A wealth of molecules has been implicated in GB, but while illustrating the heterogeneity of the disease, they fall short of providing effective treatment. Several genetic mutations and pathway alterations have been implicated in GB development, which may contribute to the inter- and intra-heterogeneity of the tumors. As depicted in Table 1, these include genetic mutations of the epidermal growth factor receptor (EGFR), over-expression of platelet-derived growth factor subunit A (PDGFA), and loss of heterozygosity of phosphatase and TENsin homolog on chromosome 10q23 (PTEN). The mutations lead to downstream alterations in tumor-suppressor pathways, including the small GTPase Ras, the guardian of the genome tumor suppressor p53, and the cell cycle regulator retinoblastoma (RB) protein [1]. Interestingly, a subset of human GB cells without p53 mutations were reported to over-express the proto-oncogene mouse double minute 2 (MDM2), a negative regulator of p53 [15]. Further alterations include the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor, and the Sonic Hedgehog (Shh) signaling pathway [16]. Typically, GB displays dysfunction of genes, proteins, or pathways that control cell proliferation, cell-cell adhesion, and apoptosis (Table 1); which we will discuss in more detail below.
EGFR houses a combination of transmembrane (ligand-binding) and tyrosine kinase domains that control several downstream cellular pathways [17]. Thus, mutations in EGFR alter the downstream phosphatidylinositol-3-kinase (P13K/Akt) and the mitogen-activated protein kinase (MAPK) pathways [18]. The mammalian target of rapamycin (mTOR/P13K/Akt pathway) regulates cell cycle, apoptosis, cellular stress, and cell growth. In turn, Akt1 controls mTOR, which promotes protein biosynthesis of cyclin D1 [19]. The MAPK component ERK1/2 triggers a signaling cascade of the MAPK superfamily proteins that regulate cell cycle and cell proliferation [16, 20]. Research has also shown that EGFR colocalizes with the IQ motif-containing GTPase Activating Protein 1 (IQGAP1), a scaffold oncoprotein that regulates a plethora of cellular functions, including cell-cell contacts, cell motility, cell division and proliferation, protein traffic, and apoptosis [21, 22]; however, its underlying mechanisms in these various events are just emerging as discussed later below. PTEN is a tumor suppressor that has been identified as a frequent GB target because its mutations promote uncontrolled and rapid tumor progression [23]. Like EGFR, PTEN also modulates the mTOR/PI3k/Akt signaling through the conversion of PIP3 to PIP2 where the formation of PIP3 triggers the activation of mTOR/Akt, which activates key cell proliferation pathways [24] and modulates cell proliferation, apoptosis, and DNA repair [25]. PDGFA is a subunit that forms homo-, or hetero-dimers that are involved in embryogenesis, glioma cell development, and hematopoiesis [26]. In vivo studies in mice and rats have also shown that PDGFA can trigger oligodendrocyte precursor cells [27]. Further, overexpression of PDGFA in mouse models has led to GB development [28].
The retinoblastoma (RB) protein regulates the cell cycle through the arrest of the G1/S phase [16, 29]. RB proteins, when phosphorylated, do not bind to E2F, a transcription factor that promotes cell proliferation [29]. Conversely, when RB is not phosphorylated, the protein binds to E2F, thus inhibiting cell cycle progression into the S phase [29]. The RB pathway is altered in GB in many different ways, including homozygous deletion, promoter methylation, or mutation of pathway component proteins (16). The tumor suppressor p53 protein that controls cell proliferation and cell cycle progression is also altered in GB [30]. The p53 protein contributes to preventing damaged cells from propagating through the cell cycle [31]. Typically, p53 is altered in GB through deletions of the CDKN2A/ARF locus [30, 32]. Gene deletions within CDKN2B and CDKN2C, which encode tumor suppressor genes CDK4 and CDK6, promote uncontrolled cancer cell proliferation [18, 33]. The JAK/STAT signaling pathway regulates tumorigenic functions like angiogenesis and anti-apoptosis along with mediating cell responses to growth factors or cytokines [34, 35]. JAK proteins, activated by cytokine stimulation, phosphorylate STAT proteins to initiate pathway activation [35]. The STAT component includes a collection of transcription factors in the cytoplasm that are activated by phosphorylation [34]. In GB, secretion of interleukin (IL)-6, IL-8, and growth factors activate the STAT proteins and increase tumor proliferation [34, 36].
The transcription factor NF-κB controls cell proliferation, motility, and differentiation through downstream effector activation that includes EGFR, PGFR, and receptor tyrosine kinases [37, 38]. GB tumors exhibit increased NF-κB activation accompanied by tumor cell proliferation and macrophage-induced inflammation [38, 39]. The Shh is a signaling pathway that functions in embryonic development and tissue homeostasis [16]. The Shh mechanism of action includes the release of a glioma-associated oncogene homolog (GLI1) [16]. GLI1, a zinc finger protein, is stabilized in promoting tumorigenic pathways in coordination with Shh [40].
Table 1
Known Molecular Targets in Glioblastoma.
Molecule | Function | Reference(s) |
Epidermal Growth Factor Receptor (EGFR) | Receptor with tyrosinase kinase and ligand-binding activity; induces downstream cell proliferation | [18] |
Platelet Derived Growth Factor A (PDGFA) | Subunit of the PDGF gene umbrella (six subunits that form ligand and tyrosine kinase receptors); functions in neuroprotection, glial cell development, and hematopoiesis | [26] |
Phosphatase and TENsin homolog on chromosome 10q23 (PTEN) | Tumor suppressor gene; regulates cell proliferation, apoptosis, and DNA repair | [25] |
Rat Sarcoma Virus (Ras) | Collection of G-proteins; regulate intermediates with signal transduction and cell proliferation pathways | [113] |
Retinoblastoma Protein (RB) | Tumor suppressor protein; targets G1/S cell cycle checkpoint, and negatively regulates apoptosis | [114] |
Tumor protein p53 (p53) | Tumor suppressor protein; prevents malignant transformation of cancer cells and eliminates damaged cells | [115] |
Janus kinase/Signal transducer and activator of transcription (JAK/STAT) | Signaling pathway that activates transcription; triggers pro-tumorigenic functions: anti-apoptosis, cancer cell proliferation, and immune suppression | [34] |
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) | Collection of five transcription factors; control and trigger cell proliferation, motility, and differentiation | [37, 38] |
Sonic Hedgehog (Shh) | Signaling pathway within the hedgehog (Hh) domain; assists in organogenesis, cell homeostasis, and neural cell type specification | [116] |
Clearly, the normal cellular functions of these variable molecules are intertwined, and when one of them becomes aberrant, their function converges to induce or sustains the GB disease state. However, key questions remain, including whether the altered protein/pathway is a cause or a consequence of the tumor progression, and how the heterogeneity in GB evolves. These questions are particularly significant because the GB heterogeneity evolves over time, and the treatment itself induces further heterogeneity in the tumor [14]. For this purpose and others, several animal models have been developed that provide advantages as well as exhibit limitations in replicating the features of human GB, and that we consider below.