**4. The cancer stem cell model**

miR34s are induced after genotoxic stress in a p53-dependent manner *in vitro* and *in vivo*. miR-34b and-34c are clustered at chromosome 11, whereas miR-34a is located in a separate genomic locus. p53 directly activates both pri-miRNAs. The miR-34s seem to be critical downstream effectors of p53, as ectopic expression of the miR-34s recapitulate the phenotype of p53 activation. The miR-34s promotes repression of several direct targets, such as Bcl-2, Cdk4, hepatocyte growth factor receptor (MET), and other, resulting in cell cycle arrest, apoptosis, and senescence [179] (Figure 3). Several other laboratories corroborated the finding that miR-34s are critical components of the p53 network [180-182]. Taken together, these results

144 Tumors of the Central Nervous System – Primary and Secondary

support a pivotal downstream role of miRNAs in the regulation of the p53 pathway.

**Figure 3.** Representation of p53 and the miR-34 family interactions. The p53 protein stimulates the transcription of

As cell cycle arrest, senescence, and apoptosis are tumor suppressive mechanisms, the inactivation of members of the miR-34 family, which induce these cellular responses, may be a selective advantage for cancer cells. Besides decreased expression of MiR-34 due to inacti‐ vating mutations of p53, the miR-34 encoding genes themselves may be targets for mutational or epigenetic inactivation in cancer. For example, loss of miR34a expression was observed in

miR34s, which inhibits oncoproteins and leads to cell senescence, apoptosis and cell cycle arrest.

Until a few years ago, the brain was thought to lack a stem cell population, but actually, it is now known that there are two regions of the adult human brain that contain neural stem cells (NSCs) (a group of self-renewing cells in the nervous system that can generate both neurons and glia): the dentate gyrus of the hippocampus and the subventricular zone. NSCs can form neurons, astrocytes and oligodendrocytes *in vitro*, although their normal physiological role in the adult human brain is disputed [190].

lation of p21 expression. These data reinforce the p53 role as a suppressor of tissue and cancer

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Armesilla-Diaz *et al.* [203] demonstrated that p53 controls the chromosomal stability, prolif‐ eration and differentiation patterns of embryonic mouse olfactory bulb stem cells. The group reported that the absence of this protein increases the number of neurosphere-forming cells and the proliferation of these stem cells, and observed that differentiation of p53 knockoutderived neurospheres was biased toward neuronal precursors. Moreover, the relevance of p53 in maintaining chromosomal stability in response to genotoxic insult was demonstrated, and additionally, the results showed that neurosphere stem cells are highly resistant to long-term epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) deprivation in a p53-

While the role of p53 in apoptosis of neuronal cells was well elucidated [202, 204, 205], its function in astrocytes, oligodendrocytes and their precursors is poorly understood. Oligoden‐ drocyte precursors cultured *in vitro* can undergo p53-dependent differentiation although the cells appear to have a low basal level of p53 expression [206]. Both oligodendrocytes and astrocytes can undergo apoptosis following infection with an adenovirus expressing p53 [207].

CSCs exhibit genetic or chromosomal alterations in addition to aberrant differentiation properties, unlike the normal stem cells [208]. It is important to highlight the fundamental differences between normal stem cells and CSCs. The first are known for the vigilance with which their proliferation is controlled and for the care with which their genomic integrity is maintained, while CSCs lack such ability [209]. The cell-of-origin of CSCs remains elusive, however evidences indicate that CSCs may originate from malignant transformation of normal stem cells, because of the perennial nature and high proliferative potential characteristics of stem cells. Some studies have shown that oncogene activation or tumor suppressor gene inactivation increased the frequency of tumor formation in primitive nestin-expressing cells but not in the more differentiated glial fibrillary acidic protein (GFAP)-expressing astrocytes [210, 211] while other researches indicate that differentiated astrocytes and NSCs may be

equally permissive to transformation when genomic alterations are introduced [212].

invasion or differentiation program in the absence of p53 [214].

The role of p53 in BTSC has not been well established, however based on current understanding of its function in neural precursors available in the literature, several hypotheses may be develop. First, loss of p53 may increase the self-renewal and proliferation of neural stem and/ or lineage-restricted progenitors, thereby expanding the pool of cells available for additional mutations in specifics oncogenes. Another hypothesis is that, depending on cellular context, p53 can both inhibit or promote cell differentiation, as well as influence cell fate decisions, so the differentiation program of neural precursors can be changed by p53 mutation. Lastly, accumulated evidences support a role for p53 in the suppression of cell migration, although much focus on p53 is directed at its growth inhibitory properties [213]. The neurogenic niche has been shown to be important for the maintenance of stem cells in an undifferentiated state, and the premature exit of NSCs from the neurogenic niche may alter their capacity for tissue

independent fashion, and they preserve their differentiation potential.

stem cell self-renewal.

With the accumulation of knowledge concerning the stem cell and the mechanisms regulating their behaviour, it was noted that many of the characteristics of stem cells were also present in cancer. These findings reinforce the "cancer stem cell model", which states that the cellular heterogeneity within the tumor is ascribed entirely to the differentiating tumor cells that derive from the cancer stem cells (CSCs), that can be defined as cells that possesses the capacity to self-renew and to originate the heterogeneous lineages of cancer cells that comprise the tumor. The term ''tumor-initiating cell'' also has been used to describe a cell with the potential to initiate a tumor. This term are essentially functionally equivalent to CSCs if it is used to refer to the subclones of cells within an established tumor that gives rise to a new tumor when transplanted [190-192].

CSCs were first observed by John Dick's group in acute myeloid leukemia and posteriorly other researchers reported CSCs in solid tumors, including those formed by breast, colon, prostate, pancreatic, lung, liver and brain [193-196], Subsequently there has been a large amount of work to identify the cancer stem cell population, and to study its role in progression of disease and resistance to treatment, allowing many experimental therapies targeting cancer stem cells can be developed and tested in preclinical models [190, 195, 196].

CSCs have been isolated from a wide range of CNS neoplasms, including adult and pediatric, anaplastic oligodendrogliomas and malignant medulloblastomas [197]. For gliomas, several researchers isolated brain tumor stem cells (BTSC) from primary tumors based in the ability to form neurospheres NSCs do and other criteria: ability to be serially transplanted; unique ability to engraft; ability to recapitulate the tumor of origin morphologically and immuno‐ phenotypically in xenografts [198].

#### **4.1. P53 role in neuronal and brain tumor stem cell**

In the ependymal cell lining of the lateral ventricle wall as well as most cells of the subven‐ tricular zone, including astrocytes and progenitors, abundance of nuclear p53 is evident and in agreement with the down-regulation of p53 in differentiating cells observed during embryogenesis. The nuclear p53 immunoreactivity is absent or found at low levels in the majority of the mature brain, including differentiating cells in the rostral migratory stream, suggesting that p53 is preferentially expressed in neural precursors [113]. Several studies show the important role(s) of p53 in the regulation of mammary [199], hematopoietic [200], embry‐ onic and neuronal stem cells [201] by regulating self-renewal, symmetric division, quiescence, survival, and proliferation.

Meletis *et al.* [202] demonstrated that *TP53* is expressed in the neural stem cell lineage in the adult brain and negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analyses of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregu‐ lation of p21 expression. These data reinforce the p53 role as a suppressor of tissue and cancer stem cell self-renewal.

and glia): the dentate gyrus of the hippocampus and the subventricular zone. NSCs can form neurons, astrocytes and oligodendrocytes *in vitro*, although their normal physiological role in

With the accumulation of knowledge concerning the stem cell and the mechanisms regulating their behaviour, it was noted that many of the characteristics of stem cells were also present in cancer. These findings reinforce the "cancer stem cell model", which states that the cellular heterogeneity within the tumor is ascribed entirely to the differentiating tumor cells that derive from the cancer stem cells (CSCs), that can be defined as cells that possesses the capacity to self-renew and to originate the heterogeneous lineages of cancer cells that comprise the tumor. The term ''tumor-initiating cell'' also has been used to describe a cell with the potential to initiate a tumor. This term are essentially functionally equivalent to CSCs if it is used to refer to the subclones of cells within an established tumor that gives rise to a new tumor when

CSCs were first observed by John Dick's group in acute myeloid leukemia and posteriorly other researchers reported CSCs in solid tumors, including those formed by breast, colon, prostate, pancreatic, lung, liver and brain [193-196], Subsequently there has been a large amount of work to identify the cancer stem cell population, and to study its role in progression of disease and resistance to treatment, allowing many experimental therapies targeting cancer

CSCs have been isolated from a wide range of CNS neoplasms, including adult and pediatric, anaplastic oligodendrogliomas and malignant medulloblastomas [197]. For gliomas, several researchers isolated brain tumor stem cells (BTSC) from primary tumors based in the ability to form neurospheres NSCs do and other criteria: ability to be serially transplanted; unique ability to engraft; ability to recapitulate the tumor of origin morphologically and immuno‐

In the ependymal cell lining of the lateral ventricle wall as well as most cells of the subven‐ tricular zone, including astrocytes and progenitors, abundance of nuclear p53 is evident and in agreement with the down-regulation of p53 in differentiating cells observed during embryogenesis. The nuclear p53 immunoreactivity is absent or found at low levels in the majority of the mature brain, including differentiating cells in the rostral migratory stream, suggesting that p53 is preferentially expressed in neural precursors [113]. Several studies show the important role(s) of p53 in the regulation of mammary [199], hematopoietic [200], embry‐ onic and neuronal stem cells [201] by regulating self-renewal, symmetric division, quiescence,

Meletis *et al.* [202] demonstrated that *TP53* is expressed in the neural stem cell lineage in the adult brain and negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analyses of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregu‐

stem cells can be developed and tested in preclinical models [190, 195, 196].

the adult human brain is disputed [190].

146 Tumors of the Central Nervous System – Primary and Secondary

transplanted [190-192].

phenotypically in xenografts [198].

survival, and proliferation.

**4.1. P53 role in neuronal and brain tumor stem cell**

Armesilla-Diaz *et al.* [203] demonstrated that p53 controls the chromosomal stability, prolif‐ eration and differentiation patterns of embryonic mouse olfactory bulb stem cells. The group reported that the absence of this protein increases the number of neurosphere-forming cells and the proliferation of these stem cells, and observed that differentiation of p53 knockoutderived neurospheres was biased toward neuronal precursors. Moreover, the relevance of p53 in maintaining chromosomal stability in response to genotoxic insult was demonstrated, and additionally, the results showed that neurosphere stem cells are highly resistant to long-term epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) deprivation in a p53 independent fashion, and they preserve their differentiation potential.

While the role of p53 in apoptosis of neuronal cells was well elucidated [202, 204, 205], its function in astrocytes, oligodendrocytes and their precursors is poorly understood. Oligoden‐ drocyte precursors cultured *in vitro* can undergo p53-dependent differentiation although the cells appear to have a low basal level of p53 expression [206]. Both oligodendrocytes and astrocytes can undergo apoptosis following infection with an adenovirus expressing p53 [207].

CSCs exhibit genetic or chromosomal alterations in addition to aberrant differentiation properties, unlike the normal stem cells [208]. It is important to highlight the fundamental differences between normal stem cells and CSCs. The first are known for the vigilance with which their proliferation is controlled and for the care with which their genomic integrity is maintained, while CSCs lack such ability [209]. The cell-of-origin of CSCs remains elusive, however evidences indicate that CSCs may originate from malignant transformation of normal stem cells, because of the perennial nature and high proliferative potential characteristics of stem cells. Some studies have shown that oncogene activation or tumor suppressor gene inactivation increased the frequency of tumor formation in primitive nestin-expressing cells but not in the more differentiated glial fibrillary acidic protein (GFAP)-expressing astrocytes [210, 211] while other researches indicate that differentiated astrocytes and NSCs may be equally permissive to transformation when genomic alterations are introduced [212].

The role of p53 in BTSC has not been well established, however based on current understanding of its function in neural precursors available in the literature, several hypotheses may be develop. First, loss of p53 may increase the self-renewal and proliferation of neural stem and/ or lineage-restricted progenitors, thereby expanding the pool of cells available for additional mutations in specifics oncogenes. Another hypothesis is that, depending on cellular context, p53 can both inhibit or promote cell differentiation, as well as influence cell fate decisions, so the differentiation program of neural precursors can be changed by p53 mutation. Lastly, accumulated evidences support a role for p53 in the suppression of cell migration, although much focus on p53 is directed at its growth inhibitory properties [213]. The neurogenic niche has been shown to be important for the maintenance of stem cells in an undifferentiated state, and the premature exit of NSCs from the neurogenic niche may alter their capacity for tissue invasion or differentiation program in the absence of p53 [214].

The number of studies concerning the cellular, molecular, and environmental factors that regulate p53 function in NSCs has increased drastically and brought a better understanding of these factors, and together with the advances in molecular biology techniques, provided much valuable information about the role of p53 in BTSCs. This scenario stimulates future studies exploring the significance of p53 alterations for prognosis and prediction of treatment response that would help development of individual treatment strategies as well as help clarifying the clinical importance of cancer stem cell biology.

progenitor cells for the delivery of immunostimulatory genes, cytotoxic genes and genes

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The fact that p53 pathway is activated in tumor cells, but not in normal cells, provides a potentially important therapeutic selectivity, indifferent of which signal in the tumor cells activates p53 following its restoration [225]. In this context, the evidences that tumor cells, but not normal cells, have a cellular environment that activates the p53 pathway would create a setting of an advantageous therapeutic index, whose main objective is the development of

Different approaches to achieve this goal are already in various stages of development and a diversity of small druglike molecules targeting the p53 system have been developed and several are now in clinical trials. Of critical importance has been the development of: agents which can increase active p53 in tumor cells by interfering with the p53–MDM2 interaction are therefore considered to have therapeutic utility in sensitizing tumor cells for chemo-or radiotherapy, such as the Nutlins [226, 227]; molecules that activate p53 via direct interaction with p53 itself, as PRIMA-1, of which there is evidence of induction of expression of mediators of p53-dependent apoptosis such as Puma, Noxa, and Bax in cells with mutant p53 [228, 229]; small molecules activating p53 family members in a p53 mutant or deficient background; molecules activating p53 by inhibiting class III histone deacetylases, nuclear export, transcrip‐ tional and nucleolar distuption. These screens in combination with RNAi based approaches are of utmost importance for the discovery of new targets for therapy in the p53 pathway [215]. Transfection of wild-type p53 in order to normalize function in mutant p53-containing tumors has been a long-pursued goal of gene therapy. Mercer *et al.* [230] initially demonstrated that plasmid-mediated transfection of the p53 gene is capable of suppressing cell growth in gliomas by inhibition of G0/G1 progression into S phase. Kock *et al.* [231] and Gomez-Manzano *et al.* [232] were among the first to demonstrate that delivering the p53 gene using an adenovirus vector (Ad-p53) resulted in high levels of apoptosis in glioma cell lines, by elevation of the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. Frederick *et al.*[233] undertook a phase I trial of Ad-p53 in the treatment of patients with recurrent malignant gliomas with the purpose of determine the clinical toxicity of Ad-p53 and obtain molecular information regarding the expression and distribution of the p53 protein after intratumoral treatment of human gliomas with Ad-p53. Thus, their results conclude that Intratumoral injection of Ad-p53 allowed the exogenous transfer of the p53 gene and expression of func‐

To the generation of an effective systemic anti-tumor immune response, it is necessary the development of strategies that promote the GBM tumor cell death, which is essential not only to kill tumor cells and reduce tumor burden, but also to induce the release of inflammatory molecules from dying tumor cells [234]. Drug combinations have been developed to selectively kill cancer cells that lack p53 function while protecting normal cells. The potential to explore the defective checkpoint status of cells with inactive *TP53* genes has also been largely recog‐ nized and in part stimulated the search of drugs that can inhibit PLK1, AURKB, and other proteins that regulate the G 2 /M checkpoint [235]. Shchors et al. [236] used a preclinical model of GBM in combination with a switchable p53 allele to model the therapeutic effect of p53

interventions that selectively kill tumor cells instead normal cells [225].

modulating the tumor microenvironment [216].

tional p53 protein, with minimaltoxicity observed.
