Antagonizing the biological effects of TGFβ has become a potential experimental strategy to treat glioblastoma, one of the most devastating human cancers. Several anti-TGFβ therapies have shown promise in both preclinical and early clinical trials [
39]. The current rationale for TGFβ antagonism includes its role in tumor promotion, migration and invasion, metastasis, and tumor-induced immunosuppression. Numerous reports suggest aberrant TGFβ activation in glioblastoma and other high-grade gliomas. This includes abnormal expression of the ligands, more specifically TGFB2 and higher levels of phosphorylated SMADs. However, to date, none of these reports has systematically examined the components of TGFβ signaling to gain a comprehensive view of TGFβ activation in a large cohort of human glioma patients. In this study, we adopted an alternative approach. By examining the transcriptional responses induced by TGFβ activation in publicly available microarray data, we identified two subgroups of glioblastomas that showed distinct patterns of TGFβ activation in two independent studies. Combining the two independent microarray studies of high-grade gliomas, we found that the grade IV glioblastomas showed stronger TGFβ induced transcriptional response than the grade III tumors. In addition, among glioblastomas, 48 out of 78 (62%) showed strong TGFβ activation, while the remaining 38% showed a much weaker TGFβ transcriptional response. How effective the anti-TGFβ therapies would be in the two subgroups of glioblastomas showing distinct TGFβ activation patterns is an open question for future clinical trials. Nevertheless, this study confirmed the previous notion that TGFβ activation occurs commonly in a large portion of glioblastomas, and anti-TGFβ therapies are likely to be beneficial for those patients.
By examining the genes differentially expressed between the two identified subgroups of glioblastomas that showed different TGFβ transcriptional responses, we found that the ligands TGFB1, TGFB2 and their receptors were expressed significantly higher in the strong TGFβ response group (Additional file
3) compared to those in the weak TGFβ response group, suggesting that increased expression of the ligands and receptors contributed to TGFβ activation. THBS1, an activator of TGFβ, was shown to have a higher level in the strong TGFβ response group in one study, suggesting that TGFβ activation may also result from increased bioavailability. In contrast, SMAD7, a negative regulator of TGFβ pathway that often was induced upon TGFβ stimulation
in vitro (Additional file
1), was downregulated in the strong TGFβ response group (fold change -1.48, p < 0.0007), suggesting the tumor-specific escape of the negative feedback mechanism may also contributed to TGFβ activation in glioblastomas. In addition, genes involved in antigen presentation were upregulated in the TGFβ strong response glioblastomas. These included the genes encoding class I major histocompatibility complex proteins HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, class II major histocompatibility complex proteins HLA-DMA, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DRB1, MHC class I binding protein CANX, immunoproteosomal subunits PSMB8 and PSMB9, and MHC peptide transport protein TAP1. The upregulation of antigen presentation molecules in the TGFβ strong response glioblastomas suggests that the reported tumor-mediated immunosuppression in glioblastoma occurs through other mechanisms. One study suggested direct targeting of cytotoxic T cell functions by TGFβ and downregulation of the expression of five cytolytic molecules perforin, granzyme A, granzyme B, Fas ligand and interferon γ in T lymphocytes [
40]. Strong TGFβ response glioblastomas identified in this study also showed higher expression of many molecules involved in integrin signaling (
ACTA2,
ACTN1,
ACTN4,
ARPC4,
COL1A1,
COL1A2,
COL4A1,
COL4A2,
DIRAS3,
FN1,
ITGA2,
ITGA3,
ITGA4,
ITGA7,
ITGB1,
ITGB2, ITGB4,
ITGB5,
LAMA4,
LAMB1,
LAMB2,
LAMC1,
MRCL3,
RAP2B,
RHOC,
RHOJ,
RRAS,
SHC1,
VASP, and
ZYX). Integrins have been shown to mediate the activation of TGFβ [
41] and TGFβ is known to regulate the expression of cell adhesion molecules including integrins [
42,
43]. Interestingly, the glioblastoma group that showed a strong TGFβ response also showed higher expression of the molecules involved in angiogenesis, such as
VEGF,
FLT1,
NRP1,
NRP2,
ANGPT2,
JAG1,
ARTS1,
TNFRSF12A. Also the gene expression of a group of insulin-like growth factor binding proteins, including
IGFBP2,
IGFBP3,
IGFBP4,
IGFBP5, and
IGFBP7 were significantly higher in TGFβ strong response glioblastomas. Interestingly,
IGFBP2, one of the most significant gene changes between the two subgroups of glioblastomas showing different TGFβ responses (fold change 7.37, p < 1.27 × 10
-9), has been shown to enhance glioblastoma invasion [
44]. In contrast, the molecules involved in GABA receptor signaling (
GABBR1,
GABRA1,
GABRA5,
GABRB1,
GABRB3,
GABRG2,
GAD1,
GPR51) and glutamate receptor signaling (
GLS,
GRIA2,
GRIA4,
GRM1,
GRM5,
GRM7,
SLC17A6,
SLC17A7,
SLC1A1) were downregulated in the TGFβ strong response glial tumors.
BMP2, a member of TGFβ superfamily that has been shown to promote GABAergic neuron differentiation [
45], was also downregulated in the TGFβ strong response glioblastomas (Fold change -2.43, p < 0.0013). These genes differentially expressed between the two identified subgroups of glioblastomas that showed different TGFβ transcriptional responses provide insights into the potential mechanisms of TGFβ-mediated tumor progression and invasion in glioblastomas.
EGFR amplification and PTEN mutations/10q LOH are frequent genetic alterations observed in glioblastomas. Recently a gene signature generated from autocrine platelet-derived growth factor (PDGF) signaling in gliomas has been used to classify gliomas, and it was shown that EGFR amplification and PTEN mutation/10q LOH were largely enriched in the cluster showing weak autocrine PDGF signaling [
46]. Using the same signature, we found the TGFβ strong response cluster overlapped with the weak autocrine PGDG signaling subgroup extensively (data not shown), suggesting potential collaboration between EGFR/PTEN/PI-3K pathway and TGFβ pathway in glioblastoma development and progression. Numerous evidence
in vitro also showed the collaborating roles of EGFR and TGFβ in inducing epithelial to mesenchymal transition, an event that contributes to cell migration, invasion, cell survival and angiogenesis [
47‐
50]. Future studies will be needed to examine if EGFR amplification and PTEN mutation/10q LOH were enriched in the subgroups of glioblastomas that showed strong TGFβ transcriptional response.