**2.4.1 Stress-induced stem cell markers**

Although, at present, there are no universally accepted markers of GCSCs, this section will cover known NSC markers along with frequently studied glioma stem cell markers (Fig. 3). Small side populations (SP) of glioma stem cells (0.01-5% of total cells) exist that rarely divide despite elevated proliferation potential (Hirschmann-Jax et al, 2004). These were first found through flow cytometry studies where a small SP of tumor cells could be sorted and differentiated from the rest of the population. These cells were shown to efflux the fluorescent nucleic acid-staining dye, Hoechst 33342 (Pattrawala et al, 2005). When isolated, this small percentage of cells was able to generate neurospheres and xenografts, which are

of its induction of autophagy. The glycolytic inhibitor activated EF-2K and thus autophagy in a PTEN-independent manner. Inhibition of EF-2K blocked autophagy induction by 2-DG, thereby sensitizing the cells to 2-DG cell death through caspase-3 apoptosis. Cells under additional stress like hypoxia were further sensitized by concurrent treatment of 2-DG and

Thus, it appears that glioma cells have used autophagy to resist a wide range of current glioma therapies. Although originally thought to be a mechanism of cell death, autophagy obviously plays a major role in protecting glioma cells from therapeutic intervention. Studies do indicate, however, that inhibiting autophagy may re-sensitize cancer cells to

Neural stem cells (NSCs) are specific to the central nervous system and are multipotent able to generate neurons, astrocytes, and oligodendrocytes. Like other stem cells, they are selfrenewing, proliferative, and quiescent until needed. NSCs are common during human embryonic development but are reduced in number and sequestered to specific regions of adult brains. These tiny subpopulations of cells can be recognized by their CD133+ status. In recent years, there has been a new consensus that gliomas contain a glioma cancer stem cell (GCSC) population in addition to other precursor and differentiated cancer cells. Thus, gliomas can express both neuronal and glial markers. There is accumulating evidence that NSCs are key players in tumor initiation and progression along with angiogenesis and dissemination. Thus, their presence is starting to redefine how therapy outcomes are determined and understanding their role in tumor progression and therapy resistance may

For years it was thought that humans were born with all the brain cells that they were ever going to have and that mitosis of neural and glial cells only occurred during early development. While most cells in the CNS do exit the cell cycle as terminally differentiated cells early in life, it has come to light that neurogenesis continues throughout life in small areas of the brain including the subventricular zone (Lois & Alvarez, 1993) and the dentate gyrus (Kuhn et al., 1996). These locations are home to NSCs that exhibit the normal stem cell markers and are capable of migration and multipotency. The existence of these NSCs that are normally present in the brain provides precedence for the idea of mutipotent cells in the CNS and gliomas. As gliomas are known to be highly heterogeneous tumors with cells from multiple neural lineages, cancerous neural stem cells could explain this finding. Poor prognosis has been linked to glioma tumor heterogeneity (Pallini et al., 2008), which could

Although, at present, there are no universally accepted markers of GCSCs, this section will cover known NSC markers along with frequently studied glioma stem cell markers (Fig. 3). Small side populations (SP) of glioma stem cells (0.01-5% of total cells) exist that rarely divide despite elevated proliferation potential (Hirschmann-Jax et al, 2004). These were first found through flow cytometry studies where a small SP of tumor cells could be sorted and differentiated from the rest of the population. These cells were shown to efflux the fluorescent nucleic acid-staining dye, Hoechst 33342 (Pattrawala et al, 2005). When isolated, this small percentage of cells was able to generate neurospheres and xenografts, which are

currently used treatments, providing a way around tumor resistance.

EF-2K inhibition (Wu et al, 2009).

**2.4 Stress and glioma cancer stem cells** 

be pivotal in improving patient prognoses.

**2.4.1 Stress-induced stem cell markers** 

be the result of GCSCs.

key stem cell properties. The SPs were able to efflux both the dye and chemotherapeutic agents through up-regulation of an ATP-binding cassette (ABC) member, BCRP, which is involved in multi-drug resistance (Eramo et al., 2006). These SPs were the first indicator that there was probably a cancer stem cell population in gliomas.

The two most common markers of neural stem cells are CD133 and nestin. CD133, also known as prominin-1, is a cell membrane glycoprotein which is present on different types of stem cells and cancer cells while being down-regulated on differentiated cells (Uchida et al., 2000). CD133+ cells can be isolated from human brain tumors and are able to demonstrate stem cell properties *in vivo* like accelerated tumor growth and invasion (Singh et al., 2004). They pass the gold standard for determining stem cell properties, which is that cells must be able to initiate formation of tumor similar to the patient's and is able to undergo serial transplantations. These cells have increased levels of stem cell genes such as nestin, Msi-1, MELK, and CXCR4 (Lui et al., 2006). Recent studies indicate that CD133 expression may be linked to periods of angiogenesis or times of stress. In fact, hypoxia can induce a CD133+ brain tumor stem cell population. CD133+ cells are resistant to drug treatment and apoptosis with increased expression of several ABC transporters and DNA repair machinery (Eramo et al, 2006). These cells also show an increase in chemokine receptor CXCR4 that directs NSC migration and thus GSCS movement (Lui et al., 2006). Even though it appears that CD133+ status is indicative of a NSC, CD133- cells can still exhibit stem cell properties (Wang et al., 2008). Therefore, CD133 status is not the final determinant of stem cellness for glioma cells.

The another common marker, nestin, is an intermediate filament protein produced by NSCs that controls cellular morphology, proliferation, and adhesion. Differentiated cells downregulate nestin and increase other neurofilaments involved in neurons and glial cells that have exited the cell cycle (Zimmerman et al., 1994). Nestin expression increases during stress like ischemia, traumatic brain injury, inflammation, and tumor progression (Holmin et al., 1997). As a glioma marker, it indicates an increased malignant potential in invasion and motility abilities associated with poor prognosis (Stronjnik et al., 2007). It was one of the first discovered NSC markers but is not ideal due to its cytoplasmic location. Thus, sorting methods like flow cytometry cannot be used to separate stem cells from non-stem cells according to nestin status.

Other stem cell markers may be more useful due to their location on the cell surface. A2B5 is cell surface marker of neural progenitor cells. It is a ganglioside normally found in cells in the subventricular zones that host NSCs (Nunes et al., 2003). It has recently been reported that cells in GBM have been found that are A2B5+ and exhibit stem cell properties like tumor initiation. In fact, A2B5+/CD133- cells are also capable of initiating tumors and forming neurospheres (Tchoghandjian et al., 2009). Thus, A2B5 status is a potential useful marker for stem cells in gliomas and should be added to initial screenings. Stage-Specific Embryonic Antigen -1 (SSEA-1), which is also known as CD15 or Lewis-X Antigen, is another cell surface antigen. It is a carbohydrate moity that associates with glycoproteins and glycolipids. SSEA-1+ cells have increased stem cell gene expressions and properties. The majority of GBM tumors analyzed for the marker were SSEA-1+, and SSEA-1+ cells are highly tumorigenic while SSEA-1- cells are not (Son et al., 2009). Therefore, these two surface markers may play an important role in determining a GCSC population.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 41

It remains a matter of debate whether GCSCs are derived from original NSCs or other progenitor cells. Some argue that cancer cells can actually transition from a differentiated cell back into an undifferentiated cell through epithelial-mesechymal transition (EMT) (Singh & Settleman, 2010). This would mean that signals that trigger EMT could also trigger cancer stem cell formation. Either way, it has been shown that stem cell pathways including Wnt/ β-catenin, SSH, and Bmi-1 can all be activated by common treatments used in glioma therapy, like ionizing radiation and TMZ, which may lead to resistance to radiation and

Thus, regardless of the origins of the GCSCs, the presence of cancer stem cells would explain the seemingly inevitable recurrence of advanced gliomas. If cancer cells can either revert to a de-differentiated state or if original mutated stem cells from a tumor could survive a therapy, then the cancer could proliferate again. Stem cells are designed to survive assaults. They are a form of cellular dormancy that can wait until the cell encounters a more favorable environment, and then self-renew, proliferate, and create differentiated progeny that are suited to the present conditions (Hambardzumyan et al., 2008). GCSCs could shed light on why patients seem to have been cured of their cancer only to have it return months or even decades later after having undergone numerous surgeries, chemotherapies, and radiation treatments. Mounting evidence indicates that cancer stem cells are key to the survival or recurrence of glioma. In fact, current treatment regimens could worsen matters by putting the cancer cells under the exact stresses that actually select for GCSCs (Tetyana et

Stresses in different areas of a tumor could select for distinct subpopulations of normal tumor cells and CSCs. Since CSCs by definition are plastic, they are able to adapt to their current environment, at times lying dormant and at others, proliferating and differentiating cancer cells that have adapted through genomic instability that is inevitable with cancer progression. These cells are even able to create stromal support layers recruiting host cells to make some of the necessary growth factors and signals (Bao

The GCSCs either migrate or produce their own local microenvironments or niches that created by cells and an ideal extracellular matrix (ECM) (Fig. 3). Niches shield the GCSCs from harsh environments present in the rest of the brain (Valshi et al., 2009). They support a specific mix of necessary growth factors, signaling molecules, and nutrients to sustain a stem cell population. Perivascular niches are also commonly home to healthy NSCs (Calabrese et al., 2007). In GBM, the niche is composed of vasculature that contacts the cells and allows secretion of factors that help to maintain stem cell quiescence. This increased density of microvessels is highly associated with GCSCs niches, which tightly regulates the availability of oxygen and nutrients while allowing the cells a means of migration to other areas if necessary. The stems cells continue to modulate this extracellular environment – they secrete VEGF and increase the number of endothelial cells, which in turn leads to increased GCSC and tumor growth (Gilbertson & Rich, 2007). Niches are abnormal in

Protected by their ideal environment, GCSCs are easily able to resist common treatments. This is conceivable since even normal stem cells need to survive years of stress under normal circumstances. As mentioned previously, GCSCs express adenosine triphosphate

GCSCs because they cause the stem cells to renew and proliferate.

**2.4.2 Glioma cancer stem cells are obstacles to effective treatment** 

chemotherapy (Bell & Miele, 2011).

al., 2010).

et al, 2006).

Fig. 3. Glioma cancer stem cell population in the perivascular niche. Common markers are listed.

Developmental pathways and other signaling pathways are also up-regulated in SPs, which later became indicators of stem cell activity (Hadnagy et al., 2006). The Wnt/β-catenin and the sonic hedgehog (SHH)/Gli1 pathways are both upregulated in GCSCs (Rich, 2007). Wnt signaling increases β-catenin activity. Over-expression of Gli1 and β-catenin have recently been shown to be correlated with poor prognosis (Pu et al., 2009), while EGFR and p53 were not predictive (Rossi et al., 2011). Some GCSCs have increased Notch-1 signaling, as do their healthy NSCs counterparts. Even PDGF status is linked to stem cells, as signaling of the growth factor is upregulated during oligodendrocyte proliferation and differentiation (Nait-Oumesmar et al., 1997).

Other stem cell pathways and molecules have been found in these populations. The Notch receptor pathway has also been implicated in GCSCs, as this signaling cascade mediates differentiation and proliferation (Koch and Radtke, 2007). Over-expression of Notch-1 leads to formation and proliferation of neurosphere-forming stem cells that are nestin-positive, while down-regulation leads to apoptosis (Hitoshi et al., 2002). Additional traditional stem cell markers such as Sox2, Bmi-1, PCNA, NANO, Msi-1, and OCT4 have all been found in these cells, indicating that gliomas do in fact have populations of cancerous cells that have stem-cell like properties and are probably GCSCs (Hemmati et al., 2003). This finding has a tremendous impact of glioma cell survival and important implications for new therapies regimens to treat patients with glioma.

## **2.4.2 Glioma cancer stem cells are obstacles to effective treatment**

40 Advances in the Biology, Imaging and Therapies for Glioblastoma

Fig. 3. Glioma cancer stem cell population in the perivascular niche. Common markers are

Developmental pathways and other signaling pathways are also up-regulated in SPs, which later became indicators of stem cell activity (Hadnagy et al., 2006). The Wnt/β-catenin and the sonic hedgehog (SHH)/Gli1 pathways are both upregulated in GCSCs (Rich, 2007). Wnt signaling increases β-catenin activity. Over-expression of Gli1 and β-catenin have recently been shown to be correlated with poor prognosis (Pu et al., 2009), while EGFR and p53 were not predictive (Rossi et al., 2011). Some GCSCs have increased Notch-1 signaling, as do their healthy NSCs counterparts. Even PDGF status is linked to stem cells, as signaling of the growth factor is upregulated during oligodendrocyte proliferation and differentiation (Nait-

Other stem cell pathways and molecules have been found in these populations. The Notch receptor pathway has also been implicated in GCSCs, as this signaling cascade mediates differentiation and proliferation (Koch and Radtke, 2007). Over-expression of Notch-1 leads to formation and proliferation of neurosphere-forming stem cells that are nestin-positive, while down-regulation leads to apoptosis (Hitoshi et al., 2002). Additional traditional stem cell markers such as Sox2, Bmi-1, PCNA, NANO, Msi-1, and OCT4 have all been found in these cells, indicating that gliomas do in fact have populations of cancerous cells that have stem-cell like properties and are probably GCSCs (Hemmati et al., 2003). This finding has a tremendous impact of glioma cell survival and important implications for new therapies

listed.

Oumesmar et al., 1997).

regimens to treat patients with glioma.

It remains a matter of debate whether GCSCs are derived from original NSCs or other progenitor cells. Some argue that cancer cells can actually transition from a differentiated cell back into an undifferentiated cell through epithelial-mesechymal transition (EMT) (Singh & Settleman, 2010). This would mean that signals that trigger EMT could also trigger cancer stem cell formation. Either way, it has been shown that stem cell pathways including Wnt/ β-catenin, SSH, and Bmi-1 can all be activated by common treatments used in glioma therapy, like ionizing radiation and TMZ, which may lead to resistance to radiation and chemotherapy (Bell & Miele, 2011).

Thus, regardless of the origins of the GCSCs, the presence of cancer stem cells would explain the seemingly inevitable recurrence of advanced gliomas. If cancer cells can either revert to a de-differentiated state or if original mutated stem cells from a tumor could survive a therapy, then the cancer could proliferate again. Stem cells are designed to survive assaults. They are a form of cellular dormancy that can wait until the cell encounters a more favorable environment, and then self-renew, proliferate, and create differentiated progeny that are suited to the present conditions (Hambardzumyan et al., 2008). GCSCs could shed light on why patients seem to have been cured of their cancer only to have it return months or even decades later after having undergone numerous surgeries, chemotherapies, and radiation treatments. Mounting evidence indicates that cancer stem cells are key to the survival or recurrence of glioma. In fact, current treatment regimens could worsen matters by putting the cancer cells under the exact stresses that actually select for GCSCs (Tetyana et al., 2010).

Stresses in different areas of a tumor could select for distinct subpopulations of normal tumor cells and CSCs. Since CSCs by definition are plastic, they are able to adapt to their current environment, at times lying dormant and at others, proliferating and differentiating cancer cells that have adapted through genomic instability that is inevitable with cancer progression. These cells are even able to create stromal support layers recruiting host cells to make some of the necessary growth factors and signals (Bao et al, 2006).

The GCSCs either migrate or produce their own local microenvironments or niches that created by cells and an ideal extracellular matrix (ECM) (Fig. 3). Niches shield the GCSCs from harsh environments present in the rest of the brain (Valshi et al., 2009). They support a specific mix of necessary growth factors, signaling molecules, and nutrients to sustain a stem cell population. Perivascular niches are also commonly home to healthy NSCs (Calabrese et al., 2007). In GBM, the niche is composed of vasculature that contacts the cells and allows secretion of factors that help to maintain stem cell quiescence. This increased density of microvessels is highly associated with GCSCs niches, which tightly regulates the availability of oxygen and nutrients while allowing the cells a means of migration to other areas if necessary. The stems cells continue to modulate this extracellular environment – they secrete VEGF and increase the number of endothelial cells, which in turn leads to increased GCSC and tumor growth (Gilbertson & Rich, 2007). Niches are abnormal in GCSCs because they cause the stem cells to renew and proliferate.

Protected by their ideal environment, GCSCs are easily able to resist common treatments. This is conceivable since even normal stem cells need to survive years of stress under normal circumstances. As mentioned previously, GCSCs express adenosine triphosphate

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 43

plans. The key is to make the most of the information available to personalize the therapies given to each patient. Therefore, a compilation of an assortment of markers and screening

structure inhibition

lysosome fusion

lysosome fusion

lysosome fusion

increase stress

therapy

Blocks translation of autophagic response

Targets mitochondria for destruction to sensitize to autophagy inhibition

Anti-angiogenic to sensitize stem cell population to

Increase cellular pH to sensitize stem cell population to therapy

Bafilomycin A1 Block autophagosome and

HCQ Block autophagosome and

Monensin Block autophagosome and

SSH, and Notch-1 pathways Reduce stem cell signaling

NH125 Inhibits EF-2K

**Inhibitors** 3-MA Pre-autophagosomal

siRNA against BECN1,ATG5,

**Enhancers** Rapamycin Inhibit mTOR pathway to

ATG7, ATG10, etc.

Arsenic trioxide

Sodium Bicarbonate

siRNA against β-catenin/wnt,

Since the majority of current glioma therapies induce autophagy in glioma cells, including radiation and chemotherapy, inhibition of autophagy would decrease the survival of these cells. Tumor cells may undergo apoptosis when autophagy is started and then disrupted. Adding the autophagy inhibitor bafilomycin A1 (H+-APTase inhibitor) to TMZ lead to cell death via apoptosis through caspase-3 and mitochondrial permeabilization. Autophagy can be blocked at multiple levels, allowing for adjustment according to whether cells are using it as a protective or cell death mechanism. The P13K inhibitor, 3-methyladenine (3-MA), can be used to inhibit autophagy before formation of the autophagosome (Kanzawa et al., 2004). Other agents like bafilomycin A1 blocks the fusion of the autophagosome with the lysosome, as do several other drugs like hydroxychloroquine (HCQ) and the proton exchanger, monensin. The natural product arsenic trioxide targets mitochondria and induces autophagy in glioma cells, and when combined with bafilomycin A1, is able to

Table 3. Proposed therapies for sensitization of gliomas to current treatments.

panels needs to be created to fit into the era of personalized medicine.

**Category Compound Target**

**Autophagic** 

**Autophagic** 

**Agents Affecting** 

**Tumor Stem Cells** Bevacizumab

(ATP)-binding cassette transporters (ABC-transporters) such as MDR1 and breast cancer resistance protein (BCRP) that are able to efflux chemotherapeutic drugs. This allows stem cells to survive regardless of the type of chemotherapy delivered. Even when the cells cannot efflux all of the agent, GCSCs derived from patient tumors, when treated with common therapies, are able to show resistance within 48 hours with continued ability to proliferate (albeit at a lower rate) in the presence of the drugs (Eramo, 2006). Many of these cells also show increases in DNA-mismatch repair with over-expression of methyl guanine methyl transferase (MGMT) which is common in resistance to alkylating or alkylating-like agents (Jullierat-Jeanneret et al., 2008). Anti-apoptotic proteins like Bcl-2 and Bcl-XL were found to be over-expressed in the increased population of CD133+ cells GCSC treated with TMZ, carboplatin, or taxol, as were members of the inhibitor of apoptosis (IAP) family such as surviving (Lui et al., 2006). Numerous mechanisms and pathways have been associated with treatment resistance of GCSCs.

Not only are GCSCs able to survive therapeutic insults, but many interventions actually select for the cancer stem cells. Radiation has been shown to enrich for GCSC. These stem cells have increased survival advantages over their non-radiotreated counterparts. Radioresistant tumors showed increases in CD133+ status. Notch-1 signaling has also been shown to be activated upon radiation exposure (Scharpfenecker et al., 2009). All cells had the same amount of initial damage to DNA and organelles, but GCSCs were able to repair damage more quickly than matched non-stem cells (Bao et al., 2006). CD133+ cells are able to evade radiation damage by preferential activation of DNA damage checkpoints. Bao determined that the resistance to radiation can be partially circumvented by inhibiting cell cycle proteins, Chk1 and Chk2. Treatment with alkylating agents also increased GCSC populations, as determined by stem cell markers (Kang and Kang, 2007).

Due to the ability of gliomas to either induce stem cell dedifferentiation or to select for already present GCSC populations during treatment and stress, glioma cancer stem cells present a considerable problem for future therapeutic regimens. While common therapies can reduce the size of gliomas to microscopic levels, these small populations of stem cells remain within the tissues, resistant to therapies and selected due to their ability to survive. Recurring tumors will therefore be more malignant and progress more quickly than the original glioma. Therefore, focus on eliminating GCSCs should be pursued during brain cancer research treatment.

#### **2.5 Targeting stress response as glioma therapy**

The preceding sections have focused on how glioma cells manage to evade the deleterious effects of both intrinsic and extrinsic stressors. Future developments in glioma therapy should take into account these survival pathways. New treatment options should take advantage of the stress response and new survival mechanisms such as autophagy, while others could target GCSCs in order to completely, and hopefully, permanently eliminate glioma tumors. Novel treatments can be used to sensitize glioma cells to traditional therapies by exacerbating the stresses the cells are under (Table 3).

Most importantly, gliomas need to be characterized for the proteins that they express. This can then inform clinicians what therapies with which that particular tumor may be treated. For example, glioma cells or tumors that are shown to be deficient in autophagic proteins might be more susceptible to traditional apoptotic therapies or radiation. Tumors void of any stem cell markers might be less likely to recur, and those patients can be offered more simple treatment

(ATP)-binding cassette transporters (ABC-transporters) such as MDR1 and breast cancer resistance protein (BCRP) that are able to efflux chemotherapeutic drugs. This allows stem cells to survive regardless of the type of chemotherapy delivered. Even when the cells cannot efflux all of the agent, GCSCs derived from patient tumors, when treated with common therapies, are able to show resistance within 48 hours with continued ability to proliferate (albeit at a lower rate) in the presence of the drugs (Eramo, 2006). Many of these cells also show increases in DNA-mismatch repair with over-expression of methyl guanine methyl transferase (MGMT) which is common in resistance to alkylating or alkylating-like agents (Jullierat-Jeanneret et al., 2008). Anti-apoptotic proteins like Bcl-2 and Bcl-XL were found to be over-expressed in the increased population of CD133+ cells GCSC treated with TMZ, carboplatin, or taxol, as were members of the inhibitor of apoptosis (IAP) family such as surviving (Lui et al., 2006). Numerous mechanisms and pathways have been associated

Not only are GCSCs able to survive therapeutic insults, but many interventions actually select for the cancer stem cells. Radiation has been shown to enrich for GCSC. These stem cells have increased survival advantages over their non-radiotreated counterparts. Radioresistant tumors showed increases in CD133+ status. Notch-1 signaling has also been shown to be activated upon radiation exposure (Scharpfenecker et al., 2009). All cells had the same amount of initial damage to DNA and organelles, but GCSCs were able to repair damage more quickly than matched non-stem cells (Bao et al., 2006). CD133+ cells are able to evade radiation damage by preferential activation of DNA damage checkpoints. Bao determined that the resistance to radiation can be partially circumvented by inhibiting cell cycle proteins, Chk1 and Chk2. Treatment with alkylating agents also increased GCSC

Due to the ability of gliomas to either induce stem cell dedifferentiation or to select for already present GCSC populations during treatment and stress, glioma cancer stem cells present a considerable problem for future therapeutic regimens. While common therapies can reduce the size of gliomas to microscopic levels, these small populations of stem cells remain within the tissues, resistant to therapies and selected due to their ability to survive. Recurring tumors will therefore be more malignant and progress more quickly than the original glioma. Therefore, focus on eliminating GCSCs should be pursued during brain

The preceding sections have focused on how glioma cells manage to evade the deleterious effects of both intrinsic and extrinsic stressors. Future developments in glioma therapy should take into account these survival pathways. New treatment options should take advantage of the stress response and new survival mechanisms such as autophagy, while others could target GCSCs in order to completely, and hopefully, permanently eliminate glioma tumors. Novel treatments can be used to sensitize glioma cells to traditional

Most importantly, gliomas need to be characterized for the proteins that they express. This can then inform clinicians what therapies with which that particular tumor may be treated. For example, glioma cells or tumors that are shown to be deficient in autophagic proteins might be more susceptible to traditional apoptotic therapies or radiation. Tumors void of any stem cell markers might be less likely to recur, and those patients can be offered more simple treatment

populations, as determined by stem cell markers (Kang and Kang, 2007).

with treatment resistance of GCSCs.

cancer research treatment.

**2.5 Targeting stress response as glioma therapy** 

therapies by exacerbating the stresses the cells are under (Table 3).

plans. The key is to make the most of the information available to personalize the therapies given to each patient. Therefore, a compilation of an assortment of markers and screening panels needs to be created to fit into the era of personalized medicine.


Table 3. Proposed therapies for sensitization of gliomas to current treatments.

Since the majority of current glioma therapies induce autophagy in glioma cells, including radiation and chemotherapy, inhibition of autophagy would decrease the survival of these cells. Tumor cells may undergo apoptosis when autophagy is started and then disrupted. Adding the autophagy inhibitor bafilomycin A1 (H+-APTase inhibitor) to TMZ lead to cell death via apoptosis through caspase-3 and mitochondrial permeabilization. Autophagy can be blocked at multiple levels, allowing for adjustment according to whether cells are using it as a protective or cell death mechanism. The P13K inhibitor, 3-methyladenine (3-MA), can be used to inhibit autophagy before formation of the autophagosome (Kanzawa et al., 2004). Other agents like bafilomycin A1 blocks the fusion of the autophagosome with the lysosome, as do several other drugs like hydroxychloroquine (HCQ) and the proton exchanger, monensin. The natural product arsenic trioxide targets mitochondria and induces autophagy in glioma cells, and when combined with bafilomycin A1, is able to

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 45

brain cancers. Both intrinsic and extrinsic stresses impact the development and progression of glioma. Stresses like nutrient deficiency, hypoxia, acidity, and the immune response are present during normal tumor growth and throughout treatment. Tumor cells that survive these stresses are more adept at surviving hostile conditions and are more resistant to current therapies. Stress, therefore, shapes the tumor cell population. The autophagic response and glioma cancer stem cells are two of the prevalent survival and resistance

New treatment regimens should take advantage of various stresses and stress responses present in glioma. Stresses like hypoxia or acidity could be exacerbated and sustained with new agents, allowing traditional therapies, such as TMZ or radiation, to permanently eliminate the sensitized cells. Autophagy is a fragile state for cancer cells, and inhibition of the autophagic process at the level of EF2K, beclin-1, and other proteins may combat this glioma cell survival mechanism. Targeting the autophagy pathway has already been shown to render glioma cells and other types of cancers more susceptible to currently available treatments. Recognition of GCSC markers could lead to better diagnostic and prognostic tools in addition to targeted cancer stem cell therapy. Combining current therapy with inhibitors that interrupt the stress response of glioma cells is an attractive approach for

Research was supported by grants from the US Public Health Service and NCI with

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Ailles LE, & Weissman IL. (2007). Cancer stem cells in solid tumors. *Curr Opin Biotechnol,*

Azad MB, Chen Y, Henson ES, Cizeau J, McMillan-Ward E, Israels SJ, & Gibson SB. (2008).

Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, &

Benhar M, Dalyot I, Engelberg D, & Levitzki A. (2001). Enhanced ROS production in

Brat DJ, & Van Meir EG. (2004). Vaso-occlusive and prothrombotic mechanisms associated

activation of the DNA damage response. *Nature*, Vol. 444, pp. 756-60. Bell D, & Miele L. (2011). A magnifying glass on glioblastoma stem cell signaling pathways.

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mechanism involving BNIP3. *Autophagy*, Vol. 4, pp. 195–204.

In: *Autophagy*. Daniel Klionsky, Landes Bioscience, ISBN: 1-58706-203-8, University

Hypoxia induces autophagic cell death in apoptosis-competent cells through a

Rich JN. (2006). Glioma stem cells promote radioresistance by preferential

oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress.

with tumor hypoxia, necrosis, and accelerated growth in glioblastoma. *Lab Invest*,

mechanisms in glioma, and both can be induced by cellular stresses.

future glioma treatment.

**4. Acknowledgment** 

of Michigan

Vol. 18, No. 5, pp. 460-6.

*Mol and Cell Bio*, Vol. 21, pp. 6913-26.

Vol. 84, pp. 397-405.

RO1CA135038.

**5. References** 

eliminate all remaining tumor cells (Kanzawa et al., 2005). Drugs like rapamycin could be used to further the stress caused by nutrient starvation, activating autophagy, thereby leading to cell self-digestion. Synergistic killing of glioma cells has been shown with treatment of rapamycin and either Akt or PI3K inhibitors (Takeuchi et al., 2005). These are just a few examples of how therapies can be combined to sensitize and eliminate glioma cells through autophagic inhibition or enhancement.

In the future, genetic modification of cells will be more feasible, so eventually the autophagic response could also be targeted with small interfering RNA (siRNA) against autophagy proteins such as BECN1, ATG5, and ATG10 along with many others. EF-2K presents a novel target for inhibiting autophagy and sensitizing cells to other therapies. NH125 is the preclinical inhibitor of EF-2K and could be developed into a bioavailable agent for humans or an siRNA could be used against the kinase as well.

Glioma cancer stem cells are another cell type that could be targeted as glioma therapy for complete abolishment of the malignancy. Bmi-1 is an E3-ubiquitin ligase that is up-regulated in GCSCs and other cancer stem cells. GSCSs have low proteosome activity, which can be used to track stem cells through Bmi-1 degradation (Vlashi et al., 2009). Eliminating GCSCs by targeting Bmi-1 expressing cells was sufficient for causing regression of the solid glioma indicating that ridding the tumor of cancer stem cells may actually be curative.

Several possibilities exist for targeting stem cells. As mentioned previously, anti-VEGF treatments like bevacizumab might help to normalize the vasculature and allow for efficient delivery of chemotherapies (von Baumgarten et al., 2011). In xenotranpslants, bevacizumab synergizes with radio- and chemo-therapy to effectively kill glioma cells (Vredenburgh et al., 2007). Anti-angiogenic therapy could help reduce the stem cell niches. Bevacizumab blocked the GSCSs ability to induce the migration of endothelial cells necessary for neoangiogenesis and cell migration (Ailles & Weissman, 2007). Thus, anti-VEGF treatment might work in multiple ways.

GSCSs could be targeted by additional approaches. Treatment of tumors with sodium biocarbonate increased tumor pH and reduced invasion, while reducing stem cell markers in breast cancer (Robey et al., 2009). While not yet used in glioma, this could restore the nonacidic pH environment, targeting the tumor microenvironment. Also, developing agents against the stem cell signaling pathways will be important in eliminating tumors. The βcatenin/wnt and SHH pathways along with Notch-1 signaling are all good candidates for targeting. Markers, CD133 and nestin, could be used to identify potential responders.

Overall, new therapies should be used to take advantage of the stress that glioma cells already encounter, in addition to targeting their means of survival. Those gliomas that use autophagy as a protective mechanism could be treated with autophagic inhibitors to enhance efficacy of therapy. These therapies could be used to sensitize tumors to treatment with standard radiation and chemotherapies. Also, any gliomas that have markers indicative of cancer stem cells might be considered to be treated with agents that target stem cells to circumvent that avenue of cancer cell survival. Autophagy and GCSCs represent attractive, novel targets for future glioma therapy that can be combined with conventional therapy to fully eradicate glioma cells.

### **3. Conclusions/perspectives**

The preceding chapter sought to introduce the concept that glioma cells are constantly under stress, a factor which needs to be taken into consideration during the treatment of brain cancers. Both intrinsic and extrinsic stresses impact the development and progression of glioma. Stresses like nutrient deficiency, hypoxia, acidity, and the immune response are present during normal tumor growth and throughout treatment. Tumor cells that survive these stresses are more adept at surviving hostile conditions and are more resistant to current therapies. Stress, therefore, shapes the tumor cell population. The autophagic response and glioma cancer stem cells are two of the prevalent survival and resistance mechanisms in glioma, and both can be induced by cellular stresses.

New treatment regimens should take advantage of various stresses and stress responses present in glioma. Stresses like hypoxia or acidity could be exacerbated and sustained with new agents, allowing traditional therapies, such as TMZ or radiation, to permanently eliminate the sensitized cells. Autophagy is a fragile state for cancer cells, and inhibition of the autophagic process at the level of EF2K, beclin-1, and other proteins may combat this glioma cell survival mechanism. Targeting the autophagy pathway has already been shown to render glioma cells and other types of cancers more susceptible to currently available treatments. Recognition of GCSC markers could lead to better diagnostic and prognostic tools in addition to targeted cancer stem cell therapy. Combining current therapy with inhibitors that interrupt the stress response of glioma cells is an attractive approach for future glioma treatment.

#### **4. Acknowledgment**

Research was supported by grants from the US Public Health Service and NCI with RO1CA135038.

#### **5. References**

44 Advances in the Biology, Imaging and Therapies for Glioblastoma

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In the future, genetic modification of cells will be more feasible, so eventually the autophagic response could also be targeted with small interfering RNA (siRNA) against autophagy proteins such as BECN1, ATG5, and ATG10 along with many others. EF-2K presents a novel target for inhibiting autophagy and sensitizing cells to other therapies. NH125 is the preclinical inhibitor of EF-2K and could be developed into a bioavailable agent

Glioma cancer stem cells are another cell type that could be targeted as glioma therapy for complete abolishment of the malignancy. Bmi-1 is an E3-ubiquitin ligase that is up-regulated in GCSCs and other cancer stem cells. GSCSs have low proteosome activity, which can be used to track stem cells through Bmi-1 degradation (Vlashi et al., 2009). Eliminating GCSCs by targeting Bmi-1 expressing cells was sufficient for causing regression of the solid glioma

Several possibilities exist for targeting stem cells. As mentioned previously, anti-VEGF treatments like bevacizumab might help to normalize the vasculature and allow for efficient delivery of chemotherapies (von Baumgarten et al., 2011). In xenotranpslants, bevacizumab synergizes with radio- and chemo-therapy to effectively kill glioma cells (Vredenburgh et al., 2007). Anti-angiogenic therapy could help reduce the stem cell niches. Bevacizumab blocked the GSCSs ability to induce the migration of endothelial cells necessary for neoangiogenesis and cell migration (Ailles & Weissman, 2007). Thus, anti-VEGF treatment

GSCSs could be targeted by additional approaches. Treatment of tumors with sodium biocarbonate increased tumor pH and reduced invasion, while reducing stem cell markers in breast cancer (Robey et al., 2009). While not yet used in glioma, this could restore the nonacidic pH environment, targeting the tumor microenvironment. Also, developing agents against the stem cell signaling pathways will be important in eliminating tumors. The βcatenin/wnt and SHH pathways along with Notch-1 signaling are all good candidates for targeting. Markers, CD133 and nestin, could be used to identify potential responders. Overall, new therapies should be used to take advantage of the stress that glioma cells already encounter, in addition to targeting their means of survival. Those gliomas that use autophagy as a protective mechanism could be treated with autophagic inhibitors to enhance efficacy of therapy. These therapies could be used to sensitize tumors to treatment with standard radiation and chemotherapies. Also, any gliomas that have markers indicative of cancer stem cells might be considered to be treated with agents that target stem cells to circumvent that avenue of cancer cell survival. Autophagy and GCSCs represent attractive, novel targets for future glioma therapy that can be combined with conventional

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**3** 

*1Sweden 2,4,5,6USA 3Denmark*

**Key Principles in Glioblastoma Therapy** 

*1Department of Neurosurgery, Karolinska University Hospital, Stockholm 2Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA*

*4Department of Neurology, Moores Cancer Center, UCSD, San Diego, CA 5Center for Theoretical and Applied Neurosurgery, UCSD, San Diego, CA 6Division of Neurosurgery, Beth Israel Deaconess Medical Center, Boston, MA* 

Glioblastoma is the most common form of primary brain tumor. The incidence of this tumor is fairly low, with 2-3 cases per 100,000 people in Europe and North America 1. It is one of the most aggressive forms of cancer 2. Without treatment, the median survival is approximately 3 months 3. The current standard of treatment involves maximal surgical resection followed by concurrent radiation therapy and chemotherapy with the DNA alkylating agent, temozolomide 4. With this regimen, the median survival is approximately

The best available evidence suggests that glioblastomas originate from cells that give rise to glial cells5, 6. These glial derived tumors are graded by the World Health Organization (WHO) into 4 categories, termed WHO grade 1 to grade 4. The higher grade denotes histologic features of

Studies carried out over the past three decades suggest that glioblastomas, like other cancers, arise secondary to the accumulation of genetic alterations. These alterations can take the form of epigenetic modifications, point mutations, translocations, amplifications or deletions and modify gene function in ways that deregulate cellular signaling pathways leading to the cancer phenotype 8. The exact number and nature of genetic alterations and deregulated signaling pathways required for tumorigenesis remains an issue of debate9, although it is now clear that CNS carcinogenesis requires multiple disruptions to the normal cellular circuitry. The genetic alteration results in either activation or inactivation of specific gene functions that contribute to the process of carcinogenesis 9. Genes, that when activated, contribute to the carcinogenesis are generally termed proto-oncogenes. The mutated forms of these genes are referred to as oncogenes. Genes, that when inactivated, contribute to the

14 months. For nearly all affected, the treatments available remain palliative.

increased malignancy. WHO 4 glioma is essentially synonymous with glioblastoma7.

carcinogenesis are generally termed tumor suppressor genes.

**1. Introduction** 

*3Institute of Cancer Biology and Centre for Genotoxic Stress Research* 

Bartek Jiri Jr.1, Kimberly Ng2, Bartek Jiri Sr.3,

*Danish Cancer Society, Copenhagen* 

Santosh Kesari4, Bob Carter5 and Clark C. Chen1,6

Warburg, O. (1956). On Respiratory impairtment in cancer cells. *Science*, Vol. 124, pp. 269-70.

