Gene Mutation and Epigenetic Modification Origin in Glioma Initiation: Are the GDNF and SOX1 Overexpression the Causes of Its Initiation?

The extrinsic and intrinsic factors are essential in glioma initiation. Many extrinsic factors (UV, radiation, food, etc.) and intrinsic factors (proteins, hormones, ageing, DNA and RNA damages, etc.) was reported to being responsible for glioma initiation and progression. However, the cell responsible for glioma origin is still unknown. Many research papers have reported that glioma stem cells, senescent cells, injured cells, and death neurons are the cells of glioma origin. However, gene mutation and oncogene protein overexpression doesn’t occur only in cancer but during life evolution. The source of genetic mutations has become a fundamental issue in understanding its role in the initiation of glioma. The glioma is the precise coordination of several distant factors that work together in the initiation and development of glioma. However, the role and effects of the genes (GDNF and SOX1) on cancer cells are well known, but their gene mutation origin is controversial. Several models and theories have been proposed to explain the origins of GDNF and SOX1 genetic mutations and epigenetic modification related to cancer. Our aim in this review Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 May 2020 doi:10.20944/preprints202005.0388.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. is to clear that incertitude about glioma origin (gene mutation and epigenetic modifications) and those factors involved in glioma initiation and recurrence.


INTRODUCTION
Glioma is the deadliest tumour of the central nervous system. The increasing number of patients suffering from glioma and the complex risk factors have shown that this disease evolves and changes form [1]. Despite the advanced discovery on cancer initiation and their manifestations in the study of cancer stem cells, the mechanism of action and the origin of glioma is still a holy grail of cancer research [1][2][3][4]. However, many brain cells was reported as being responsible for glioma initiation through a strange physiological and genetic phenomenon that occurs in one cell and induces the transformation of other multitude cells [5]. Genetic materials in the cells undergo a constant modification for the implementation of some tasks depending on the body's needs. These modifications guide, direct, and attribute the cells towards a specific function. Gene modification can be physically mutation (deletion, addition) of sequence or nucleic base during DNA and RNA maturation (splicing), and epigenetic changes especially the addition of molecules without altering the DNA or RNA sequence how a single cell accumulate, all genetic and epigenetic mutations are still not well known. The overexpression of oncogenes during embryogenesis, inflammations, and other diseases (meningitis, wound, etc.) during life processes not always lead to glioma or other cancers, prompting questions about the initiation of the glioma. Can glioma be due to an exaggerated increase in a specific oncogene expression or due to niche modification? In this review, we will briefly discuss and highlight the origin of gene modification and epigenetic modifications on GDNF and SOX1 genes during embryos development to adults in which their overexpression affects the brain neurons function due to a perpetually changes in cell protein levels according to the body needs.

Eukaryotic cell and genetic mutation origin
Eukaryotes are the type of cells characterized by the presence nucleus in which the chromosome containing the genetic materials. The main molecular systems and machinery of Eukaryotes are found in Prokaryotes, most especially, Archaea.
The simplest interpretation is to consider the Eukaryotic cell as the result of successive symbioses between Archaea and Bacteria. The eukaryotic cell, like we know today, has originated for many centuries as a result of the symbiotic association between a host cell and microorganism according to the evolution theory. This symbiosis includes the fusion of genetic information and physical elements of two cells into a single cell. After the apparition of the first eukaryotes, life started to evolves from a single cell to an association of many cells to form one organism (metazoans) such as a human. During this process of evolution, many modifications occur in the management of the DNA and RNA sequence of a species, which is essential for the adaptation of such animals and plants concerning their environment [6][7][8][9]. All metazoans originate from a single pluripotent eukaryotic stem cell in which many phenomena occur. The genetic materials fusion promote the apparition of novel characteristic and behavior that the first eukaryote cells don't have such as the cell structure modification, ability of cells to communicate each other, novel proteins expression and fecundation [9].
It was reported that genetic mutations and disorders occur during the process of cancer initiation [1,4]. The mechanism related to the accumulation of all these genetic mutations in a single cell is still not clear. A lot of oncogenes are activated in glioma through DNA or RNA damages, gene sequence depletion, and epigenetic modifications (methylation, acetylation) [4]. All these genetic matters showed clearly that glioma initiation, aberrant oncogene amplification, and proteins overexpression, uncontrollable proliferation, malignancy, evolution plasticity of glioma cells, and their resistance to immune reaction are associated to genetic alteration. The complexity of genetic mutation in cancer is so much that even two daughter cells derived from the same cancer cell have a different genetic sequence; This explains the heterogeneity within tumour cells [10] and the various results derived from the same experiment (technology).
Sometimes the researcher has to repeat the experiment many times before to get the expected results even sometime without success. These genetic modifications depend on the type of cells and the microenvironment where the cells are located.
Moreover, chow et al., 2011; Alcantara et al., 2016 [5,11] reported that the glioma initiated cells would be a Neuronal stem cell (NSC) that can differentiate into other types of brain cancer cells. The reasoning is logical because neuron cells are all derived from a single stem cell capable of differentiating into various kinds of neuron cells, but it is not sufficient to conclude the origin of glioma cells.
The first initiated cell is not always a stem cell or a neuron stem cell. All the cells can become a cancer cell, but the persistent and strong resistance of cancer stem cells is generally due to their ability to differentiate into several types of cells. Glioma cell origin or initiation largely depends on their microenvironment and extrinsic factors rather than intrinsic factors. However, all these factors are linked and influence each other. Cancer-associated fibroblasts (CAFs) and supporting cells such as glial cells are more exposed to extrinsic factors because they have more contact with other cells because their environment is prone to perpetual change and also benefit immune cells protection. The concept of glioma cell origin depends on a specific point that the researcher wants to highlight. There are no criteria to determine the cell responsible for the origin of gliomas. Stem cells are targeted for more perspective (resistance, differentiation) towards an eventual therapy. To understand glioma origin, we should go back to the source of the first eukaryotic cell. The gene modifications during the transformation of a host cell into eukaryotic cell trigger a new behaviour "heterotrophy" which push eukaryotic cells to communicate and organize themselves to increase their survival rates. The necessity of this communication and survival behavior leads to a symbiosis between the primary eukaryotes and the bacteria, which drives the mitochondria to perform the metabolic machinery [6,9]. This novel function and the abundance of nutrients trigger the necessity of cellular division for more dissemination and to mimic the extinction of species [9]. It is this necessity of the cell to divide that will cause the "boom of gene mutation" because of the old way of cell division "scissiparity," which is the separation of a single cell into two identical clones, which was not the secure and efficient way for eukaryotes dissemination. To compensate these scissiparity limits, eukaryotic cells have to neglect asexual reproduction for sexual reproduction, which is a boon to their survival with a very high rate of spread and heterogeneity. This mixing of genetic pieces of information has reinforced communication between cells because sexual reproduction requires the communication and the fusion of two cells of the opposite sex, which was not the case with scissiparity. For this fusion of sex cells to be effective, eukaryotes have developed a new strategy called "chemotactic," which makes it possible to attract gametes to each other through genetic manipulation and protein synthesis. The success of this process has been the key to the development and evolution of species through gene manipulation and also marks the beginning of diseases linked to defective genes (cancer) [12] and malformation (trisomy 21).

b. Genetic mutation and gene overexpression during fecundation and embryo development is a precursor of glioma initiation
Fecundation is a crucial stage of life evolution and improvement in living beings [13,14]. An error in these processes can lead to fatal or vital consequences in an individual. The increase in the expression of specific genes does not necessarily initiate cancer but participates step by step in its establishment throughout life by increasing the risk factors.
The fecundation and embryonic machinery development are activated and regulated by many genes and hormones that have oncogenic proprieties to let the egg cells to proliferate into an entire independent organism. In the early stage of embryonic development, many genes are activated, and their expression increases. Glial cell line-derived neurotrophic factor (GDNF) and sexdetermining region box 1 (SOX1) are among the first proteins expressed in embryonic development during ectoderm and neural tube differentiation [1,2,15]. GDNF and SOX1 play an essential role in the nervous system (central and peripheral nervous system) differentiation. It was reported that GDNF and SOX1 are highly overexpressed in glioma and promote glioma stem cell proliferation, invasion, and migration [2,15]. However, the increase in the expression of GDNF or SOX1 is not necessarily followed by glioma or other types of cancer but can be a distant cause of its initiation. During embryonic development, the

c. Genetic mutations and epigenetic modification of glioma cells regulate genes (GDNF and SOX1) expression.
• GDNF/SOX1 methylation and histone acetylation GDNF gene is localized on chromosome 5 at p12-p13.1 and contains two promoters (I and II) and five exons [1]. SRY (SOX1) gene is localized on chromosome Y, but other researches also reported SOX1 gene to be located on the biggest chromosome 13 (chr13:112,067,599-112,071,706) and have 4,108 bases in its sequence [2,15,18]. Recent studies demonstrated that epigenetic silencing of the tumour suppressor's gene promotes the overexpression of oncogenes to regulate cancer geneses, maintenance, and progression [18,19].
The high expression of GDNF and SOX1 is due to epigenetic modifications that occur in the genome.
The methylation is a process in which a "methyl" group attaches itself to the carbon chain of another main molecule. In the case of epigenetic modifications, the "methyl" group binds itself to histone and lysine residues K4, K9... sometimes also to arginine [20,21]. Methylation, in general cases, always occurs in chromatin in collaboration with some specific enzymes such as histone methyltransferases, which play a crucial role in the methylation process. Many types of research done in our laboratory and other research articles demonstrated the epigenetic modifications (methylation and acetylation), which occur in GDNF high expression in glioma [1,22,23]. In SRY superfamily, especially SOXB1, the case of methylation is almost the same as in TGFβ.
Methylation or hypermethylation of the SOX1 gene is involved in glioma initiation and progression [24][25][26][27]. Lai, H. C. et al. [27] demonstrated that the methylation of PAX1/SOX1 in human papillomavirus (HPV) also increases the risk of cancer, especially in glioma and promotes GSC proliferation [28,29]. Histone H3K9 is highly involved in GDNF gene methylation. In the case of SOX1, the Histone H3K4 methylation/acetylation is responsible for SOX1 overexpression and GSC initiation and proliferation by interacting with OCT4 [11,30]. SOX1 gene methylation is not well known because some details remain not evident. The epigenetic modifications involved in GDNF and SOX1 are almost the same, but only the location of methylation is different, H3K9 for GDNF and H3K4 for SOX1.
Glioma initiation is a continuous process of transformation in the brain cells.
During nervous system differentiation, the brain neurons inherit the mutations from the neuronal stem cells that originate from the fecundation via mitosis.
However, the origin of mutations and epigenetic modification in the brain cells All brain cells are susceptible to become brain cancer cells depending on the brain cell microenvironment. However, the genetic sequence of the body cells is different; the only factors that can change is the cell niche, microenvironment, which determines the big part of the function, metabolism, and future (differentiation, proliferation, apoptosis). The brain senescence cell stimulates the other brain neuron to produce more GDNF and SOX1 for their survival and proliferation, therefore trigger glioma initiation. During oxidative stress response (ROS), NFkβ, and interleukin one alpha (Il-1α) overexpressions promote inflammation to modify the cell microenvironment changes leading to the initiation of neurons senescence and death. During the senescence, GDNF and SOX1 play an essential role in reviving the apoptotic cells to initiate glioma [17]. This explains the fact that the increase in GDNF and SOX1 during embryogenesis doesn't promote glioma but just ectoderm and neural tube differentiation. The factors which regulate glioma initiation is more extrinsic (microenvironment, niche) rather than intrinsic (genetics); however, both factors are essential in glioma initiation.

Genes mutations: one of the main trigger of glioma initiation a. The gene mutation promotes niche modification and cancer-associated fibroblast (CAFs) activation
The usual niche is composed of fibroblast, immune, endothelial and perivascular cells, extracellular matrix (ECM), cytokine network, and growth factor [31,32].
The niche composition contributes to tumour cell heterogeneity and tumour progression, genetic diversity, and epigenetic modifications [33,34] In glioma, GDNF, a growth factor expression significantly increases [17]. The overexpression of GDNF promotes the recruitment and activation of Cancer-Associated Fibroblast (CAFs), leading to the initiation of inflammation and activation of an immune response [35]. These promote the synthesis of interleukine-6/8 (IL-6/8), and CD113 by T-cells to overcome and heal the injury [36]. CAFs will activate immune reaction and promote the overexpression of growth factors like CXCL12, IL-6, IL-8, Wnt /Notch to induce the differentiation of epithelial non-stem cells into tumor-initiating stem cells (TISC) and changed their phenotypes [37,38]. The proliferation of CAFs cells affects glioma stem cell morphology, modification microenvironment, mesenchymal stem cells, and their implications in glioma stem cell proliferation and metastasis [37,39].
Glioma stem cell proliferation promotes immunosuppression in the niche by adapting themselves to cytokine, chemokine, IL-6, and Transforming growth factor β (TGFβ), which will inhibit the immune cell receptors [36,38]. Through these extensions, CAFs will bind to the epithelial-mesenchymal cells and initiate their transformation into tumour cells [39,41]. During that period, we also found that the increase in the expression of GDNF and SOX1 is also followed by the overexpression of other associated genes in which the expression depends on the GDNF and SOX1 gene [2,15]. So glioma cell initiation started earlier since the apparition of eukaryotes and during embryo development (neurectoderm differentiation) in which genes mutation and epigenetic modification begin. During and after embryo development into an adult organism, many phenomena occur (disease due to virus, bacteria, infection, wound healing, vaccination, etc.) promoting genetic mutation and cell structure modification. During that period, the contracted disease, virus, bacteria RNA, and DNA can be associated with cell DNA leading to genomic mutations. All these genetic disruptions associated and accelerated by extrinsic factors such as UV, dugs, food, age will induce gliomas. Glioma and cancers have their origin and cause so far, but their development depends on triggers and accelerator factors.
The healed neuron cells after injury or inflammation can also transfer their mutations and epigenetic modifications (histone acetylation and methylation) to their daughter cells via mitosis. This will affect the neuroendocrine signaling leading to the overexpression of the oncogenes that was previously promoting glioma maintenance. The neuroendocrine system plays a critical role in glioma recurrence due to epigenetic modifications and genetic mutation, which enable feedback control of oncogenes expression and glioma initiation via the regulation of protein vesicles and neurotransmitters synthesis. Aging neurons can also cause the disorder and troubles in neuroendocrine and synapses system.
The proliferation of aging neuron cells and the spread of damaged DNA via mitosis and the alteration of the central nervous system synapses is also one of the causes of tumour recurrence. The alteration of the neuroendocrine and synapses significantly affect neuron microenvironment and cancer-associated fibroblast initiation. This environment is favorable for glioma initiation or tumour recurrence after surgical resection.

CONCLUSION
Glioma is a long process of transformation and modification of brain neurons, and the gene mutation is one of the triggers of its initiation and malignancy. The there is no cell of origin of glioma [42]. However, all the body cells are potent cancer cells, but only the trigger factors expose some cells more than others to become tumour initiated cells. This explains the recurrence of tumour cells after therapy or clinical resection and the ability of cancer cells to adapt themselves to the environment and drug [17]. The role of GDNF and SOX1 respective family groups is vital in the nervous system differentiation but also cancer initiation and progression [2,15]. The protective effect of high GDNF and SOX1 on neurons and neuron progenitor cells is a solid proof that all the cells are defined to cancer, and cancer initiation is more a gradual than spontaneous process [17]. Both extrinsic and intrinsic factors mutually influence each other, but external factors are more involved in cancers (glioma) initiation. The best way to overcome glioma and cancers is to develop a better therapy approach targeting the damage cancer cell (DNA & RNA) and its microenvironment (fibroblast, support cells, immune cells) simultaneously. The treatment of cancer cells only by using stem cells without treating the external factors (niche/microenvironment) such as fibroblast cells and extracellular elements would be a failure.