A total of 3810articles were identified using database searching, 3494 were recorded after duplicates removal. Three thousand sixty-six (3066) were excluded after screening of title/abstract, 202 were finally excluded (because when many separate articles were present with similar conclusions, only those were selected to be included which mainly focused on genes/ signaling pathways involved in gliogenesis and neurogenesis in relation to GBM development), 3 articles were excluded during data extraction.Finally 223 articles were included (based on the objectives of the study).
There are also limitations of this study as mentioned in the ‘Methodology’ ‘Study Design’ and other respective sections. The TP53, RB1, MDM2, NF1 or other such factors that already have well established role in glioblastoma development , they are not the primary focus of this study.
Based on the results of this study, it also addresses in the ‘Discussion’ section the increased predisposition of glial cells towards oncogenesis compared to neurons. This will help to understand the factors that put the glial cells such as astrocytes more towards at risk of oncogenesis such as glioblastoma development.
This study investigates the gliogenic genes and signaling pathways to trace the origins of GBM, it also focuses on related neurogenic genes/signaling pathways that play role in GBM oncogenesis. It focuses on the signaling pathways and genes that work in the form of combinatorial codes in cell type specific programming in gliogenesis and neurogenesis. This study also tries to map the landscape of genetic switches that lead to the origin of Glioblastoma, also illustrated in Fig. 1 (landscape of GBM onset and development).
GLIOGENIC GENES/SIGNALING PATHWAYS AND THEIR ROLE IN GLIOBLASTOMA DEVELOPMENT:
Key gliogenic genes having the ability to control oncogenesis in glioblastoma cells: p300, BMP, PAX6, HOPX, NRSF/REST, LIF, TGF beta.
- TGF beta: It has very important gliogenic effect. TGF beta family also includes BMP. The SMADS working with TGF beta, they inhibit G1 to S transitions and cause cytostasis. They also inhibit Myc  [92, 93, 94, 95, 96].
BMP: BMP-mediated PS-SMAD1/5/8 plays significant gliogenic role. BMPs switch the progenitor cells towards gliogenesis. It also downregulates the gene expression of EGFR. The CTNF, BMP and JAK-STAT mediated astroglial differentiation is disrupted in GBM development. The role of BMP signaling is anti-oncogenic as it causes the cell cycle to exit. CTNF and BMP dysregulations have downstream effects [29, 30]. It inhibits proliferation in GBM but stemness remains intact . Several studies have found that BMP signaling has cell-differentiation effect in GBM . BMP also works with STAT3 for astroglial differentiation. This regulation of STAT3 is very crucial as STAT3 over-activation is involved in GBM oncogenesis .
BMP with SMAD proteins: They contribute to gliogenesis in CNS development. The SMAD/TGF-beta together regulates the cell cycle and cause cytostasis by downregulating gene expression of c- Myc. This prevents G1 to S transitions . The importance of dysregulations in the regulators of cell cycle can also be best estimated by the fact that SMAD/TGF beta becomes oncogenic in GBM and fails to prevent G1 to S phase transition. The dysregulations in SMAD alters role of PDGF-B in GBM development. When Smad, PI3K and FoxG1 signaling pathways are dysregulated, they switch the TGF-beta to become oncogenic . EGFR-Akt-Smad signaling is also another way via which SMAD contributes to GBM development. TGF-β1 works with Smad, p38 MAPK, and PI3K/Akt signaling pathways in GBM development . TGF beta interacts with VEGF and FGF. This process is negatively regulated by PI3K pathway which is an inhibitor of FoxO localization in the nucleus. PI3K also becomes dysregulated in GBM development .
- PAX6 is neurogenic in development but with Nkx6.1 it contributes to astrocytogenesis:
PAX6 role is fascinating at many levels as it controls the cell proliferation and also plays role in regulating the cell cycle. There is inverse relationship of PAX6 with GBM development. When PAX6 is upregulated in GBM, it acts as a tumor suppressor [67, 68]. It is the combinatorial effect of multiple genesworking at specific timings that regulate the establishment and homeostasis of the cell fate. PAX6 is primarily neurogenic and works with Nkx6.1 in establishing some types of astrocytes . PAX6 also suppresses the invasiveness of GBM and controls the expression of matrix metalloproteinase-2 gene . PAX6 also controls the VEGF expression in GBM and prevents angiogenesis. It stops the cells from entering S-phase of cell cycle and also induces apoptosis. It also interacts with PTEN signaling pathways . The transcription factor PAX6 exerts a very strong neurogenic role. It has been proposed to be a regulator of balance between astrocytes and other glial cells . PAX6 also interacts with neurogenins to swing the stem cells towards neuronal commitment state.
In a 2018 study, CRISPR-CAs9 was used to study the role of PAX6 in GBM development. PAX6 has very strong neurogenic role and its expression has been found to work as tumor suppressor gene in GBM. The role of PAX6 is also pleiotropic in nature. Its signaling is involved in the differentiation processand is also capable of exertingimpact on GBM oncogenesis . Pax6 interacts with regulators of neural progenitor cells including Neurod1/4, Nestin, Neurog1/2, and Notch pathway components such as Dll1 and Hes6 .
It is very important to note that neuronal cells are the cell types with the least risk of tumor development while glial cells are most likely the cell types in which one of the most aggressive tumors originates, GBM. It is postulated here that there are many genes whose expression may provide a different effect in different cell types. This variability may have origin in cell fate specification based on the differentiation of cell types, such as PAX6 plays different roles in neurons and astrocytes. This is also evident in different disease states such as in Alzheimer’s disease and glioblastoma, in both diseasesthe genes such as p53 have different roles. In Alzheimer’s diseases p53 is upregulated while in GBM it is downregulated. PAX6 and Sox2 are involved in gene regulation of neural stem cells in neurogenesis. Both are also contributors in the commitment of ectodermal cells towards neuronal differentiation. Their contributions also go to the extent of inducing transcription factors that contribute to neurogenesis. They work in the form of gene regulatory circuitry. PAX6 is deeply interlinked with notch signaling in inducing neurogenesis. It is not properly understood yet the level of complexity with which they work together. One of their key roles that are of immense significance is the repression of non-neuronal cell types.
P300 is a very strong regulator of gliogenesis. It is also the repressor of nestin which is also involved with stemness (sox2). Its activation in GBM cell lines induces the cells more towards GFAP expression. The impact of P300 on the cell fate can be estimated from this fact. It is important to note here that c-myc gene overrides p300 and then GFAP, leading to upregulation of nestin. This plays very important role in GBM oncogenesis .
- Neuronal Restrictive Silencing Factor (NRSF)/REST:
It halts neurogenesis and inducesgliogenesis. Initially it was considered to function only to repress neuronal genes in non-neuronal cells. Now its presence in many different cell types has emerged. It is expressed in neural progenitor cells (NPCs) but negatively regulates neurogenesis. It has also been found to be involved in many cancers . It is considered to be contributory towards the neurodegeneration in Alzheimer’s disease as it is downregulated in Alzheimer’s disease neurons. It has been found that REST remains in quiescent state in differentiated neurons but its expression increases after neuronal injury. With aging, there is always present low level of REST gene expression in the neurons because its absence may fuel Alzheimer’s disease. REST also reduces beta amyloid toxicity and apoptotic signaling . In GBM, it contributes to more invasive and proliferative properties of GBM. It also contributes to renewal of GBM stem cells and is oncogenic in GBM landscape .
REST amplification is also implicated in GBM and has been found to suppress apoptosis in GBM cells. It also promotes stemness in GBM. Though NRSF/REST is considered to have more profound role in gliogenesis but in medulloblastoma where neuronal pathways are involved, its over-expression has been detected as it is linked to enhancing proliferative capabilities. REST based stemness has been detected in both neuronal tumors such as medulloblastoma and glial tumors such as GBM. REST also interacts with shh, wnt and PI3K signaling pathways. All of them have well-established role in GBM oncogenesis. In adult neurons, the gene expression of REST remains low so as to promote the gene expression of neuron specific genes which REST can downregulate. But REST is upregulated many folds in medulloblastoma cells, the role of REST overexpression is very important as blocking the REST gene expression results in revival of neuronal genes and also promotes apoptosis . REST overexpression is also part of GBM. It also provides a key finding about pro-neural type of GBM as when P53 and REST are deleted; it switches the cells to change into pro-neural type of GBM . REST in normal cells contributes to genomic integrity, but in cancer development it is oncogenic. It also contributes to invasion, stemness and also regulates apoptosis . NRSF/REST is highly upregulated in NSCs to maintain stemness and prevent neuronal differentiation. Its gene expression downregulation contributes to drive the neuronal differentiation. But after neuronal differentiation, a basal level of NRSF/REST expression is maintained all the life and increases with aging. There is aging related increase in REST expression in brain cells including neurons and astrocytes. REST has strong interactions with WNT signaling pathways and both have significant oncogenic role in GBM. It also downregulates the genes involved in apoptosis .
The differences in REST gene expression are likely one of the key reasons of different proliferative potentials between two cell types, i.e. neurons and glial cells. It is postulated here that as REST is gliogenic and is not similarly expressed in neuronal cells which are considered permanent cells, hence the direction of cell circuitry guided by REST tends to have high proliferative potential. The REST based proliferative potential is so strong that it is also involved in tumor development in neuronal cell type (medulloblastoma) by silencing the key neuronal cell type specific genes. NRSF/REST works as one complex developmental expression program. It has been found to be highly expressed in almost all non-neuronal cells. Hence, it is also considered to be a master regulator of transcription. One of its most significant roles is to regulate gene networks that are involved in maintaining pluripotency in embryonic stem cells.
- Leukemia inhibitor factor (LIF): Normally it inhibits differentiation but its low levels cause differentiation. In GBM, TGF-beta signaling causes it to become pro-oncogenic. Although when LIF is applied alone to cell lines, it causes growth inhibition in GBM .
Genes that have gliogenic roles but in GBM development contribute to the oncogenesis: IL-6 family, FGFR 3, JAK-STAT pathway, STAT3, S100, hey1, HES1, DTX, NF-κB, Neuregulin-1, MAPK, MEK, E2F, TCFL2, NFIX TF, Ephrins (EFNB1) and Netrins (NTN).
- FGFR signaling pathway: FGFR3 is one of the mitogenic drivers in GBM development. It also contributes to further driver mutations. In GBM disease development, it activates very crucial pathways such as AKT, Ras-Raf-MEK-ERK pathway . FGFR signaling pathway is profoundly involved in cancers including brain cancer such as GBM . In GBM, their role is complex and is based on their ability to contribute towards glial differentiation. FGFR signaling pathway has strong interactions with MAPK which is involved in gliogenesis and also in GBM development. FGFR2 is involved in gliogenic differentiation, its activation in glioma cells causes differentiation of brain glioma cells . FGFR 1 and 2 are expressed in neurons and astrocytes respectively. Their expression decreases with grade of glioma [13, 14, 15]. It is important to mention here that IGF-1, FGF, PDGF and EGF, all are based on receptor tyrosine kinase and they all have significant roles in GBM development. FGFswitches NSCs towards glial fate in embryogenesis. The cell fate based switching is through MAPK pathway. FGF is also involved in GBM. FGF also works with IGF to regulate MAPK pathway. The role of IGF-1 in cancer development is well established. IGFR signaling pathway interacts with AKT to further GBM proliferation .
- JAK-STAT pathway: It is a key gliogenic pathway. It interacts with PI3K/AKT/mTOR Pathway. It has ability to integrate with MAPK/ERK pathway. There are negative regulators that keep JAK-STAT in check such as tumor suppressor genes. In GBM, JAK-STAT becomes oncogenic. It also contributes to the stem cell maintenance. The JAK-STAT pathway downstream targets include Bcl-xL, Bcl-2, cyclin D1, and c-Myc, and EGFR amplification which lead to further dysregulated STAT3 signaling. Dysregulated EGFR, FGF, PDGF, c-MET, also activate STAT-3 signaling. The EGFR based negative regulatory mechanisms become dysregulated in GBM [16, 17]. The dysregulations in JAK/STAT signaling lead to upregulation of pluripotency related genes including oct4, c-Myc, Nanog, Sox2 [18, 19, 20, 21, 22]. Sox2 is also involved with JAK-STAT signaling pathway in maintaining pluripotency. TGF Beta, SMAD, and IL6 family also interact significantly with JAK-STAT pathway [23, 24, 25, 26, 27, 28].Damage to RB2/p130 diminishes its tumor suppressor effect and contributes to GBM progression .
The role of JAK-STAT pathway and STAT3 is well established in gliogenesis. Oncogenic mutations also dictate the role of STAT3 in relation to its interactions such as with PTEN-Akt-FOXO axis (suppressive) and with Leukemia inhibitory factor receptor beta (oncogenic). It also interacts with EGFR which plays key role in GBM development [42, 43]. The knockdown of STAT3 or SRF significantly suppressestumor invasive properties. It also interacts with HOPX which is gliogenic and also expresses tumor suppressive effects .
It is postulated here that genes and pathways including STAT3 have multiple biological roles which are also guided by the timing and gene expression of other factors that regulate the cell cycle. In tumor microenvironment, a physiologic pathway may go on to become oncogenic. Their integrated relationship with one another decides the maintenance of cell fate or deviation of cells from their cell fate.
- IL- 6 family: In GBM, it works with STAT3 in promoting pro-oncogenic pathways . It is to note that cytokines IL-2, IL-6, IFN, all involve JAK-STAT based non-receptor tyrosine kinase signaling. With age, the gene expression of inflammatory cytokines increase in the body. In GBM, CTNF and its receptor have been found in GBM .
CTNFR alpha is pro-glioma and is linked to the grade of glioma. It is considered to have role in initiation or GBM maintenance [7, 8]. CNTF-mediated JAK-STAT pathway contributes to astrocytogenesis as well.
- GFAPand S100:
They are involved in astrocytogenesis and are also upregulated in GBM, indicating the cell type specific contributions to GBM development. GFAP also regulate astrocyte neuronal interactions .
- Notch targets Hey1:
One of the key targets of Notch signaling is Hey1 and this is also upregulated in GBM. Notch signaling in later stages stimulates gliogenesis [50, 51, 52, 53]. It is postulated here the timings of gene activity and microenvironment impact profoundly in the process of maintaining cellular homeostasis and in disease development. The dysregulations in gene expression of Notch also induce profound impact on the genes it regulates and the pathological effect goes further in downstream pathways.
- Interactions of Hes1 with STAT3 and JAK2:
These interactions play very important role in gliogenic developmental mechanisms. But dysregulated expression of even the key genes including Notch1, Hes1, and DTX1 contributes to GBM pathogenesis [54, 55, 56]. Dysregulated Hes1 plays role in stemness and EMT induction in GBM development. Shh also upregulates Hes1 gene expression . SHH will be discussed later.
In understanding GBM, it is of immense significance to understand interconnectedness of signaling pathways and genes. The interactions of Notch are very diverse as it also interacts with Hes3 signaling axis and STAT3, and plays physiologic role of being the regulator of Neural Progenitor Cells (NPCs). In GBM, Notch, STAT3, Hes3 axis all become dysregulated.
- NF-κB harms neural stem cells (NSCs) and gliogenesis potential:
The role of inflammation in GBM oncogenesis and its impact on the GBM genetic landscape is of immense significance. It is already well established that the levels of inflammation in body increases with aging. NF-κB which also acts as one of the prime regulators of inflammation, it gets upregulated. And NF-κB has been found to play very important role in GBM oncogenesis as it also plays role in EGFR amplification. The EGFR amplifications also contribute to the pyruvate kinase m2 (PKM2) dysregulations, resulting in the upregulation of this rate limiting enzyme of glycolysis in GBM. In GBM, NF-κB and EGFR interactions contribute to GBM development, invasiveness and progression .
There is also depletion of negative regulators of NF-κB such as KLF6. The upregulation of NF-κB in GBM also contributes to Epithelial Mesenchymal Transition (EMT) and GBM stemness. The NF-κB is linked with TNF alpha and IL1 expression. It interacts with MAPK, Tyrosine Kinase-R, EFGR, PDGF and AKt signaling pathways. They are also part of GBM landscape. The NF-κB signaling promotes IL-6, IL-8 and VEGF, further fueling GBM development. It also interacts with PI3K/AKT pathway. With loss of PTEN and NF1 which are well established contributors to GBM development, the PI3K/AKT becomes upregulated. Similarly like other tumor suppressors such as PTEN, NF1 and others, also loss of P53 contributes to GBM oncogenesis. The dysregulations in P53 also contribute to NF-κB upregulations. NF-κB also interacts with cascades of genes that are involved in cell survival including Bcl-xL, Bcl2, inhibitor of apoptosis proteins and survivin,cyclin D1 and others. The NF-κB in GBM has strong interactions with EFGR, PDGFR and AKT signaling pathways. The loss of tumor suppressor in NF1 also causes RAS over-activation . PTEN regulates PI3k and blocks AKT signaling. The dysregulations in P53 also contribute to Mdm amplifications in GBM oncogenesis. The NF-κB also disrupts the notch signaling and promotes the cell signaling through cell proliferation with CyclinD1 .
It is important to note here that NF-κB signaling upregulation is also involved with loss of tumor suppressors. It is also postulated here that inflammatory signaling cascades have more profound role in GBM oncogenesis than previously assumed. As aging also increases inflammation and most cases of GBM have onset with increased age, hence role of aging is of immense significance in GBM development.
- Neuregulin-1 is a gene of EGF family and contributes to astrocytogenesis:
Nrg1 and erb receptor signaling pathways interact with each other and also with PI3K, contributing to the growth of GBM. The role of EGFR amplification is already well established in GBM [78, 79, 80, 81]. Nrg1, TGF alpha, EGFR all have profound interactions with one another and impact PI3k/AKT pathway, MAPK and JAK/STAT pathway .
It is postulated here that Nrg1 is very significant in GBM landscape as it is involved in astrocytogenesis during embryonic development but in GBM landscape, it becomes oncogenic. It signifies the role of cell fate specific genetic programming that regulates the unique gene transcripts in a specific manner. In GBM, when dysregulations in signaling pathways and genes accumulate beyond a specific threshold then the role of many factors such as Nrg1 may become dysregulated. This further fuels GBM development and progression. The FGFR3 has major gliogenic contributions, but in GBM development it also loses the cell fate specification related effects. As the cell fate is determined by combinatorial code based specific unique set of gene programming, this loss of cell type specific gene expression is able to swing FGFR3 towards becoming oncogenic.
- MAPK: In GBM, it interacts with EGFR, mTOR/PI3K/Akt and RAS. It also works with PDGFR. MAPK is also involved In Insulin resistance. The pathway mTOR stimulates glucose uptake in GBM and works with Akt pathway.
- MEK and E2F: RAS over-expresses MEK in GBM. Rb1 interacts with CDKs in inhibiting E2F. This pathway becomes damaged in GBM and CDKs cause E2F based G1 to S transitions [98, 99].
- TCFL2: TCF/LEF transcription factors of WNT signaling works with beta catenin and have been detected in GBM samples [100, 101, 102].
- Ephrins (EFNB1) and Netrins (NTN): These pathways related proteins are involved in gliogenesis. Their role also includes angiogenic activity including angiopoietin-2 (ANGPT2), EFNB1 and FGF. The dysregulations in Ephs and Ephrins are involved in GBM development.Their downstream signaling includes MAPK, ERK, RAS, AK, FGF, MEK, PI3k/Akt/mTOR and others .
Netrins over-expression contributes to the increased proliferation of GBM cell lines. It is also involved in gain of stemness in GBM. NTN-1 activates Notch signaling in GBM and contributes to oncogenesis. This increases the expression of CD133, nestin and Sox2 in GBM stem cells . Netrins also interact with EGFR.This reduces partially the GBM cell senescence that occurs from DNA damage. This EGFR based role is mediated by AKT and ERK signaling pathways.
- Transcription factors NFIX and HOPX with GFP control: This is involved in gliogenesis and is dysregulated in GBM.
NFIX TF interacts with STAT3 and has been found to be upregulated in GBM. NFIX works with Ezrin protein that is involved in cross-linking of cytoskeleton and plasma membrane; both are dysregulated in GBM .
HOPX is primarily astrocytogenic and also works as tumor suppressor in time dependent manner. HOPX is also involved in neural stem cells and is downregulated in solid tumors. This downregulation promotes tumor invasiveness and growth. It is important to note that HOPX over-expression is involved in suppression of cancer cell proliferation and metastasis. Key genes of interest with which HOPX interacts include ASCL2, NKX2-1. HOPX contributes to regulation of NOTCH and also causes senescence by activating Ras and MAPK pathway. It is also involved in upregulating klf4 in promoting tissue homeostasis. Klf4 is involved in pluripotency and time dependent switching of astrocytes into neurons. It also regulates apoptosis in stem cells. When it becomes dysfunctional, stem cell apoptosis decreases and more neurons are produced. Its upregulated gene expression promotes apoptosis. HOPX also contributes to neurogenesis by modulating NOTCH pathway signaling but it plays more profound role in gliogenesis .
It is postulated here that role of HOPX is far different than other gliogenic factors that become oncogenic when dysregulated including Notch, FGFR3 and others. Its effect on cancer cells is tumor suppressive. It keeps NSCs in quiescent stage, also regulates proliferation and differentiation . GBM lacks expression of HOPX and its induced expression causes tumor suppressor effect in many GBM cell lines by decreasing survival but proliferation remains unaffected .
Key genes that contribute to gliogenesis but also contribute to the stemness in GBM: Notch (cancer stem cells), Sox9, Sox4, SHH.
- Notch signaling pathway: In gliogenesis, Notch effector protein (NIFA) binds to GFAP promotor and contributes to astrocytogenesis. EGF motifs and notch signaling are interlinked. In GBM, EGFR amplifications are one of the most significant parts of GBM oncogenesis. GBM cells also have presence of cancer stem cells . Notch2 expression is involved with proliferating cells that function as progenitors. Notch1 gene expression is involved in post-mitotic cells that are in differentiated state. In GBM, Notch1 promotes Akt signaling, also causesβ-catenin and NF-κB upregulation. In GBM, Notch2 is also upregulated but contributes to GBM stemness. Notch signaling pathway also interacts with stemness genes (nestin, SOX2), with vimentinand GFAP that are involved in astrocyte fate. It also inhibits apoptosis . Dlls are linked with Notch signaling. They are upregulated in GBM and contribute to oncogenesis [124, 125, 126]. Nestin upregulation contributes to GBM stem cells [127, 128, 129].
Notch signaling is regulator of EGFR and EGFR amplifications are also modulated by dysregulations in TP53. Notch signaling and EGFR, both are upregulated in GBM oncogenesis. Importance of notch signaling in developmental mechanisms and its impact on GBM development both are very significant part of GBM research. They both work like a positive feedback signaling loop. EGFR interacts with RAS-RAF-MEK-ERK and the PI3K-AKT-mTOR cascades.It also interacts with CDKs. Inhibition of notch also promotes apoptosis in GBM cells. Notch inhibition also decreases gene expression of Akt and Stat3. Delta/Notch-like epidermal growth factor-related receptor (DNER) is involved in GBM progression. The notch signaling pathway also regulates neural stem cells (NSCs). Though Notch is also involved in cell fate decisions in gliogenesis but in GBM microenvironment, it makes tumor cells more invasive and increases de-differentiation. Higher grade of gliomas have been linked with increased gene expression of ASCL1, Dll1, notch1, notch3, notch4, and hey1. The ASCL1 is also used as a transcription factor in the switching of astrocytes to neurons. Dysregulated Notch also interacts with other important developmental genes and gene expression levels of Notch1, Notch4, Dll1, Dll4, Jagged1, CBF1, Hey1, Hey2 and Hes1 are upregulated in GBM oncogenesis. There is also simultaneous decrease in PTEN expression, resulting in upregulation of Akt and VEGF. The notch mediates overexpression of genes including Zeb1, and Snail, vimentin. This enhances invasiveness of GBM. The notch signaling also interacts with Hey1, PI3K/AKT/mTOR and ERK/MAPK pathway. All of them also act as proliferative and survival signaling . Hey1 enhances GBM survival and interacts with dysregulated Notch and E2F signaling. It is considered that the notch signaling is one of those key pathways whose inhibition can promote GBM cells to differentiate and promote apoptosis. Despite having such crucial role, still inhibition of notch signaling pathway doesn’t reverse GBM oncogenesis . Notch signaling is an important player in gliogenesis. The oncogenic alterations in notch signaling pathway sets in motion a cascade of pathological downstream dysregulation such as hey1 gene which interacts and works with notch signaling, its gene expression is increased in GBM development .
NOTCH works with FGF to keep NSCs in proliferative stage. Neural progenitor cells (NPCs) are regulated by notch signaling, it is also involved in their maintenance and renewal. But later in developmental, NOTCH contributes to glial cell differentiation.
- SOX9 and SOX4:
It is involved in gliogenesis. The pathways Shh and Notch use Sox9 as regulator of morphogenesis. It also works with SOX10 for maintenance multi-potency of neural crest cells. It also differentiates NSCs into non-neuronal cells. Sox9 upregulation contributes to oncogenesis in GBM and interacts with wnt/beta catenin for progression of GBM. Knockdown of Sox9 in GBM impairs the proliferation of glial cell types and cause apoptosis [39, 40]. Sox9 interacts with EGFR, BMI-1, PTEN. Other SOX including Sox5, Sox6, and Sox17 also contribute to GBM. SOX4 in Glioblastoma sustains stemness and is regulated by TGF-β. It also modulates SOX2. The SOX2 in glioblastoma maintains stemness and oncogenic properties. Sox members in GBM also interact with TGF beta and are involved in reprogramming of GBM stem cells .
It is postulated here that factors that swing the stem cells towards non-neuronal cell types which are not permanent in nature and have proliferative capacity, they are at risk of oncogenesis later in life. Many genes such as Sox9 that are gliogenic in embryonic development, they may become contributor to GBM oncogenesis because of dysregulations in cell circuitry at the time of GBM development. Such dysregulations change the direction of cell fate and they become more and more undifferentiated. It is important to note that sox9 and NIFA together work as regulator of key gliogenic genes in embryonic development. Their over-expression cause astrocytic differentiation in GBM cells. Many pathways that work in a combinatorial manner, they may get dysregulated with aging. Such dysregulations in cell circuitry lead to disease development such as GBM. The transcription factor NIFA, key initiator of gliogenesis works with Notch and Hes signaling pathway and pathological dysregulations in one key pathway lead to further downstream damage. It is further discussed in later parts of the study.
Astrocytogenesis is linked with inhibition of neurogenic signals in Neural Stem Cells (NSCs). While SHH is secreted by neurons and work more as a connecting bridge between neurons and glial cell. It works with Nkx2.2 in defining the identity of neural progenitor cells. But still it also contributes to the development of glial cells as well . Sox9 is downstream effector of Notch and SHH. The role of SHH in GBM becomes more significant as Hes1 that is also involved in stemness of GBM, is also upregulated by SHH [59, 60, 61].
The role of PTEN is very significant as it also contributes towards controlling the SHH and PI3K expression. When PTEN becomes defective in GBM, then SHH and PI3K become dysregulated and both contribute to GBM oncogenesis .
SHH works as a morphogen but its sensitivity in neural cells is determined by BMPs. SHH have more profound role in neurogenesis compared to gliogenesis. SHH regulates and induces a variety of regulatory genes including 2, Olig2, Nkx6.1, Nkx6.2, Dbx1, Dbx2, Irx3, Pax6, and Pax7. This is mentioned here to show the far reaching impact of SHH dysregulation in GBM development. Cross talk of SHH with many key pathways and genes, play major role in regulating the activity of all of them. Such major interactions include GSK3 beta, mitogen activated protein kinase (Mek1), PKA and protein kinase C (PKC), Phosphoinositide-3 kinase (PI3K), CK1 or dual specificity Yak1-related kinase (DYRK1) and others. SHH is also modulated by these interactions. SHH also has major interactions with key signaling pathways including TGF-beta, wnt/beta catenin and notch.It also interacts with K-RAS, PKA and EGFR. The signaling pathway SHH has profound regulatory interactions with GSK3 beta as it can act both as negative regulator and positive regulator of SHH depending on the microenvironment. Many major signaling pathways and key genes have regulatory interactions with shh, notch,wnt signaling, ERK signaling pathway, wnt/β-catenin and KRAS, TGF-β/TGF-βR, EGFR, and platelet-derived growth factor receptor α (PDGFRα). In GBM development, their dysregulation plays key role in progression of the oncogenesis [63, 64].
EGFR that is considered to play key role in GBM development, its cross–talk with SHH in GBM further fuels oncogenesis. SHH dysregulations are capable of inducing the profound impact on the gene expression of EGFR/RAS/RAF/MEK/ERK in different cancer cell lines . The EGFR signaling downregulates the gene expression of negative regulators of Shh signaling, further fueling cascade of downstream changes by impacting the gene expression of Akt, ERK and others .
It is postulated here that as cell fate is determined by unique combinations of transcription factors and regulatory genes. When such key genes and signaling pathways including SHH become dysregulated, the cells switch towards de-differentiation. This downstream cascade of damage works like a positive feedback loop that keeps switching the cells towards higher grade and more aggressiveness. There exists a very delicate balance among the expression of different genes which exert their effect on one another. For example, PTEN acts as tumor suppressor and it also impacts SHH and PI3K. When PTEN is damaged in GBM development, then it produces number of downstream pathological changes. The role of SHH in inducing and determining the cell fate is concentration and time dependent. The dysregulations in signaling pathways such as SHH in GBM landscape and development, they can set in motion a cascade of oncogenic changes. This cascade is based on the interactions of genes involved and their roles in cell circuitry. The negative and positive regulatory effects of genes and signaling pathways are of major significance as dysregulations in one can set in motion a cascade of pathologic changes. Such changes unfold step by step and in time dependent manner influenced by their microenvironment.
RELATED NEUROGENIC GENES/SIGNALING PATHWAYS AND THEIR ROLE IN GBM ONCOGENESIS:
GBM cells maintain low or null expression of neurogenic genes.Here we investigate their significance in GBM .
Key neurogenic genes having the ability to control oncogenesis in Glioblastoma cells: PAX6,Neurogenins including Ngn1, NeuroD1, NeuroD4, Numb, NKX6-1 Ebf, Myt1, ASCL1.
- Neurogenins including Ngn1: It prevents the interaction between p300/CBP complex and STAT3, thus preventing gliogenesis and promoting neuronal differentiation. In GBM, its expression causes mitotic arrest [111, 112].
- NeuroD: It is also the most potent key regulator of neurogenesis and is involved in the differentiation of neuron. In GBM, its induced gene expression also blocks the proliferation. NeuroD1 is so potent that it is able to switch reactive glial cells into neurons. Key interactions include Cyclin D1, MAFA and MAP3K10.The NeuroD1 and NeuroD4 are key neurogenic pathways. Their upregulation cause arrest of cell cycle in GBM cells.
But the BDNF works in close association with NeuroD and activates the neurogenic SHP2-Ras-Raf-MEK-ERK pathway. This interferes with gliogenic JAK-STAT signaling. The neurogenic SHP2 is linked with potentiation of MEK-ERK signaling and inhibits gliogenic JAK-STAT signaling. In GBM, SHP2 becomes dysregulated and oncogenic .
- BMP in neuronal development:
It is involved primarily in the induction of neurogenin and ASCL1. The BMPS are involved in both gliogenesis and neurogenesis, but play a major role in switching NSCs towards astrocytogenesis. And its expression is also present postnatally in astrocytes. BMP upregulation is considered to play role in halting GBM progression [130, 131].
- PAX6: It is neurogenic but in GBM developmentits expression is like a tumor suppressor. It interacts with neurogenins to swing the neural stem cells towards neural commitment state and is involved in the repression of non-neuronal cell types. When weak Shh signaling combines with strong TGF-beta signaling, the PAX6 becomes upregulated. PAX6 expression is maintained in many areas of brain throughout life [67, 68] .
- Numb gene negates Notch signaling and contributes to neuronal differentiation:
Upregulation of Numb gene contributes to halting the GBM growth and progression. It also upregulates the Pax6 and Sox2. It also interacts with EGFR. But the functions of Numb are so diverse and complex as it has also been found to be highly upregulated in mesenchymal GBM cells . But this may be due to the fact that despite gene expression upregulation GBM oncogenesis is beyond the control of numb. But still its tumor suppressor role is controversial .
- Nkx6.1: Increasing evidence indicates that it suppresses tumor development and metastasis [117, 118].
- ASCL1: It is strongly neurogenic. In GBM, its expression causes cascade of downstream changes and switch the cells towards neuronal cell fate. It also suppresses oncogenesis in GBM cells .
- Ebf: its loss in GBM contributes to oncogenesis because EBF3 downregulates the gene expression of proliferation and survival related genes including cyclins, CDKs, Mcl-1 and Daxx. It also upregulates genes involved in cell cycle arrest including p21, p27 .
- Myt1: It is neurogenic transcriptions factor. In GBM, its expression is involved in downregulating proliferation .
Genes that have neurogenic roles but in GBM development contributes to oncogenesis: PDGF, NT3, Pax7, Dbx2, hes6, Runx1 and Runx2.
- PDGF and NT3: Both are neurogenic, but in GBM landscape they play oncogenic role .
- CNS neural progenitor markers:Pax7 and Dbx2
Pax7 becomes upregulated in GBM with PTEN deficiency  and high DBX2 in GBM is linked with low survival .
- Hes6: Upregulated in GBM .
- Runx1 and Runx2: In GBM, there role is oncogenic [122, 123].
Key genes that contribute to neurogenesis but also contribute to the stemness in GBM: klf4, myc, oct2, sox4, TLF TF, wnt signaling, sox2, sox4, Notch/STAT3-Ser/Hes3 Axis, GSK3beta also works with PI3K/FGF signaling pathway and contributes to stability of c-myc.
- Wnt signaling: It is kept in check by tumor suppressors and is more involved in neurogenesis. It is also involved in insulin based increase in glucose transporters. This increase is mediated by activation of wnt/beta catenin signaling pathway. It also plays role in diabetes type2 pathogenesis. In neurogenesis, wnt acts on Pax6-Ngn2-Tbr2-NeuroD-Tbr1 based neurogenesis TFs cascade. The wnt signaling represses astrogliogenesis via ngn2-dependent direct suppression of astrocyte gene expression. When wnt is inhibited, this promotes gliogenesis.Wnt1 works as antagonist of neural differentiation and promotes the proliferation of neural stem cells (NSCs).
But in GBM landscape, it becomes highly oncogenic. Cancer Stem cells are heavily associated with WNT signaling. Dysregulated wnt signaling causes activation of CyclinD1 and c-myc, causing G1 to S phase transition. It also contributes to Epithelial to Mesenchymal transitions [145, 146, 147].
It has already been discussed in other sections too. Here we point out the integrated nature of GSK3beta interactions in normal cells and cancer cells. It also focuses on interconnectedness of beta catenin and GSK3-beta in development of GBM. The GSK3 and more specifically GSK3beta play a very significant role in normal cells and cancer cells. In normal cells, it acts as negative regulator of Epithelial-Mesenchymal-Transitions (EMTs) and many proto-oncogenes.But dysregulated GSK3beta is oncogenic .
Even in Alzheimer’s diseases, it plays a very important role by forming a complex with p53 and contributes to neurodegeneration . In developmental biology, GSK3 deletions contribute towards the enhanced proliferations of neural progenitor cells with simultaneous increase in SOX2 and beta-catenin expression. The GSK3 activation is involved in neuronal differentiation . GSK3-beta has important interactions with beta-catenin which is capable of inhibiting neuronal differentiation. It also works with PI3K/FGF signaling pathway and contributes to the stability of c-Myc. In GBM, all of them become dysregulated . In GBM, dysregulated GSK3-beta also acts to downregulate BMP that has significant gliogenic roles.It also downregulates CDKN1A and other important genes. GSK3 has interactions with many genes and pathways that are involved in GBM such as PI3k/AKT/mTOR pathway, wnt, notch, shh, ras, raf, mek, erk, APC, axin and others. It also interacts with key tumor suppressors including p53, PTEN and others [160, 161]. Though its main role is with neural progenitor cells and neurogenesis, but it is estimated that it may also have contributions towards gliogenesis .
There may be a possible relationship between GBM and diabetes type2. The GSK3 plays significant role in both the diseases. Inactivation of GSK3-Beta contributes to the insulin resistance. As astrocytes are glycogen storing cells and GSK3beta also has major association with PI3k/AKT pathway in diabetes type 2 and in GBM, this should be evaluated in great detail in future studies. This may point to some possible significance or relationship between diabetes type2 and GBM as the risk of both increases exponentially with age. Similarly, wnt signaling dysregulations have also been associated with diabetes type2. The MAPK, PI3k, AKT, mTOR and many other common genes/signaling pathways that are involved in disease development mechanisms of both GBM and Diabetes type 2,they should be investigated for any possible profound relationship. Investigating it any further is beyond the scope of this study.
- Myc: It is also involved in neurogenesis but is oncogenic in GBM development . It is important to note here that myc gene overrides p300 and then GFAP, leading to upregulation of nestin. This plays very important role in GBM oncogenesis . GSK3beta also works with PI3K/FGF signaling pathway and contributes to stability of c-Myc.
- Pluripotency in NSCs and brief look on their role in GBM: SOX2 and SOX4
Sox2 is associated with regulation and maintenance of neural stem cells. It becomes downregulated during later stages of differentiation. The role of Sox2 in neurogenesis is very important because Sox2 also regulates the gene expression of other key genes. SOX2 induces ASCL1 and TLX TF. It alone is a very strong switch to neurons from astrocytes. In GBM, stemness is mediated by SOX2 and SOX4. They are also modulated by TGF-beta . It also has several important key interactions with LIF signaling and Klf4. The dysregulations in both of them are also involved in development of GBM stem cells and invasiveness. The role of wnt signaling has already been explored in earlier sections of this study. Nanog, oct4, myc, they are also major contributors to GBM stemness .
- Klf4: Neurogenic but acts like oncogene in GBM .
- SOX2 induces TLX TF: TLX transcription factor works like an oncogene in GBM. It inhibits TGF-beta that causes cytostasis and this leads to the progression of oncogenesis [143, 144].
- Oct4: It is involved in pluripotency and stemness. Its expression is several fold upregulated in GBM. The FGF signaling pathway is involved in neuronal cell fate determination despite presence of oct-4 gene expression. Without FGF signaling, the neural progenitor cell can revert back to embryonic stem cell like state with predominant oct-4 expression [137, 138, 139, 140, 141, 142].
- Notch/STAT3-Ser/Hes3 Axis:
This is neurogenic axis but it is linked to cancer development and diabetes. In GBM, it impacts the cascades of downstream signaling pathways. This signaling axis is important regulator of NSCs. Its major activators include insulin, Tie2 (involved in angiogenesis) and Notch. The upregulation of this axis has the ability to oppose the development of neurodegenerative diseases [150, 151, 152, 153, 154, 155].
Hes3 has role of pro-survival signaling. It works with a strong relationship to SHH, and potentiates FGF and EGFR signaling. It is one of the key regulators with ability to reprogram cells into NSCs. In GBM, this signaling axis becomes severely dysregulated.
GLIOGENIC GENES/SIGNALING PATHWAYS AND THEIR ROLE IN GLIOBLASTOMA DEVELOPMENT:
- Key gliogenic genes having the ability to control oncogenesis in glioblastoma cells: p300, BMP, PAX6(Anti-GBM override), HOPX (tumor suppressive + differentiation), NRSF/REST (astrocytogenic but capable of playing oncogenic role), LIF, TGF beta.
- Genes that have gliogenic roles but in glioblastoma development contribute to the oncogenesis: IL-6, FGFR 3, JAK-STAT pathway, STAT3, S100, hey1, HES1, and DTX, NF-κB, Neuregulin-1, MAPK, MEK, E2F, TCFL2, NFIX TF, Ephrins (EFNB1) and Netrins (NTN)
- Key genes that contribute to gliogenesis but also contribute to stemness in GBM: Notch (cancer stem cells), Sox9, Sox4, SHH.
Note: Other stemness related genes such as nanog, oct4, FGF2 and others also get upregulated in GBM and contribute to the stemness in glioblastoma oncogenesis. But the prime focus of this study is to focus on key gliogenic and neurogenic genes/signaling pathways that are involved in cell fate maintenance and differentiation. This study focuses on investigating their role in GBM development and progression.
RELATED NEUROGENIC GENES/SIGNALING PATHWAYS AND THEIR ROLE IN GBM ONCOGENESIS:
- Key neurogenic genes having the ability to control oncogenesis in glioblastoma cells: PAX6, neurogenins including Ngn1, NeuroD1, NeuroD4, Numb, NKX6-1 Ebf, Myt1, ASCL1.
- Genes that have neurogenic roles but in GBM development contribute to the process of oncogenesis: PDGF, NT3, Pax7, Dbx2, hes6, Runx1 and Runx2.
- Key genes that contribute to neurogenesis but also contribute to the stemness in GBM: klf4, c-myc, oct2/4, TLF TF, wnt signaling, sox2, sox4, Notch/STAT3-Ser/Hes3 Axis, GSK3-beta works with PI3K/FGF signaling pathway and contributes to the stability of c-Myc.
GLIOBLASTOMA CORE PATHWAY ABNORMALITIES:
Furthermore, glioblastomas are broadly divided into the following core pathway abnormalities. They are focused here to a limited extent because further focus is beyond the scope of this study.
1. RTK/RAS/PI3K signal alteration
- Receptor Tyrosine Kinase in GBM and gliogenesis/neurogenesis:
The most significant RTKs involved in glioblastoma include EGFR, IGFR, PDGFR and VEGFR .
EGFR: Its amplification is considered to play a key role in GBM development. In gliogenesis, JAK-STAT pathway interacts with EGFR. Dysregulated EGFR also activates STAT3 signaling. Both are considered to be important players in the glioblastoma development [16, 17]. The negative regulatory mechanisms of EGFR become dysregulated in GBM .
BMPs act as gliogenic regulator in the process of gliogenesis.They act as negative regulator of EGFR. It is important to remember that BMPs cause the cell cycle to exit [29, 30].Notch signaling is involved in astrocytogenesis. The EGFR is also interlinked with NOTCH signaling. Notch signaling is regulator of EGFR and this EGFR amplification is also modulated by dysregulations in TP53. The notch signaling and EGFR, both are upregulated in GBM. EGFR interacts with RAS-RAF-MEK-ERK and the PI3K-AKT-mTOR cascades.It also interacts with CDKs.
SOX9 is involved in gliogenesis and its dysregulation contributes to stemness in GBM. SOX9 interacts with EGFR in GBM oncogenesis .EGFR-AKT-Smad signaling is also another way via which SMAD contributes to GBM .
EGFR is considered to play key role in GBM development. Its cross–talk with SHH in GBM, further fuels oncogenesis. The EGFR signaling down-regulates the gene expression of negative regulators of Shh signaling pathway. This further fuelsthe cascade of downstream pathological changes such as by impacting the gene expression of Akt, ERK and others [42, 43].
NF-κB has been found to play very important role in GBM oncogenesis as it also plays role in EGFR amplification. EGFR amplifications also contribute to Pyruvate kinase M2 (PKM2) dysregulations, resulting in the upregulation of this rate limiting enzyme of glycolysis in GBM. In GBM, NF-κB and EGFR interactions contribute to GBM oncogenesis. In GBM, EGFR amplifications impact the MAPK, PI3k/AKT pathway, JAK/STAT pathway and contribute to the progression of GBM development.
Netrins play very important role in gliogenesis and their role is also crucial in GBM oncogenesis. Netrins also interact with the EGFR. This reduces partially the GBM cell senescence that occurs from DNA damage. This EGFR based role is mediated by AKT and ERK signaling pathway [63, 64].
In neurogenesis, EGFR interacts with the numb gene to modulate the process of neuronal differentiation. Numb gene negates Notch signaling and contributes to neuronal differentiation . Upregulation of Numb genes contributes to halting GBM growth and progression  [78, 79, 80, 81].
Another very important role of EGFR in neurogenesis includes its involvement with Notch/STAT3-Ser/Hes3 Axis. Hes3 has role of pro-survival signaling. It has strong relationship with SHH, and potentiates the FGF and EGFR signaling. It is one of the key regulators with the ability to reprogram cells into NSCs. In GBM, this signaling axis becomes severely dysregulated .
PDGFR: It plays diverse role in the gliogenesis and neurogenesis. It plays very important role in neurogenesis but in the process of GBM development it contributes to oncogenesis. Such as PDGF and NT3, they both are neurogenic in neuronal development, but in GBM landscape they play oncogenic role [63, 64].
It is important to mention here that IGF-1, FGF, PDGF, EGF, Insulin are based on receptor tyrosine kinase and they all have significant roles in GBM development. Like EGFR, dysregulated PDGFR also activate STAT3 signaling.
The SMAD proteins which are involved in gliogenesis, they are of key significance. Dysregulations in SMAD alters the role of PDGF-B in GBM. When Smad, PI3K and FoxG1 signaling pathways are dysregulated, they switch the TGF-beta to become oncogenic.
In GBM development, when SHH becomes deregulated it also contributes to dysregulated PDGFR signaling. NF-κB is linked with TNF alpha and IL1 expression. It has major interaction with PDGFR in GBM oncogenesis. In gliogenesis, PDGFR works with MAPK pathway.When PDGFRbecomes dysregulated in GBM, it also impacts the MAPK signaling .
IGF-1: It works with FGFR3 in gliogenesis but it becomes one of the key oncogenic drivers in GBM development. FGF also works with IGF-1 to regulate the MAPK pathway. The role of IGF-1 in cancer development is well established. IGFR signaling pathway interacts with AKT to further the GBM proliferation  [13, 14, 15].
VEGFR: When Notch signaling that plays key role in gliogenesis becomes dysregulated, this results in upregulation of Akt and VEGF signaling and contribute to GBM development. There is also simultaneous decrease in PTEN expression .PAX6 and TGF beta also control the VEGF expression in GBM and prevents angiogenesis. They both stop the cells from entering S-phase of cell cycle and also induce apoptosis. The NF-κB promotes IL6, IL8, VEGF, and this further fuels the GBM development[76, 71].
- RAS pathway in GBM and gliogenesis/neurogenesis:
It becomes affected by alterations in FGFR3 which is one of the key gliogenic pathways. The alterations in Notch and SHH also impact the EGFR which in turn also impacts the RAS signaling. The loss of tumor suppressor in NF1 also causes RAS over-activation. Dysregulations in RAS also contribute to MAPK signaling alterations and contribute to GBM oncogenesis   .
MEK signaling is involved in gliogenesis.The RAS over-expression also dysregulates the MEK signaling in GBM.
HOPX contributes to the regulation of NOTCH signaling and also causes senescence by activating the Ras and MAPK pathway[63, 64].
Ephrins which are involved in gliogenesis. In GBM, the dysregulation of ephrins contribute to the RAS over-activation  .In neuronal differentiation, RAS interacts with key neuronal differentiation factor NeuroD. GSK3 beta has important contributions towards GBM oncogenesis. It also contributes to the GBM progression by contributing to RAS over-expression [160, 161].
JAK-STAT pathway, MAPK, EGFR and NOTCH have major interactions with PI3K/AKT/mTOR Pathway in process of gliogenesis [16, 17]. SMAD dysregulations also impact PI3K/AKT/mTOR and cause the TGF beta to become oncogenic.EGFR-AKT-Smad signaling is also another way through which SMAD contributes to the GBM. The TGF-β1 works with Smad, p38 MAPK and PI3K/Akt signaling pathways in GBM development .Neuregulin-1 is a gene of EGF family and contributes to the astrocytogenesis. Nrg1 and erb receptor signaling pathway interact with each other and also with PI3K pathway, thus contributing to the growth of GBM [78, 79, 80, 81].
NRSF/REST halts the neurogenesis and induces gliogenesis. It works with PI3k in GBM oncogenesis . The GSK3 also works with PI3K/FGF signaling pathway and contributes to the stability of c-Myc. In GBM, all of them become dysregulated. GSK3 has interactions with many genes and pathways that are involved in GBM such as PI3k/AKT/mTOR pathway [160, 161]. PI3k/AKT/mTOR pathway contributes to the gliogenesis and neurogenesis but also contributes to stemness in GBM .
- PTEN: The role of PTEN is very significant as it also contributes towards controlling the SHH and PI3K expression. When PTEN becomes defective in GBM, then SHH and PI3K becomes dysregulated and both contribute to the oncogenesis  .PTENacts as tumor suppressor and it also impacts SHH and PI3K pathways.
NF-κB also interacts with the PI3K/AKT pathway. With loss of PTEN and NF1, the PI3K/AKT pathway also becomes dysregulated  .PTEN regulates PI3k and blocks AKT signaling .
Notch dysregulation causes simultaneous decrease in PTEN expression, resulting in the upregulation of Akt and VEGF.
Sox9 which is also involved in maintaining pluripotency of NSCs, it also interacts with PTEN. The PTEN works as a tumor suppressor by controlling the gene expression of STAT3 [42, 43].
PAX6 stops the cells from entering S-phase of cell cycle and also induces apoptosis. It also interacts with PTEN to regulate the cell cycle . Loss of PTEN causes the PI3k/Akt pathway to become dysregulated. CNS neural progenitor marker Pax7 also becomes upregulated in glioblastomawith PTEN deficiency . GSK3 beta also interacts with tumor suppressors including PTEN [160, 161].
NF1 contributes to the regulation of NSCs proliferation and gliogenesis. Loss of NF1 leads to the drastic effects that lead to dysregulated NSCs proliferation and uncontrolled gliogenesis. This also impacts the downstream signaling and most significantly PI3K/Akt/mTOR pathway becomes dysregulated as a consequence. The NF1 negatively regulates the RAS-signaling pathway. With loss of NF1, the RAS/MAPK signaling pathways become dysregulated [170, 171].
Defects in NF1 impact both neuronal and glial cells. The impact of NF1 loss on neuronal cells is also of key significance as it contributed to abnormalities in cAMP generation in such cells. The GFAP works also by interacting with NF1 and it has capacity to impact the NF1 gene expression in order to enhance glial proliferation. The NF1 expression is essential in neuronal differentiation as well. Other key pathways that interact with NF1 include Notch, ERK, MEK, SMAD3 and Hes1 .
ERK pathway works in a delicate regulated balance with NF1. The ERK pathway works as a G1 to S transition switch when upregulated. It has key interactions with many key regulator genes/signaling pathways such as SHH, FGFR3, JAK-STAT, Notch, EGFR, ephrins, netrins, NeuroD, GSK3, CDKs .Nf1 inactivation leads to the increased gliogenesis and this declines the neurogenesis. The MEK/ERK pathway becomes upregulated and hyperactive. Thisdysregulates NSCs proliferation and increases the risk of GBM .Nf1 mutations are of prime significance in mesenchymal subtype of GBM .
- FOXO axis:
This axis promotes differentiation and negatively regulates proliferation. This axis is dysregulated in GBM. The PI3k/AKT pathway negatively regulates FOXO axis. This axis interacts with IGF1 as well. The role of FOXO is also governed by the pathways with which it interacts. In GBM oncogenesis, the dysregulated Akt cause the FOXO axis to become dysfunctional .FOXO axis plays very significant roles as it works with TGF beta/SMAD axis. This plays a vital role in the process of gliogenesis and neurogenesis. The IGF1 suppresses FOXO axis. IGF1 signaling also promotes cdkn1a expression .
PI3k/Akt, RAS, MAPK pathways inhibit the FOXO axis. They all have well established role in GBM development . FOXO axis is damaged in GBM. The physiologic role of FOXO is also to regulate cell cycle. It also enhances the expression of sirtuin1 during cellular senescence. Several studies have indicated that FOXO1 has the ability to inhibit EMT and metastasis .FOXO is also being considered a target in treating GBM [181, 182].In GBM oncogenesis, Foxo/SMAD signaling is downregulated while FoxG1 works to enhance proliferation by increasing the expression of Sox2 and Sox5 .
- P53 signal alteration:
P53 plays diverse functions ranging from neurogenesis to NSCs and brain development. P53 is dysregulated in GBM oncogenesis. The consequences of p53 loss on neuronal differentiation are still controversial as it is yet to be fully understood how it impacts NSCs. Although it is likely that p53 inhibits neuronal differentiation of NSCs .
Studies involving brain organoids have shown p53 to be a crucial player in CNS development. It also contributes to genomic stability and regulates neurogenesis .Dysregulations in P53 also contribute to Mdm amplifications in GBM. In GBM, damage to P53 also dysregulates the notch signaling, GSK3 beta, Ras/MAPK, PI3k/mTOR pathway, EGFR signaling and others.
It is important to note that deletions of P53 and REST are so significant that it causes the cells to switch into pro-neural type of GBM. Loss of p53 in Li-Fraumeni syndrome also results in GBM development. This also makes p53 a major player in GBM oncogenesis [187, 188].
Cyclin-dependent kinase inhibitor 2A (CDKN2A): It is a tumor suppressor and works with Rb1 to halt G1 to S phase transition. It encodes two tumor suppressor proteins P16 and ARF. CDKN2A mutations are associated with Glioblastoma development. ARF functions as stabilizer of P53 and sequesters the MDM2 which is a degrader of p53.CDKN2A mutation is also linked with EGFR amplification that is a major part of GBM oncogenesis . MDM2 and MDM4 both have structural similarities and are p53 binding proteins. MDM4 is capable of reversing MDM2-based degradation of p53, but the apoptotic functions of p53 remain suppressed. MDM2 becomes over-expressed in GBM resulting in increased degradation of p53 . Aging is also considered to play important role in the dysregulation of EGFR, Mdm2,andp53 .
- RB signal alteration:
Rb1 is a tumor suppressor. It is also involved in neuronal differentiation. Rb1 interacts with CDKs in regulating cell cycle. This pathway becomes damaged in GBM and CDKs cause E2F based G1 to S transition . It also contributes to differentiate the NSCs into astrocytes. Mutated Rb1 is considered a major player in GBM oncogenesis. P16-CDK4-RB pathway works in an interconnected manner and control cell cycle progression . Rb1 knockout mouse models have been shown to increase the neurogenesis as a result. Rb1 interacts with CDKs in inhibiting E2F. This pathway becomes damaged in GBM and CDKs cause E2F based G1 to S transition .
Cyclin-dependent kinases and inhibitors: CDKN2B forms complex with CDK4 and CDK6.This halts the activation of CDKs. CDKN2B regulates the progression of G1 phase of cell cycle. CDKN2C also forms same complex but it works more with Rb1 expression. TGF-beta induces CDKN2B. It regulates the cell cycle and is also key player in gliogenesis. TGF beta works with SMAD and CDKN2B to regulate cell cycle and gliogenesis. They also inhibit c-myc. CDKN2B also plays role in neurogenesis while working with PAX6. It is important to mention here that Sox2 and sox4 are involved in the stemness of GBM cells, they cause the downregulation of CDKN2B [196, 197].
CCND2 forms complex with CDK4 and CDK6. This contributes to G1 to S cell transitions and inactivates the Rb1 for this purpose .
SOME OTHER MUTATIONS IN GBM: briefly explored here as deeper investigation into all of them is beyond the scope of this study.
IDH1: Mutant IDH1 contributes to the GBM development and ultimately contributes to oncogenesis through epigenetic mechanisms as well . IDH1 mutation alters the neurogenic niche and promotes glioma formation. Resultant accumulation of 2-hydroxyglutarate alters DNA methylation and histone binding. This contributes to the oncogenic changes in neuronal and glial cell types . Similarly, IDH2 has also emerged to play similar role in GBM oncogenesis.
IDH mutations are considered to be among the initial mutations in GBM development. They are also common in diffuse gliomas. IDH mutations are more common in secondary GBM and far less common in primary GBM . 1p/19q loss has been found to occur with IDH1 mutations in oligodendrogliomas .
TERT: It is upregulated in stem cells including cancer stem cells. It is also related to the pluripotency of cells. It interacts with STAT3 which also has major involvement in the process of gliogenesis. The dysregulated STAT3 signaling is involved in glioblastoma development. Another major interaction of TERT is with GSK3 beta. The GSK3-beta deletions contribute to the enhanced proliferations of neural progenitor cells with simultaneous increase in SOX2 and beta-catenin. In normal cells, it acts as negative regulator of Epithelial-Mesenchymal-Transitions (EMTs) and many proto-oncogenes. But dysregulated GSK3-beta is oncogenic.TERT plays a wide array of roles ranging from cellular aging to epigenetic clock [202, 203]
Similarly, ATRX is important component of chromatin remodeling complex. Its dysfunction also causes immense genomic instability. This alteration is present in 44% of GBM .
FUBP1: In undifferentiated NSCs, FUBP1 induces the expression of c-myc. The c-myc interacts with JAK-STAT and is downregulated by SMAD/TGF beta pathway in gliogenesis. The c- myc is also involved in neurogenesis but plays the oncogenic role in GBM development. Cancer stem cells are heavily associated with the Wnt signaling. The dysregulated Wnt signaling causes the activation of cyclinD1 and c-myc, causing G1 to S phase transition. It also contributes to Epithelial to Mesenchymal transitions [205, 206].
GLIOBLASTOMA SUBTYPES AND VAST HETEROGENIETY:
The Cancer Genome Atlas identified 840 genes involved in GBM, leading to the ultimate classification of glioblastoma into three subtypes [207, 208, 209].
- Pro-neural subtype:
This subtype is present mostly in younger patients. They tend to survive longer. It has the highest subtype shifting and most favorable survival .IDH1 is one of the key genes involved in this subtype. It is also considered the reason for favorable prognosis. IDH1 mutant form is present with 1p/19q co-deletion . Other important mutations include p53 dysfunction, PDGFR amplifications, upregulated Nkx 2-2 and Olig2. Most low grade glioblastomas and secondary glioblastomas belong to pro-neural subtype.Some most common GBM abnormalities such as chromosome 7 amplification and chromosome 10 deletions have low occurrence in this GBM subtype . The EGFR, PTEN and Notch are normal in this subtype .
- Classical subtype:
It is the most common subtype and key genetic alterations include EGFR amplifications, homozygous deletion of CDKN2A, chromosome 7 amplification and others .Abnormalities in IDH1, TP53, PDGFR and NF1 are mostly absent in this subtype .
- Mesenchymal subtype:
It is the most stable subtype. Signature mutations include NF1, NF-κB and upregulated gene expression of S100A1, CHI3L1, MET, VEGFR2, CD31, fibronectin, COX2. There is increased inflammation and necrosis in this subtype .
There are fewer alterations of EGFR in this subtype. Pro-neural markers are dysregulated in this subtype. The alterations in TGF-beta and STAT3 play key roles in the transition from pro-neural to mesenchymal subtype. The proneural-mesenchymal transition upon tumor recurrence has been suggested as a mechanism of tumor resistance to multimodal therapy [217, 218].
The relationship between neurogenesis/gliogenesis and the key genes/signaling pathways involved in the subtypes has beeninvestigated in the earlier sections.
SIGNIFICANCE OF HETEROGENIETY IN GLIOBLASTOMA:
Glioblastomas are one of the most aggressive tumors with multiple subtypes and are widely known to have vast heterogeneity in nature. This heterogeneity is also evident among the pro-neural, classical and mesenchymal subtypes of GBM. The genetic landscape of GBM is so diverse that there is lots of intra-tumoral heterogeneity as well. The landscape of GBM has vast heterogeneity that key epigenetic alterations such as those emerging because of IDH1/2 mutations, they are different in 3 subtypes and among different samples of GBM. Multiple studies have pointed out that landscape of GBM mutations vary to such an extent that RTK/RAS/PI3Kalterations have been detected in 83% of samples, the TP53 alterations in 87%, the RB1 alterations in 78% of the samples. Still there is lots of variability in even in core mutations among different samples [219, 220, 221]
There may be multiple factors that are responsible for this vast heterogeneity in GBM. Based on the results of this study, it is hypothesized here that this genetic variability may have origin in the differences between the genetic landscape of gliogenesis and neurogenesis. NSCs are the progenitors of both neurons and glial cells. The initial mutations that occur in three subtypes of GBM may drive the direction of subsequent mutations in tumor development. The internal genetic and epigenetic homeostasis that is established at the time of establishment of cell fate, when this homeostasis is dysregulated then the risk of glioblastoma oncogenesis increases. Aging plays a very significant role in GBM development as majority of the GBM cases are in old age. But the pro-neural subtype of GBM affects mostly younger patients and here different set of mutational landscape impacts GBM development. The timing and sequence of initial driver mutations also play very important role in altering the internal cell circuitry and direction of further mutational changes involved in GBM progression.
In depth further discussion of GBM core pathways, heterogeneity and subtypes is beyond the scope of this study.