arm of gliomas [67]. SEPW1 has been shown to be involved in the control of cell cycle progression [23] and to regulate expression, activation and degradation of EGFR [1].
C1orf144 (SZRD1) located on 1p was underexpressed in oligodendrogliomas in comparison to normal brain tissue. C1orf144 has recently been reported as a potential tumor suppressor in cervical cancer involved in the regulation of cell cycle arrest in G2 and induction of apoptosis [84].
MAG located on 19q was underexpressed in oligodendrogliomas compared to normal brain tissue. MAG encodes for a membrane protein involved in myelination of oligodendrocytes, protection of neurons against apoptosis, and inhibition of neurite outgrowth [59].
Further, only DPH5 located on 1p was overexpressed in oligodendrogliomas compared to normal brain. DPH5 encodes for a specific methionine-dependent methyltransferase involved in diphthamide synthesis. Diphthamide, a post-transcriptionally modified histidine, is required for eEF-2, which is essential for protein biosynthesis. Further, two underexpressed high-impact genes, RPL22 and EIF3K, known to be important for protein synthesis were found. Strong impacts of genes involved in protein synthesis might represent a byproduct of increased transcription in tumors. In addition, COX6B1 located on 19q and ATP5F1 located on 1p were underexpressed in oligodendrogliomas in comparison to normal brain. Both genes have functions in the respiratory chain.
In addition, we also used our network propagation algorithm to further predict potential regulatory downstream effects of high-impact genes from Fig. 4b on individual metabolic pathways (Additional file 3: Figure S6b). Interestingly, six genes were predicted to contribute to a downregulation of the oxidative phosphorylation. Detailed information to specific impacts of individual high-impact genes are summarized in Additional file 3: Text S1.
Again, genes like SLC17A7, SDHB, SEPW1, or SZRD1 may support oligodendroglioma growth and other genes like PARK7 or HBXIP may restrict the speed of tumor growth. Such counteracting impacts could contribute to a better prognosis [69].
Impact of rare gene copy number mutations on cancer-relevant signaling and metabolic pathways
We further used our network-based impact quantification strategy to determine if potential candidate genes with high-impact on signaling or metabolic pathways are located on chromosomal arms that were rarely affected by deletions or duplications in oligodendrogliomas with 1p/19q co-deletion (Table 1; deletions: 4q, 9q, 13q, 15q, 18q; duplications: 7p, 7q, 11q; Additional file 3: Figure S1). All these additional mutations have previously been observed in the POLA cohort [33] and several of these mutations were also observed in copy number profiles of single oligodendroglioma cells [71]. These additional copy number mutations occurred more frequently in oligodendrogliomas of WHO grade III than in grade II tumors suggesting that they are associated with tumor progression and may impact on survival [35, 74]. See Additional file 3: Text S2 for further details to subgroups of oligodendrogliomas with additional chromosomal arm mutations. We first determined for each subcohort of oligodendrogliomas with a specific chromosomal arm mutation all differentially expressed genes in comparison to normal brain tissue (q-value ≤0.05). We next analyzed all differentially expressed genes of a mutated chromosomal arm to identify those genes that were predicted to have a strong impact on the expression of cancer-relevant signaling (Additional file 7: Table S6) and metabolic pathways (Additional file 8: Table S7) utilizing network propagation. We predicted 15 differentially expressed genes with strong impact on signaling pathways on the chromosomal arms 4q, 9q, 7p, 7q, 11q, and 18q (Fig. 5a–f) and 12 genes with strong impact on metabolic pathways on the chromosomal arms 7p, 11q, 15q, and 18q (Fig. 5g–i, 7p not shown) at a q-value cutoff of 0.1 (less stringent than before because of much smaller sample sizes). Functional annotations and literature searches of all predicted high-impact genes are summarized in Additional file 3: Text S3 for signaling pathways and in Additional file 3: Text S4 for metabolic pathways. Next, we only briefly highlight some findings.
Considering genes with high-impact on signaling pathways (Fig. 5a–f), we identified several overexpressed genes in subcohorts of oligodendrogliomas with additional chromosomal arm mutations that were previously found to be involved in tumorigenesis. For example, EMCN located on the q-arm of chromosome 4 was overexpressed in oligodendrogliomas with 4q deletion. EMCN encodes a glycoprotein that can inhibit adhesion of cells to the extracellular matrix [34]. EIF3B located on the p-arm of chromosome 7 was overexpressed in oligodendrogliomas with 7p duplication. EIF3B encodes a subunit of the eukaryotic translation initiation factor. A knockdown of EIF3B inhibited cell proliferation and increased apoptosis in a glioblastoma cell line [43]. CALD1 located on the q-arm of chromosome 7 was overexpressed in oligodendrogliomas with 7q duplication. CALD1 is involved in the regulation of the neovascularization of gliomas [85] and has been associated with tamoxifen resistance of breast cancer [17]. Also DNAJB6 located on the q-arm of chromosome 7 was overexpressed in oligodendrogliomas with 7q duplication. Overexpression of DNAJB6 has been reported to promote invasion of colorectal cancer [83]. In addition to these putative oncogenes, we also observed two overexpressed genes with potential tumor suppressor functions that may counteract oligodendroglioma development. DMTF1 located on 7q encodes a transcription factor with a cyclin D-binding domain that has been shown to inhibit cell growth and cell cycle progression in bladder cancer [57]. Further, FAU located on 11q encodes a fusion protein that has been reported to be involved in the regulation of apoptosis of breast cancer [58].
Considering genes with high-impact on metabolic pathways (Fig. 5g–i), we identified four underexpressed genes in subcohorts of oligodendrogliomas with additional chromosomal arm mutations with functions in cellular energy metabolism and impacts on cell migration, apoptosis, or blood vessel development in cancer (deletion of 15q: COX5A, PKM2; deletion of 18q: ATP5A1; duplication of 11q: PYGM; Additional file 3: Text S4). In addition, UBXN1 located on the q-arm of chromosome 11 was overexpressed in oligodendrogliomas. UBXN1 encodes a ubiquitin-binding protein and has been reported to inhibit the tumor suppressor BRAC1 [81]. Interestingly, we found that SDHD located on 11q was overexpressed in oligodendrogliomas with 11q duplication. This might represent a response to the reduced expression of SDHB discussed before. Further, activation of the expression of the tumor suppressor CDKN1A in response to the loss of SDHD expression has been reported [52]. Thus, overexpressed SDHD might counteract the expression of CDKN1A to support cell proliferation. Moreover, also ACAT1 located on 11q was overexpressed. ACAT1 encodes a mitochondrially localized acetyl-CoA acetyltransferase. Inhibition of ACAT1 by Avasimibe inhibited cell growth by inducing cell cycle arrest and apoptosis in glioblastoma cell lines [6, 55]. Further, inhibition of ACAT1 has also been shown to suppress growth and metastasis of pancreatic cancer [41].
In-depth analysis of known potential tumor suppressor genes FUBP1 and CIC
We also performed a detailed analysis of the expression behavior and corresponding network-based impacts of the potential tumor suppressors FUBP1 and CIC reported for oligodendrogliomas [8]. FUBP1 located on 1p and CIC located on 19q were both moderately underexpressed in oligodendrogliomas with 1p/19q co-deletion compared to normal brain references (Additional file 4: Table S3). Further, oligodendrogliomas with additional small deletions, insertions or point mutations within FUBP1 or CIC showed moderately reduced expression of these genes compared to oligodendrogliomas without mutations. This trend was much stronger for tumors with FUBP1 mutations (average expression 4.58 vs. 5.25 comparing 38 tumors with to 95 tumors without mutation, t-test: P=0.0001) than for tumors with CIC mutations (average expression 6.42 vs. 6.60 comparing 85 tumors with to 48 tumors without mutation, t-test: P=0.01).
Further, FUBP1 has been reported to negatively regulate the expression of MYC [8]. This relationship was also predicted by our network propagation approach. FUBP1 had a stronger impact on MYC comparing our networks to corresponding random networks (paired Wilcoxon rank test: P<0.001, see “Materials and methods” for details). Globally, FUBP1 and CIC underexpression had moderate impacts on different signaling and metabolic pathways (Additional file 3: Figure S7). Thus, reduced expression of both genes due to the 1p/19q co-deletion could contribute to tumor development, but both genes were not among the predicted putative high impact genes with altered gene expression levels. Still, other pathomechanisms triggered by small deletions, insertions or point mutations within FUBP1 or CIC could play an important role in affected tumors.