Glioblastoma multiforme (GBM) is the most widespread and aggressive type of primary brain tumor (1). Standard treatment strategies are commonly ineffective (2). Even following complex modern treatment regimens, the prognosis for patients with GBM is poor, with a median survival time of 14 months (2). The unsuccessful treatment of this aggressive disease is commonly due to late diagnosis, a lack of specific diagnostic markers, resistance to traditional treatment, and the high proliferation and metastatic potential of tumor cells (3).

The invasion and infiltration of tumor cells, as well as an extremely high proliferation rate, are significant contributors to mortality in patients with GBM (4). In order to develop effective treatment methods, it is necessary to identify novel targets involved in GBM tumorigenesis (4). Thus, there has recently been an increase in interest towards investigating the signaling pathways associated with glioma (5). Ultimately, improving the understanding of the underlying molecular mechanisms of GBM oncogenesis will aid the development of novel and more effective methods of treating the disease.

As one of the various signaling pathways associated with glioma, transforming growth factor-β (TGF-β) signaling is important for regulating the behavior of these tumors (5). It has been previously reported that TGF-β levels are high in the blood serum and tumor tissue of patients with malignant glioma, and this level was correlated with the type of malignancy, the tumor developmental stage and the patient prognosis (6). Furthermore, it was also hypothesized that the TGF-β signaling pathway is directly involved in the molecular mechanisms of glioma malignancy (6). Certain studies have reported that TGF-β is able to induce metastatic processes and tumor progression via autocrine mechanisms (7,8).

TGF-β is a member of a large cytokine family involved in regulating various biological processes, including fetal development and tissue homeostasis (9). At the molecular level, TGF-β affects numerous cellular activities, including proliferation, survival, differentiation, migration and immune activation, depending on the cell type and cellular context (9). TGF-β performs its multiple functions via a complex network of various ligands and receptors, which transmit corresponding signals (9,10). Regarding cancer, the TGF-β signaling pathway exhibits tumor suppressive and oncogenic functions (10). TGF-β is considered to be a tumor suppressor as it strongly inhibits the proliferation of epithelial cells, astrocytes and immunocytes (6). Certain tumors avoid the cytostatic response to TGF-β via the mutation of particular genes involved in the TGF-β signaling pathway (7). Several malignant tumors, including gliomas, selectively block the ability of TGF-β to inhibit proliferation, while preserving its other functions (9). In such tumors, TGF-β may induce proliferation, angiogenesis, invasion, metastasis and immune suppression. Thus, TGF-β is able to exert a dual effect on carcinogenesis depending on the stage and type of tumor; it may act as a tumor suppressor or an oncogene (11,12). This conversion from tumor suppression to oncogenic activity is known as the ‘TGF-β paradox’ (13,14). Whilst significant progress has been made in the understanding of the TGF-β signaling pathway, its importance in glioma oncogenesis remains to be elucidated.

Particular interest has been given to investigating the modulation of TGF-β-induced epithelial-to-mesenchymal transition (EMT). During this process, epithelial cells lose their intercellular connections and apical-basal polarity, reorganize their cytoskeleton, release ECM proteins, and transdifferentiate into mobile mesenchymal cells (15). EMT induction leads to the metastatic invasion of a number of carcinomas; however, the full extent of its influence remains to be elucidated, particularly with regard to glioblastomas (16). Previous studies have attributed EMT to the generation of tumor stem cells (16,17). The underlying molecular mechanisms of TGF-β-induced EMT require further investigation. However, existing data demonstrates that the TGF-β signaling pathway is involved in the late stages of glioma oncogenesis and metastasis development, suggesting it may be an important potential candidate for targeted therapy (4,17).

The present study performed comparative proteome mapping of the U87 human glioblastoma cell line, with and without TGF-β1 treatment, using the nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Proteome analysis identified various proteins associated with TGF-β1 regulation of GBM oncogenesis, which may be novel potential markers of metastasis or targets for glioblastoma therapy. The target candidates may potentially be validated and used in the clinic to develop novel approaches for the diagnosis and treatment of GBM, one of the most serious forms of cancer.

The development of malignant glioma proceeds through several stages, including transformation of healthy cells, activation of cell proliferation signals, loss of cell cycle control, development of an invasive phenotype, increased angiogenesis and additional development of clones (20). GBM is the most aggressive type of brain tumor and is characterized by diffuse infiltration of brain parenchyma, aberrant proliferation, radio- and chemotherapy resistance, and relapse following surgical resection (20,21). Advances in molecular technologies, including high-density chip microarrays and genome sequencing, have enabled the evaluation of genetic and epigenetic changes in these tumors at the genome level (22).

Currently, there is no evidence demonstrating that the TGF-β signaling pathway has anti-oncogenic activity in glioma, whereas in carcinomas, senescence is induced via TGF-β signaling (9). However, in malignant brain tumors, TGF-β expression has been previously demonstrated to promote tumor cell survival, and to increase cell proliferation, migration, invasion, angiogenesis, stem cell functioning and immune system suppression (23).

In glioma, TGF-β is released from glioma cells via autocrine mechanisms or is secreted from microglial cells. Autocrine secretion of TGF-β has been observed in cell lines and cells that have been obtained from surgically removed malignant gliomas (24,25).

Out of 2,555 identified proteins, the current study detected that 635 exhibited significantly altered expression following TGF-β1 treatment of U87 cells. The majority of bioinformatic analysis was performed on these proteins, although certain analyses were performed on the entire list of proteins in order to gain a complete picture.

TGF-β signaling stimulates a cytostatic response (26,27), which is why proteins involved in cell cycle regulation were analyzed in the present study (Table I). TGF-β1 treatment caused a significant decrease in the protein levels of ATR serine/threonine kinase, cell division cycle 16, minichromosome maintenance complex component 5 (MCM5) and MCM6 and an increase in cullin 1. Controlling the levels of cyclin-dependent kinases (CDKs) and their activation is an important mechanism of the anti-proliferative effect of TGF-β (28). As Table I demonstrates, the expression levels of CDK9 and CDK inhibitor 2D interacting protein were increased and the CDK5 regulatory subunit associated protein 1-like 1 regulatory subunit was decreased.

Integrins are heterodimeric transmembrane cellular adhesion receptors that interact with various extracellular ligands (for example ECM proteins, including collagen, laminins, vitronectins and fibronectins). These interactions activate integrins and, thus, regulate tumor cell invasion, metastasis, migration, proliferation, angiogenesis and survival (29,30). This demonstrates the importance of integrins in GBM oncogenesis (31). Immunohistological analysis previously demonstrated that integrin α3 (ITGA3) is expressed in the plasma membrane of GBM cells, particularly in invading cells and the surrounding blood vessels (21). The increased expression of ITGA3 enhances migration and invasion of glioma cells and glioblastoma stem cells (GSCs) (32). The current study detected that the expression of various proteins associated with EMT was increased following TGF-β1 treatment of U87 cells, including integrins (ITGB1, ITGB3, ITGAV, ITGA5, ITGAX, ITGA3, ITGA8) and fibronectin 1 (FN1) (Table II). It is necessary to note that ITGA8 was only detected in the TGF-β1-stimulated cells. ITGA2 and ITGB3 levels were increased by ~2-fold. FN1 expression was increased 3.78-fold.

The significance of epigenetic alterations in cancer is well-established (25). Histone deacetylases (HDACs) are able to alter chromatin structure to organize DNA and regulate gene transcription in malignant glioma (27). HDAC inhibitors have previously been used to inhibit growth and induce apoptosis of cancer cells (27). Histone acetyltransferases (HATs) and histone hyperacetylation may cause degradation of hypoxia inducible factor-1α and reduce the level of vascular endothelial growth factor (VEGF), resulting in anti-angiogenic effects (33). In a previous study, treatment with HDACs decreased proliferation of glioblastoma-derived cells and caused their differentiation (34). Analysis of the data of the present study demonstrated that HDAC1 and HDAC2 protein levels were increased (1.52- and 1.69-fold, respectively) and HAT1 levels were decreased (0.67-fold) in TGF-β1-stimulated U87 cells.

Heat shock proteins (Hsp), particularly Hsp90, are required for malignant transformation, growth stimulation, survival and invasiveness of cancer cells (27,35). They stabilize the expression of cell surface proteins epidermal growth factor receptor (EGFR)vIII and EGFRvIV, and may promote GBM invasiveness (35). In the present study, TGF-β1 stimulation marginally increased Hsp90b1, Hsp90AB1 and Hsp90AA1 expression; however, the effect was not significant. The most substantial increase was observed in Hsp90AA4P (1.46-fold).

The effect of 14-3-3 proteins on cancer development and their interaction with various oncogenes and tumor suppressor genes is complex (36). A previous study investigated the increased immune reactivity of various 14-3-3 isoforms in human astrocytoma samples, and the immunoreactivity of cells was significantly enhanced with increased disease progression (37). Furthermore, 14-3-3β and h were identified as two tumor-specific isoforms of 14-3-3 in astrocytomas, and they may be potential candidates for targeted therapies (38). However, the exact function of 14-3-3 proteins in GBM oncogenesis remains to be elucidated. In the present study, the protein expression levels of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein θ (YWHAQ), YWHA ζ, YWHA γ and YWHA β increased marginally following TGF-β treatment of U87 cells. The most substantial increases were observed in YWHA η (1.38-fold) and YWHAQ (1.46-fold).

To improve the understanding of underlying biological processes and signaling pathways involved in TGF-β-induced responses, comprehensive bioinformatic analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/kegg/) for pathway analysis and GO for biological processes. The descriptions of processes identified as potentially involved in EMT were extensive (39). The KEGG and GO search terms were ‘intercellular contacts’, ‘focal adhesion’ and ‘actin cytoskeleton’.

Breakdown of intracellular tight junctions during EMT is accompanied by decreased expression of claudin and occludin, and diffusion of zonula occludens 1 [termed tight junction protein 1 (TJP1)] from intercellular contacts (40,41). Following TGF-β1 treatment, the TJP1 expression level was increased (1.86-fold) and TJP2 was decreased (0.54-fold). EMT additionally stimulates the expression of neural cell adhesion molecules, which interact with proto-oncogene Src family tyrosine kinase Fyn (FYN) to reduce inhibition of focal adhesion, migration and invasion (42). In the current study, the FYN protein expression levels were increased 1.45-fold. ECM remodeling and changes in cellular interactions with the ECM are important for the initiation of EMT (35). Integrin complexes enable cells to receive signals from ECM proteins through interaction with signal mediators, including integrin-linked kinase (ILK) and parvin (41,43). Following treatment of U87 cells with TGF-β1 for 72 h, there was a slight increase in ILK expression (1.2-fold) and no detectable change in parvin-α levels, whereas the protein level of parvin-β was decreased (0.66-fold). Changes in the expression of integrins during EMT are correlated with increased expression of proteases, including matrix metalloproteinase 2 (MMP2) and MMP9, which increases ECM degradation and promotes invasion (44). In the present study, a significant increase in MMP2 expression was detected (2.85-fold), whereas MMP14 levels did not change. In addition to the traditional SMAD-mediated TGF-β signaling, TGF-β induces signal transduction via Rho GTPases, phosphatidylinositol-4,5-bisphosphate 3-kinase and the mitogen-activated protein kinase signaling pathways (45,46) that also promote EMT (47–49).

Activation of Rho, Rac and cell division control protein 42 homolog GTPases results in reorganization of actin, as well as lamellipodia and filopodia formation (50). A previous study demonstrated that TGF-β stimulates partitioning defective 6 homolog family cell polarity regulator via TGF-β receptor II and recruits SMAD specific E3 ubiquitin protein ligase 1, which promotes RhoA ubiquitination and degradation when tight junctions are broken (51). TGF-β additionally induces RhoA activation (52), partially via promotion of the expression of guanine nucleotide exchange factors, and Rho-associated coiled-coil containing protein kinase 1 and LIM domain kinase 1 activation (52,53). Therefore, decreased expression of RhoA or chemical inhibition of RhoA prevents actin reorganization, which is required for TGF-β-induced EMT (52–54). The results of the comparative expression analysis of proteins participating in these processes are presented in Table III.

FGF signaling enhances mesenchymal features in epithelial cells (55). In the current study, the levels of FGF18 protein were increased (1.89-fold) in the TGF-β stimulated U87 cells. EGF induces endocytosis of E-cadherin in vitro, similar to the effect of SNAIL family transcriptional repressor 1 or TWIST family bHLH transcription factor 1, which decrease the levels of E-cadherin (56,57). EGF also activates EMT in epithelial explants, resulting in increased mobility of cells and MMP2- and MMP9-mediated proteolysis, which is dependent on ILK and the extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathways (58). Activation of human EGF2 receptor (human epidermal growth factor 2; also termed ERBB2) in the epithelial cells of the mammary gland causes development of tumors with an EMT phenotype, which are able to escape immune system responses (59,60). In the current study, stimulation with TGF-β induced a significant increase in ERBB2 interactive protein expression (4.79-fold). A previous report demonstrated that PDGF induces breakdown of adhesive cellular contacts, nuclear localization of β-catenin and E-cadherin level repression in colon adenocarcinoma cells (61). Ablation of the PDGF-receptor α-subunit enhances the formation and migration of mesenchymal-like cells, and decreases MMP2 activity (62). VEGF is understood to be associated with PDGF and induces angiogenesis, which also causes EMT (63).

As previously demonstrated, TGF-β stimulates glioma growth via interactions with the PDGF-β signaling pathway (64). TGF-β is able to increase expression levels of PDGF-β mRNA and protein, and enhance the phosphorylation of the PDGF receptor; thus, increasing proliferation of U373MG glioblastoma cells (65). The results of the current study demonstrated that the expression of PDGF receptor β was significantly increased (1.74-fold) following TGF-β1 treatment of U87 cells, whereas the levels of PDGF receptor α were not significantly altered by TGF-β1 treatment (1.1-fold increase).

Bioinformatic analysis demonstrated a slight annotation enrichment of proteins involved in ECM-receptor interaction (Table IV), regulation of actin cytoskeleton, the spliceosome (Table V), DNA replication (Table VI), adherens or tight junctions and focal adhesion, which are established signaling responses to TGF-β (10,66–68). Proteins in these signaling pathways may be potential markers of metastasis and targets for glioblastoma treatment. Additional annotations corresponded to functions that are not typically associated with TGF-β signal transduction, for example DNA mismatch repair proteins (Table VII).

Cellular adhesion receptors are highly expressed in GBM cells and are important for their invasion. Glioma cells use these receptors for adhesion and migration via interaction with components of the ECM, which is specifically distributed and regulated within the human brain and spinal cord. Therefore, glioma cell invasion into adjacent brain tissue depends on the interaction between glioma cells and the ECM (69).

TGF-β positively regulates the expression of ECM components, including type I collagen and FN1, which are significant proteins involved in tumorigenesis (70). As Table VI demonstrates, TGF-β1 impact on U87 cells increases collagen, type I, α 2 and FN1 expression 3.59- and 3.78-fold, respectively. In pathological cases, intensified TGF-β signaling may cause excessive accumulation of ECM (71). During EMT, cells obtain mesenchymal markers, including FN1 and vimentin, with the levels of vimentin observed to be non-significantly increased in the current study, unlike the levels of FN1.

The ECM of the central nervous system is enriched by hyaluronate (HA). CD44 and receptor for HA-mediated motility (RHAMM) are ubiquitous receptors for HA. RHAMM mediates migration and proliferation of glioma cells, with the levels of RHAMM increasing with the progression of disease (72). The current study only detected RHAMM expression in TGF-β1-stimulated U87 cells, while the levels of CD44 did not change during the experiment.

ECM remodeling via ECM protein proteolysis is an important step in the metastatic process that enables neoplastic cells to invade through the basal lamina (BL) into the stroma (73). Proteolysis stimulates signaling pathways that regulate migratory, functional or signaling molecules, including growth factors, and the generation of biologically active fragments of the ECM (74). Laminins are ECM glycoprotein components present in the BL and have been previously identified as substrates for cell migration and adhesion (75). To the best of our knowledge, the function of laminin β1 in these processes has not been previously investigated. As demonstrated in Table IV, the levels of laminin β1 were increased significantly (7.22-fold) in TGF-β1-induced U87 cells.

Angiogenesis is crucial for supporting the growth and metastasis of tumors (27,41). Thrombospondin-1 (TSP-1) is a powerful inhibitor of angiogenesis, and negative regulation of TSP-1 may alter tumor growth by modulating angiogenesis in various tumor types. TSP-1 expression is positively regulated by tumor-suppressing gene p53, and negatively regulated by oncogenes, including Myc and Ras. TSP-1 reduces angiogenesis by inhibiting migration and proliferation of endothelial cells and inducing apoptosis. Furthermore, TSP-1 activation of TGF-β1 is important for the regulation of tumor progression. Investigating the molecular mechanisms of TSP-1-mediated suppression of angiogenesis and tumor progression may aid the development of novel diagnostic methods and cancer therapeutics (76). In the present study, TSP-1 was only detected following TGF-β1 treatment of glioblastoma cells (Table IV).

Gliomas have a subpopulation of cells that exhibit stem-like properties. This population of cells has been suggested to be involved in the initiation, metastasis, support and relapse of gliomas. Treatment methods that attempt to regulate GSC function may improve survival rates for patients with glioma (77,78).

The TGF-β superfamily is crucial for morphogenesis and the specification of cell progenies during the development of the human brain (79,80). Ikushima et al (7) demonstrated that autocrine TGF-β signaling is an important factor in supporting the stem cell-like phenotype of GSCs. The association between TGF-β and stem cell properties was also demonstrated in mammary gland epithelium, where a short-term incubation of mammary epithelial cells with TGF-β activated EMT and increased the ability of the cells to form mammospheres (81). Similarly, incubation with TGF-β increased the formation of neurospheres in a primary culture of brain tumor cells, demonstrating that TGF-β increases the self-restoration ability of GSCs (82).

Attempts to develop targeted therapies for GBM are predominantly focused on the analysis of GSCs. Previous studies have characterized the spliceosome proteins that are specifically required for GSC growth and survival compared with neural stem cells and other types of non-transformed cells (47,72,82). As demonstrated in Table V, TGF-β1 actively modulates the expression of certain spliceosomal proteins of this group in U87 cells. The protein expression of pre-mRNA processing factor 19, WW domain binding protein 11, nuclear cap binding protein subunit 1 and serine/arginine-rich splicing factor 2 was increased. Notably, LSM2 homolog U6 small nuclear RNA, mRNA degradation associated protein, survival motor neuron domain containing 1 and thioredoxin like 4A proteins were only detected in the lysates of TGF-β1-treated U87 cells.

In conclusion, the current study investigated the underlying molecular mechanisms that mediate the effect of TGF-β1 on U87 human glioblastoma cells. The intracellular processes identified to be involved in the regulation of malignant glioma oncogenesis by TGF-β1 included EMT, ECM-receptor interaction, regulation of the actin cytoskeleton, spliceosomal functions, DNA replication, adherens or tight junctions and focal adhesions, with significant patterns being discovered. The current study used comparative proteome mapping to identify candidate markers of glioblastoma metastasis and potential targets for glioma therapeutics.

TFG-1β changes the molecular phenotype of human glioblastoma cells. In response to TFG-1β, the expression of 512 proteins associated with survival, proliferation, cell migration and DNA repair is increased. In addition, the expression of 123 proteins responsible for apoptosis, interaction with the extracellular matrix and aerobic metabolism is decreased. Therefore, TFG-1β holds a critical role in glial brain tumor biology and is among the key stimulators of GBM invasive growth. This makes TFG-1β a promising target for targeted cancer therapy. Since tumor stem cells are tightly involved in GBM cancerogenesis, future studies should be focused on the impact TFG-1β has on various subpopulations of this cell type.