**2.1.1 Stresses during tumor development and progression**

While research on glioma cells in laboratories is conducted primarily under nutrient-rich conditions, the micro-environments for cancer cells are actually quite hazardous. Rarely do tumor cells find themselves in conditions of perfect nutrient balance with necessary blood flow and comfortable living spaces. More often, glioma cells are constantly inundated with a barrage of stresses (Fig. 1). The internal stresses faced by glioma cells are not unique to brain tumors, but are actually shared by the majority of solid tumors types.

changes. Reactive oxygen species (ROSs) can be produced from activation of NADPH oxidase or other ceullar oxidases in the cell's various membranes. Stressor-specific responses, on the other hand, may be induced differentially depending on the type, severity, and duration of the stress. The following sections will cover these stresses and the resulting

Although gliomas can vary by type and stage, more advanced gliomas are characterized by high rates of mitosis, hypercellularity, evidence of angiogenesis, and areas of necrosis. Gliomas are known to have relatively high cellular heterogeneity, much of which may be caused by different areas of a tumor encountering different stresses and growing conditions. Thus, stress may be a driving factor in tumor heterogeneity. Although, medical professionals, along with patients, are able to control to a certain extent the extrinsic stresses put on patients' bodies and tumors such as treatments and environmental stressors (*e.g.*

Fig. 1. Typical stresses encountered by glioma cells during tumor growth and progression.

ROS Chemo-

therapy

Glioma Cell

Hypoxia

Nutrient Deprivation

> pH Imbalance

Immune System

While research on glioma cells in laboratories is conducted primarily under nutrient-rich conditions, the micro-environments for cancer cells are actually quite hazardous. Rarely do tumor cells find themselves in conditions of perfect nutrient balance with necessary blood flow and comfortable living spaces. More often, glioma cells are constantly inundated with a barrage of stresses (Fig. 1). The internal stresses faced by glioma cells are not unique to brain

**2.1.1 Stresses during tumor development and progression** 

Radiation

Growth Factor Inhibitors

tumors, but are actually shared by the majority of solid tumors types.

smoking), intrinsic stresses still naturally affect the tumor as it progresses.

Extracellular Signals

responses by normal cells and cancerous glioma cells.

**2.1 Types of stress** 

Environmental and metabolic stress occurs during tumor growth and progression. As cancer cells divide, they take up more space. Normal cells would stop growing through contact inhibition, but malignant cells overcome the signals to inhibit growth and continue to divide. As the tumor expands, it continually outgrows its blood supply. Tumors larger than 1 mm in diameter can no longer subsist on passive diffusion of nutrients (Gimbrone, 1973). The glioma cells, thus, go through periods of severe nutrient deprivation and hypoxia until enough tumor cells are able to signal new blood vessel formation or neoangiogenesis. Even after angiogenesis, cells are still subject to stress as the new vessels are prone to collapse due to their abnormal state and harsh surrounding conditions (Vajkoczy & Menger, 2004). During this time, cancer cells must adapt to survive in conditions and intermittent periods of limited amino acids, salts, and oxygen. Some researches indicate that this is when cancer cells start to rely on glycolysis, which continues even after oxygen is available, a phenomenon known as the Warburg effect. This contributes to the high metabolic demand of proliferating tumor cells and is a relatively inefficient method of producing energy (Warburg, 1956). Therefore, the cells are put under enormous stress just to keep up with energy production needs and are subjected to further metabolic stress when nutrients become unavailable.

The metabolic demands of the glioma cells are partially responsible for the increased acidity or pH imbalance found in many tumors. Human brain tumors measured with electrodes had a mean pH of 6.8, with measurements as low as 5.9; the normal pH for the human brain is ~7.1 (Vaupel et al., 1989). Such pH imbalance is even found in well vascularized areas of gliomas, thus indicating that tumor cells reside within a highly acidic environment even when oxygen is present. It was originally hypothesized that that hypoxia caused the acid buildup, but these new findings mean that hypoxia and acidity are not always linked. The increased energy metabolism of the glioma cells produce hydrogen ions and metabolites like lactic acid and carbonic acid. All these products are actively pumped out of the cell through proton exchangers and other transporters (Chiche et al., 2010). In cases with decreased perfusion, poor circulation contributes to the buildup of an acidic extracellular environment. As the tumor grows, the extracellular environment strives to slow down the progress of the cancer. Growth inhibition signals are sent that can either activate or deactivate cellular receptors depending on the need. Cancer cells survive by undergoing mutations in receptors like EGFR and PDGFR, changes that either stop the signaling cascades or rewire the signaling pathways to actually promote cancer cell growth. In this way, cellular proliferation is dissociated from nutrient availability by stress selection of surviving cells.

Hypoxia can play a major role in glioma development, with oxygen deprivation actually being necessary for tumor progression through alteration of gene expression, genomic instability, apoptotic dysregulation, and neoangiogenesis. In glioma, hypoxia is believed to be a key player due to the evidence of tumor necrosis in highly malignant forms like glioblastoma multiforme (GBM) (Brat & Meir, 2001). Brain tumors smaller than the previously stated 1 mm cutoff are found to be highly hypoxic and ill-perfused (Li et al. 2007). The oxygen deprivation is actually responsible for the growth of elaborate microvascular networks that indicate tumor progression in GBM. Even though larger GBM tumors are more vascularized, the blood vessels present are inefficient, and parts of the tumor environment remain hypoxic (Vajkoczy & Menger, 2004). Further transformation of the tumor cells occurs as reactive oxygen species (ROSs) increase during this time due to production by the mitochondria (Lui et al., 2008). Thus, hypoxia not only deprives cells of oxygen but leads to oxidative stress as well.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 27

rare due to the specificity of programming of glial cells (Pasquier et al., 1980). The cells that do survive are again subjected to the stresses of the immune system, new ECM signals, and

With the above intrinsic stresses attacking gliomas throughout their progression, it is a testament to cancer cell adaptability that any cancerous cells survive. By adapting to these stresses, gliomas have selected for the most stress resistant cells, making cancer therapy a challenge. However, while many therapies do produce the same types of stresses already present in the body, the treatments cause more effective and sustained stress, especially

Tumor cells have already found ways to survive the numerous internal stresses covered in the previous section, so it is of no surprise that glioma cells are often able to find ways around common therapies due to the similarity of the mechanisms of action of the interventions with the mechanism of the body's natural defenses. Table 1 lists some of the common and experimental therapies used to treat glioma in addition to the type of stress they cause. While the mechanisms of action are diverse, the treatments cause the same stresses already encountered by the glioma cells during the body's intrinsic response to

Surgery is almost always used on patients who are surgical candidates. Debulking of the tumor not only allows for better brain function but also allows chemotherapies to be more effective by working on a smaller population. While surgery should be undertaken in situations where critical structures will not be disrupted, the act is extremely stressful on the brain. Small areas will be cut off from the blood supply creating a hypoxic and nutrient deprived environment leading to metabolic stress for unremoved cancer cells. The death of neighboring cells along with the immune response will cause an increase in oxidative stress

Radiation is another first-line therapy against gliomas. While there are many variations of radiotherapy, ionizing radation (IR) tends to work through two basic mechanisms that ultimately damage the DNA by either charged particles or photons. In the case of photon radiation, like in intensity modulated radiation therapy (IMRT), this technique causes indirect damage that occurs after water is ionized producing free radicals. Double-stranded DNA breaks (DSBs) are the most significant cause of cell death. Photon radiotherapy requires welloxygenated tumors to create the damaging free radicals, which requires adequate blood supply to all areas of the tumor. Because many areas of gliomas are hypoxic, this technique is often relatively unsuccessful long-term in many brain tumors (Harrison et al., 2002). Particle therapy, on the other hand, works by directly damaging the DNA by charged particles. Direct damage can occur through transfer of energy from charged particles like proton, carbon or boron ions that do not require oxygen. These particles can cause DBSB themselves. In either case, there are free radicals and ROSs produced by radiation; the ROSs are necessary for the efficacy of the treatment, further injuring cells (Dal-Pizzol et al., 2003). However, these reactive molecules are released during cell death causing increased stress to surviving cells. The body mounts an immune response to repair and clear damaged cells. Although many patients with gliomas take steroids to reduce the swelling and inflammation produced by radiotherapy, remaining neoplastic cells are still subjected to large amounts of stress, killing many while

further transforming others into radioresistant tumor cells.

lack of designated tumor blood vessels.

when combined.

aberrant cell growth.

and ROSs production.

**2.1.2 Therapeutic stresses** 

One of the hallmarks of a cancerous cell or tumor is its ability to invade through the basement membrane of one tissue into another type of tissue. The body has many stop guards in place to prevent this from happening, but somehow glioma cells overcome the challenge. Again, during this time, extracellular signals are sent to the cancer cells informing them to stop growing or to go through programmed cell death. Cell growth pathways are down-regulated by these signals causing severe stress to the cells. Without the normal nutrients or pathway activations, uncancerous cells would die, but glioma cells find a way to overcome the death signals. Cellular stress pathways that involve tumor suppressor p53 and metabolic stress pathways which activate the apoptotic protein Bim are often deregulated in human glioma (Tan et al.,2005). Therefore, the neoplastic cells are able to overcome invasion preventions.

The immune system is an added stress to cancer cells during all times of tumor progression, but is especially active during invasion and dissemination. While the immune system may ignore some cancer cells that stay in their own tissue, cells from different tissues are recognized by the markers or antigens they display. The innate immune system encounters the cancer cells first, with first-response cells like macrophages, granulocytes, and mast cells attacking foreign cells displaying unknown or altered markers. Even cancer cells that have managed to down-regulate these markers are subjected to hazardous surrounding environments due to the release of ROSs, metalloproteinases, chemokines, and cytokines created by the attack and death of neighboring cancer cells (Qian and Pollard, 2010). Dendritic cells transport the antigens from the neoplastic cells to the lymphoid organs in order to mount an adaptive response against the tumor. Yet somehow, in cases of cancer progression, tumor cells are able to survive these stresses and move to alternate locations. This is partially due to activated innate immune cells and paracrine signals from surrounding cells releasing soluble pro-survival molecules that initiated tumor cells can use to alter their levels of gene transcription, continuing the cell cycle and surviving (Egeblad et al., 2010). Even though the hazardous environment may kill some neoplastic cells, others may develop and thrive due to increased genomic instability from free radicals, creating additional, resistant cancer cells (Grivennikov et al., 2010). In fact, chronic inflammation has actually been linked to tumor development. Inflammatory cells can actually help in the angiogenesis and migration of glioma cells by promoting vasculature development and releases extracellular proteases that rebuild and mold the tumor environment (Colotta et al., 2009). The adaptive immune response eventually builds such that it can clear some of the neoplastic cells, but many of the cancer cells have further transformed so that they are not recognized by the cytotoxic T-cells. Even though the adaptive immune response may initially be helpful, as it continues it further promotes chronic inflammation and stress in the area, thereby contributing to cancer progression.

Those cells able to overcome the response of the immune system have a better chance of surviving invasion and migration into new tissues. Although very rare in glioma, occasionally cells do metastasize to other locations of the body, but more commonly disseminate to nearby areas of the brain. After breaking through the basement membrane, invasion may include entrance into nearby white matter tracks, and less frequently, blood vessels. By invading the white matter tracks, glioma cells are able to migrate along CNS developmental paths of the brain, taking up residence in new areas of ideal conditions, again through the process of invasion (Dai et al., 2001). In the uncommon case of hematogenous dissemination, the liver, lungs, pleura, lymph nodes and skeletal system are the most common sites of metastasis, although metastases outside of the brain are relatively rare due to the specificity of programming of glial cells (Pasquier et al., 1980). The cells that do survive are again subjected to the stresses of the immune system, new ECM signals, and lack of designated tumor blood vessels.

With the above intrinsic stresses attacking gliomas throughout their progression, it is a testament to cancer cell adaptability that any cancerous cells survive. By adapting to these stresses, gliomas have selected for the most stress resistant cells, making cancer therapy a challenge. However, while many therapies do produce the same types of stresses already present in the body, the treatments cause more effective and sustained stress, especially when combined.

#### **2.1.2 Therapeutic stresses**

26 Advances in the Biology, Imaging and Therapies for Glioblastoma

One of the hallmarks of a cancerous cell or tumor is its ability to invade through the basement membrane of one tissue into another type of tissue. The body has many stop guards in place to prevent this from happening, but somehow glioma cells overcome the challenge. Again, during this time, extracellular signals are sent to the cancer cells informing them to stop growing or to go through programmed cell death. Cell growth pathways are down-regulated by these signals causing severe stress to the cells. Without the normal nutrients or pathway activations, uncancerous cells would die, but glioma cells find a way to overcome the death signals. Cellular stress pathways that involve tumor suppressor p53 and metabolic stress pathways which activate the apoptotic protein Bim are often deregulated in human glioma (Tan et al.,2005). Therefore, the neoplastic cells are able to

The immune system is an added stress to cancer cells during all times of tumor progression, but is especially active during invasion and dissemination. While the immune system may ignore some cancer cells that stay in their own tissue, cells from different tissues are recognized by the markers or antigens they display. The innate immune system encounters the cancer cells first, with first-response cells like macrophages, granulocytes, and mast cells attacking foreign cells displaying unknown or altered markers. Even cancer cells that have managed to down-regulate these markers are subjected to hazardous surrounding environments due to the release of ROSs, metalloproteinases, chemokines, and cytokines created by the attack and death of neighboring cancer cells (Qian and Pollard, 2010). Dendritic cells transport the antigens from the neoplastic cells to the lymphoid organs in order to mount an adaptive response against the tumor. Yet somehow, in cases of cancer progression, tumor cells are able to survive these stresses and move to alternate locations. This is partially due to activated innate immune cells and paracrine signals from surrounding cells releasing soluble pro-survival molecules that initiated tumor cells can use to alter their levels of gene transcription, continuing the cell cycle and surviving (Egeblad et al., 2010). Even though the hazardous environment may kill some neoplastic cells, others may develop and thrive due to increased genomic instability from free radicals, creating additional, resistant cancer cells (Grivennikov et al., 2010). In fact, chronic inflammation has actually been linked to tumor development. Inflammatory cells can actually help in the angiogenesis and migration of glioma cells by promoting vasculature development and releases extracellular proteases that rebuild and mold the tumor environment (Colotta et al., 2009). The adaptive immune response eventually builds such that it can clear some of the neoplastic cells, but many of the cancer cells have further transformed so that they are not recognized by the cytotoxic T-cells. Even though the adaptive immune response may initially be helpful, as it continues it further promotes

chronic inflammation and stress in the area, thereby contributing to cancer progression. Those cells able to overcome the response of the immune system have a better chance of surviving invasion and migration into new tissues. Although very rare in glioma, occasionally cells do metastasize to other locations of the body, but more commonly disseminate to nearby areas of the brain. After breaking through the basement membrane, invasion may include entrance into nearby white matter tracks, and less frequently, blood vessels. By invading the white matter tracks, glioma cells are able to migrate along CNS developmental paths of the brain, taking up residence in new areas of ideal conditions, again through the process of invasion (Dai et al., 2001). In the uncommon case of hematogenous dissemination, the liver, lungs, pleura, lymph nodes and skeletal system are the most common sites of metastasis, although metastases outside of the brain are relatively

overcome invasion preventions.

Tumor cells have already found ways to survive the numerous internal stresses covered in the previous section, so it is of no surprise that glioma cells are often able to find ways around common therapies due to the similarity of the mechanisms of action of the interventions with the mechanism of the body's natural defenses. Table 1 lists some of the common and experimental therapies used to treat glioma in addition to the type of stress they cause. While the mechanisms of action are diverse, the treatments cause the same stresses already encountered by the glioma cells during the body's intrinsic response to aberrant cell growth.

Surgery is almost always used on patients who are surgical candidates. Debulking of the tumor not only allows for better brain function but also allows chemotherapies to be more effective by working on a smaller population. While surgery should be undertaken in situations where critical structures will not be disrupted, the act is extremely stressful on the brain. Small areas will be cut off from the blood supply creating a hypoxic and nutrient deprived environment leading to metabolic stress for unremoved cancer cells. The death of neighboring cells along with the immune response will cause an increase in oxidative stress and ROSs production.

Radiation is another first-line therapy against gliomas. While there are many variations of radiotherapy, ionizing radation (IR) tends to work through two basic mechanisms that ultimately damage the DNA by either charged particles or photons. In the case of photon radiation, like in intensity modulated radiation therapy (IMRT), this technique causes indirect damage that occurs after water is ionized producing free radicals. Double-stranded DNA breaks (DSBs) are the most significant cause of cell death. Photon radiotherapy requires welloxygenated tumors to create the damaging free radicals, which requires adequate blood supply to all areas of the tumor. Because many areas of gliomas are hypoxic, this technique is often relatively unsuccessful long-term in many brain tumors (Harrison et al., 2002). Particle therapy, on the other hand, works by directly damaging the DNA by charged particles. Direct damage can occur through transfer of energy from charged particles like proton, carbon or boron ions that do not require oxygen. These particles can cause DBSB themselves. In either case, there are free radicals and ROSs produced by radiation; the ROSs are necessary for the efficacy of the treatment, further injuring cells (Dal-Pizzol et al., 2003). However, these reactive molecules are released during cell death causing increased stress to surviving cells. The body mounts an immune response to repair and clear damaged cells. Although many patients with gliomas take steroids to reduce the swelling and inflammation produced by radiotherapy, remaining neoplastic cells are still subjected to large amounts of stress, killing many while further transforming others into radioresistant tumor cells.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 29

activation of SAPK/JNK pathway (Benhar et al., 2001). Thus, chemotherapy stresses glioma

A general chemotherapy-induced stress response is seen in many types of cancer cells. This is a response to anti-neoplastic agents that can destroy many cancer cells but induce survival and resistance mechanisms in others. In yeast, stress changed the cell cycle and lead to increases in *de novo* protein synthesis, proliferation, HSP90 expression, and proton pump levels. The first line of defense in severe shock is *de novo* synthesis of protective proteins (Miligkos et al., 2000). Increasing key membrane component proteins can up- or downregulate their efficacy to restore ionic balance. Changes in the heat shock protein (HSP) population of the cell due to chemotherapeutic stress also increase HSP27 and HSP70 in resistant cells; these cells are translocated to the nucleus in response to stress, increasing protein synthesis necessary for resistance (Nadin et al., 2003). Whole body response is also important as hormones production levels can change during the stress response; such hormones can affect the cell cycle or gene transcription. On a smaller scale, cell-to-cell interactions occur between transformed and non-transformed cells involving the transfer of survival signals, thus indicating that the extracellular environment is important. The stress response to chemotherapy-induced hyperthermia can even lead to induction of drug resistance through a general increase in MDR P-glycoprotein production (Benhar et al.,

Anti-angiogenic therapies are becoming more common in glioma and are used to combat the tumor vasculature. Most of the inhibitors, like bevacizumab, are monoclonal antibodies that work by antagonistically binding vascular endothelial growth factors (VEGFs), the factors responsible for signaling growth of blood vessels. Contrary to other cancers, it is thought that anti-angiogenic drugs in glioma could work by transiently normalizing the tumor vasculature (Nagy et al., 2010). As discussed previously, tumor blood vessels are abnormal and unstable due to the mixture of pro- and anti-angiogenic factors. An angiogenesis inhibitor would override many of these signals. Although, this might decrease blood vessel formation, it might also stabilize the existing vasculature. By normalizing the vasculature, there could be improved delivery of chemotherapeutic agents. Either way, the neoplastic cells would be subject to stress caused either by hypoxia and nutrient deprivation

Progress in targeted therapies for glioma has been made in recent years. Numerous small molecule inhibitors are being tested in clinical trials to antagonize the commonly mutated or over-expressed growth factor pathways. These inhibitors, like erlotinib which works on EGFR and imatinib for PGDFR, work by intracellularly binding the tyrosine kinase receptors, interrupting the downstream PI3K and MAPK signaling cascades. Monoclonal antibodies like cetuximab and nimotuzumab (EGFR inhibitors) work similarly, except they bind extracellulary to the growth factor receptors. Most of the stress caused by these antagonists is through decreased growth factor signaling and the resulting metabolic

All these therapies cause stresses already encountered by tumor formation, but the duration and severity of the stresses during therapy is more extreme. Prolonged exposure to these stresses can induce cell death programming more effectively than short, intermittent periods. However, in most cases, some cancer cells do survive. They evade the immune system and death signals, selected for by their unique mutations leading to therapy resistance. It is therefore important to determine accurate markers to identify these cells and

cells through different mechanisms.

or increased concentrations of anticancer drugs.

to classify the mechanisms through which they survive.

2001).

stress.


Table 1. Common therapies and the stresses they cause. \* Indicates experimental therapies

Chemotherapy is often used in conjunction with surgery and radiotherapy. Most of the common genotoxic chemotherapies for glioma produce their effects by disrupting the DNA strands. Alkylating agents like temozolomide (TMZ) and carmustine (BCNU) primarily work by alkylating the guanine base of DNA leading to cross-linking of the DNA strands which causes the strands to be unable to uncoil and separate. The platinum drugs, such as carboplatin and cis-platinum, work similarly by using the platinum ion to cross-link the guanine base pairs on the DNA strand. These therapies are more toxic to cells that replicate and proliferate faster, thus making cancer cells more sensitive than normal cells to genotoxic therapy. The stresses to the cell caused by chemotherapy are mostly due to interference of mitosis and induction of DNA repair mechanisms. When the cell is unable to unwind and repair its DNA, it causes apoptosis and metabolic stress. As apoptosis continues, ROSs are released into the ECM affecting nearby cells. Genotoxic stress through ROSs is dependent on

**site** 

agents

agents

or II

response

Platinum DNA crosslinkers Mitotic inhibitor

Anti-angiogenic Tyrosine kinase inhibitors

Tyrosine kinase inhibitors

Table 1. Common therapies and the stresses they cause. \* Indicates experimental therapies Chemotherapy is often used in conjunction with surgery and radiotherapy. Most of the common genotoxic chemotherapies for glioma produce their effects by disrupting the DNA strands. Alkylating agents like temozolomide (TMZ) and carmustine (BCNU) primarily work by alkylating the guanine base of DNA leading to cross-linking of the DNA strands which causes the strands to be unable to uncoil and separate. The platinum drugs, such as carboplatin and cis-platinum, work similarly by using the platinum ion to cross-link the guanine base pairs on the DNA strand. These therapies are more toxic to cells that replicate and proliferate faster, thus making cancer cells more sensitive than normal cells to genotoxic therapy. The stresses to the cell caused by chemotherapy are mostly due to interference of mitosis and induction of DNA repair mechanisms. When the cell is unable to unwind and repair its DNA, it causes apoptosis and metabolic stress. As apoptosis continues, ROSs are released into the ECM affecting nearby cells. Genotoxic stress through ROSs is dependent on

**Surgery Evacuation of tumor** 

Immunotherapies/Vaccines\* Immune system

Radiation

Brachytherapy

Vincristine

 Bevacizumab EGFR – Cetuximab, Nimotuzumab

Therapy\*

External beam radiation

Genotoxic Chemotherapy Temozolomide, Procarbazine

 *Cis-*platinum, Carboplatin

Etoposide, Irinotecan

Monoclonal Antibodies

Small Molecule Targeted

 PDGFR – Imatinib mTOR - Everolimus

EGFR – Gefinitib, Erlotinib

Carmustine (BCNU), Lomustine

**Therapy Mechanism of Action Form of Stress**

Nonclassical alkylating

Nitrosourea alkylating

Inhibits topoisomerase I

Ionizing radiation Oxidative stress, DNA

damage

ROSs

Hypoxia

Growth factor signaling inhibition leading to oxidative stress

Oxidative stress, DNA damage, metabolic stress

Growth factor signaling inhibition leading to oxidative stress

**De-vascularization with subsequent hypoxia and nutrient deprivation, immune response** 

DNA and organelle damage due to interruption of replication, induces metabolic stress and activation of SAPK/JNK pathway (Benhar et al., 2001). Thus, chemotherapy stresses glioma cells through different mechanisms.

A general chemotherapy-induced stress response is seen in many types of cancer cells. This is a response to anti-neoplastic agents that can destroy many cancer cells but induce survival and resistance mechanisms in others. In yeast, stress changed the cell cycle and lead to increases in *de novo* protein synthesis, proliferation, HSP90 expression, and proton pump levels. The first line of defense in severe shock is *de novo* synthesis of protective proteins (Miligkos et al., 2000). Increasing key membrane component proteins can up- or downregulate their efficacy to restore ionic balance. Changes in the heat shock protein (HSP) population of the cell due to chemotherapeutic stress also increase HSP27 and HSP70 in resistant cells; these cells are translocated to the nucleus in response to stress, increasing protein synthesis necessary for resistance (Nadin et al., 2003). Whole body response is also important as hormones production levels can change during the stress response; such hormones can affect the cell cycle or gene transcription. On a smaller scale, cell-to-cell interactions occur between transformed and non-transformed cells involving the transfer of survival signals, thus indicating that the extracellular environment is important. The stress response to chemotherapy-induced hyperthermia can even lead to induction of drug resistance through a general increase in MDR P-glycoprotein production (Benhar et al., 2001).

Anti-angiogenic therapies are becoming more common in glioma and are used to combat the tumor vasculature. Most of the inhibitors, like bevacizumab, are monoclonal antibodies that work by antagonistically binding vascular endothelial growth factors (VEGFs), the factors responsible for signaling growth of blood vessels. Contrary to other cancers, it is thought that anti-angiogenic drugs in glioma could work by transiently normalizing the tumor vasculature (Nagy et al., 2010). As discussed previously, tumor blood vessels are abnormal and unstable due to the mixture of pro- and anti-angiogenic factors. An angiogenesis inhibitor would override many of these signals. Although, this might decrease blood vessel formation, it might also stabilize the existing vasculature. By normalizing the vasculature, there could be improved delivery of chemotherapeutic agents. Either way, the neoplastic cells would be subject to stress caused either by hypoxia and nutrient deprivation or increased concentrations of anticancer drugs.

Progress in targeted therapies for glioma has been made in recent years. Numerous small molecule inhibitors are being tested in clinical trials to antagonize the commonly mutated or over-expressed growth factor pathways. These inhibitors, like erlotinib which works on EGFR and imatinib for PGDFR, work by intracellularly binding the tyrosine kinase receptors, interrupting the downstream PI3K and MAPK signaling cascades. Monoclonal antibodies like cetuximab and nimotuzumab (EGFR inhibitors) work similarly, except they bind extracellulary to the growth factor receptors. Most of the stress caused by these antagonists is through decreased growth factor signaling and the resulting metabolic stress.

All these therapies cause stresses already encountered by tumor formation, but the duration and severity of the stresses during therapy is more extreme. Prolonged exposure to these stresses can induce cell death programming more effectively than short, intermittent periods. However, in most cases, some cancer cells do survive. They evade the immune system and death signals, selected for by their unique mutations leading to therapy resistance. It is therefore important to determine accurate markers to identify these cells and to classify the mechanisms through which they survive.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 31

**Minimal Stress Proteome** 

**sensing/repair** 

Glutathione reductase MutL/MLH Multifunctional beta

Aldehyde reductase MutS/MSH Long-chain fatty acid ABC

Thioredoxin Topoisomerase I/III Long-chain fatty acid CoA

MsrA/PMSR **Molecular chaperones Energy metabolism** 

Proline oxidase DnaJ/HSP40 Ca2+/Mg2+-transporting

hydrolase 6 GrpE (HSP70 cofactor) Ribosomal RNA

YIM4 DnaK/HSP70 Phosphoglucomutase

regulatory subunit Inositol monophosphatase

oxidoreductase YMN1 HSP60 chaperonin Enolase (glycolysis)

Isocitrate dehydrogenase **Protein degradation Other functions** 

FtsH/proteasome-

Quinone oxidoreductase Lon protease/protease La Nucleoside diphosphate

dehydrogenase Serine protease Hypothetical protein

Protease II/prolyl endopetidase

Aromatic amino acid aminotransferase Aminobutyrate aminotransferase

These kinases are part of the larger superfamily known as mitogen-activated protein kinases (MAPKs), which control many intracellular events. SAPK is activated by SEK1 or MKK4. The SAPK/JNK pathway is activated by stresses like hypoxia, radiation, drug therapy, ROSs, and inflammatory molecules (Benhar et al., 2001). They signal through a variety of receptors, including G-protein coupled receptors (GPCRs), cytokine receptors (TNFα), death

Table 2. The minimal stress proteome as described by Kültz, 2005.

SelB Petidyl-prolyl isomerase Citrate synthase (Krebs

**Fatty acid/lipid metabolism** 

transporter

oxidation protein

ligase

cycle)

ATPase

methyltransferase

kinase

YKP1

**Redox regulation DNA damage** 

Peroxiredoxin RecA/Rad51

Superoxide dismutase

Hydroxyacylglutathione

NADP-dependent

Putative oxidoreductase

Aldehyde dehydrogenase

Succinate semialdehyde dehydrogenase

Glycerol-3-phosphate

2-hydroxyacid dehydrogenase

phosphogluconate dehydrogenase

#### **2.2 Markers of stress**

While glioma cells may be adept at surviving cellular stress, they do show indicators of the stresses they endure. These indicators or markers may eventually be exploited to determine what stresses the cancer cells are under, and thus what types of stress they may be more susceptible to if subjected further. This section will cover the most common markers of general cellular stress and the specific stresses mentioned previously.

When cells encounter stress, certain elements of the stress response are universal. There is a highly conserved minimal stress proteome that is shared among species. In a paper by Dieter Kültz, a list of the 41 proteins needed for the minimal stress proteome was compiled (Table 2).

While this table is not exhaustive for all proteins involved in the stress response, nor does it list the most reliable markers, it does indicate that cells all have a fundamental basic response to stress. The response is referred to as the conserved stress response (CSR). Various stresses may induce different proteins and markers, but certain responses are unchanged between stresses, even amongst species.

The general response of cells to stress originally focused on three types of proteins: heat shock proteins (HSPs), glucose-regulated proteins (GRPs) and ubiquitin-associated proteins, all of which are inter-related (Feder & Hofmann 1999). Of these three types of proteins, HSPs have been studied the most thoroughly. HSPs are induced during stress as a protective mechanism. While HSPs ordinarily play a more mundane role in the cell, folding proteins into their appropriate tertiary structures and facilitating steroid hormone binding, the subjection of cells to stress activates heat shock transcription factors (HSFs), allowing the transcription of stress-related HSPs like HSP27, HSP70 and HSP90 (Calderwood et al, 2006). In many gliomas and other cancers, binding of HSP90 to p53 mutants in the cytoplasm can further the damage caused by stress (Goetz et al., 2003). This is because p53 functions in the nucleus, and it leads to enhanced HSP70 transcription which allows for cancer cell growth (Ciocca & Calderwood, 2005). These HSPs, along with others, have been linked to cancer therapy resistance.

The unfolded protein response (UPR) has gained increasing coverage as a fundamental stress reaction caused by changes in the celluar redox potential, energy status, or Ca2+ levels leading to unfolded or misfolded proteins within the lumen of the endoplasmic reticulum (ER). This is also known as ER stress (Herr & Debatin, 2001). ER stress is closely linked to hypoxia and glucose depletion. Misfolded proteins can be a problem due to their propensity to aggregate together and cause harmful accumulations. The role of UPR is to stop protein translation, arrest the cell cycle, and to signal pathways that increase activation of protein folding chaperones, some of which are HSPs. Ultimately, UPR leads to cell death through apoptosis if translation is halted for a prolonged period. GPRs are related to UPR and are actually just specialized HSPs that are found in the ER of the cell. In fact, Grp78 is the protein responsible for chaperoning the misfolded proteins and signaling downstream activators of the UPR. Another GRP, grp94 or HSP90B1, is actually essential for immune responses as it is a chaperone that regulates both innate and adaptive immunity through secretory pathways (Maki et al., 1990). Upregulation of these proteins is often seen during stress, and thus could represent markers for stress induction.

Many stresses signal through the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, which is activated by numerous extracellular signals and stresses.

While glioma cells may be adept at surviving cellular stress, they do show indicators of the stresses they endure. These indicators or markers may eventually be exploited to determine what stresses the cancer cells are under, and thus what types of stress they may be more susceptible to if subjected further. This section will cover the most common markers of

When cells encounter stress, certain elements of the stress response are universal. There is a highly conserved minimal stress proteome that is shared among species. In a paper by Dieter Kültz, a list of the 41 proteins needed for the minimal stress proteome was compiled

While this table is not exhaustive for all proteins involved in the stress response, nor does it list the most reliable markers, it does indicate that cells all have a fundamental basic response to stress. The response is referred to as the conserved stress response (CSR). Various stresses may induce different proteins and markers, but certain responses are

The general response of cells to stress originally focused on three types of proteins: heat shock proteins (HSPs), glucose-regulated proteins (GRPs) and ubiquitin-associated proteins, all of which are inter-related (Feder & Hofmann 1999). Of these three types of proteins, HSPs have been studied the most thoroughly. HSPs are induced during stress as a protective mechanism. While HSPs ordinarily play a more mundane role in the cell, folding proteins into their appropriate tertiary structures and facilitating steroid hormone binding, the subjection of cells to stress activates heat shock transcription factors (HSFs), allowing the transcription of stress-related HSPs like HSP27, HSP70 and HSP90 (Calderwood et al, 2006). In many gliomas and other cancers, binding of HSP90 to p53 mutants in the cytoplasm can further the damage caused by stress (Goetz et al., 2003). This is because p53 functions in the nucleus, and it leads to enhanced HSP70 transcription which allows for cancer cell growth (Ciocca & Calderwood, 2005). These HSPs, along with others, have been linked to cancer

The unfolded protein response (UPR) has gained increasing coverage as a fundamental stress reaction caused by changes in the celluar redox potential, energy status, or Ca2+ levels leading to unfolded or misfolded proteins within the lumen of the endoplasmic reticulum (ER). This is also known as ER stress (Herr & Debatin, 2001). ER stress is closely linked to hypoxia and glucose depletion. Misfolded proteins can be a problem due to their propensity to aggregate together and cause harmful accumulations. The role of UPR is to stop protein translation, arrest the cell cycle, and to signal pathways that increase activation of protein folding chaperones, some of which are HSPs. Ultimately, UPR leads to cell death through apoptosis if translation is halted for a prolonged period. GPRs are related to UPR and are actually just specialized HSPs that are found in the ER of the cell. In fact, Grp78 is the protein responsible for chaperoning the misfolded proteins and signaling downstream activators of the UPR. Another GRP, grp94 or HSP90B1, is actually essential for immune responses as it is a chaperone that regulates both innate and adaptive immunity through secretory pathways (Maki et al., 1990). Upregulation of these proteins is often seen during

Many stresses signal through the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, which is activated by numerous extracellular signals and stresses.

general cellular stress and the specific stresses mentioned previously.

unchanged between stresses, even amongst species.

stress, and thus could represent markers for stress induction.

**2.2 Markers of stress**

(Table 2).

therapy resistance.


Table 2. The minimal stress proteome as described by Kültz, 2005.

These kinases are part of the larger superfamily known as mitogen-activated protein kinases (MAPKs), which control many intracellular events. SAPK is activated by SEK1 or MKK4. The SAPK/JNK pathway is activated by stresses like hypoxia, radiation, drug therapy, ROSs, and inflammatory molecules (Benhar et al., 2001). They signal through a variety of receptors, including G-protein coupled receptors (GPCRs), cytokine receptors (TNFα), death

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 33

Autophagy was first discovered as a Type II programmed cell death (PCD) mechanism with distinct differences from apoptosis in yeast. Most initial research was done in yeast, and later, it was found that many of the proteins involved are conserved in higher eukaryotes with human homologues. During autophagy, double membrane autophagosomes or autophagic vacuoles engulf cytoplasm along with long-lived proteins and damaged organelles through a process of self-digesting. The enveloped contents of the autophagosome are degraded by fusing with the lysosome. Their constituents of fatty acids and amino acids are recycled into new molecules or shunted into the synthesis of ATP to meet energy needs. If left unchecked, autophagy does indeed lead to cellular destruction and canabolitic cell death. Because autophagy occurs in many cells immediately preceding cell death, it was initially seen as a cell death mechanism (Levine & Yuan, 2005). However, cells can also utilize autophagy as a means to go into a cellular "hibernation" state where they have decreased energy needs as a strategy of survival (Kuma et al., 2004). Thus,

Autophagy is known to occur in the brain during neurodegenerative processes such as Alzheimer, Parkinson, and Huntington disease. However, its role as a protector or cause of disease is debated (Nixon, 2006). As for its role in glioma, cancer cells were shown to have decreased levels of autophagy compared to non-malignant cells, while nutrient deprivation upregulated its autophagic activity in cancer cells (Wu et al., 2006). This could be due to the early stages of tumorigenesis needing increased protein synthesis and proliferation, where autophagy would impede the growth of the cells. Also, since autophagy removes damaged organelles, it can decrease the mutation rate of the cancer cells, leaving them at a disadvantage during early stages of the disease. Later stages of glioma progression see an increase in autophagy to protect against the numerous cellular stresses present at this stage

An important mechanism of regulation of autophagy is through the PI3K-Akt-mTOR pathway, which is activated in many cancers. Suppression of autophagy occurs through class 1 PI3K, while class III PI3K promotes autophagosome development (Lum et al., 2005). This is because class III PI3K binds to a molecule known as beclin-1 (BECN1). Beclin-1, the homologue of yeast Atg6, is part of an early-autophagy complex that participates in the autophagosome formation (Liang et al., 1999). It is also known to interact with the antiapoptotic protein Bcl-2, binding the molecule and preventing cell death (Erlich et al., 2007). Bcl-2 may regulate the balance between autophagy and apoptosis, since it plays a role in both. Down-regulation of Bcl-2 or up-regulation of its binding protein, BNIP3, induces autophagy. Beclin-1 expression has been shown to be aberrant in cancers, including glioma (Liang et al., 2006). When beclin-1 binds class III PI3 kinase, the complex can activate autophagy. This complex is located in the cell cytoplasm and trans-Golgi network where it can sort the necessary autophagosome components. It, in turn, can be regulated through miRNA miR-30a which is able to down-regulate beclin-1 and thus regulate autophagy. miR-30a expression in glioma was initially implicated in a miRNA screen for differential expression during conditions that induce autophagy and was found to be decreased during autophagy. Further studies with the miRNA showed that expression of miR-30a in glioma cells decreased beclin-1 expression and decreased autophagy (Zhu et al., 2009). This was the first report of miRNA regulating autophagy, so it is certainly possible that other miRNA's

autophagy can also be seen as a temporary protective response of cells.

**2.3.1 Role of autophagy in stress response** 

like nutrient deficiency.

may play a role in this process.

receptors (Fas), and antigen receptors. SAPK/JNK pathways can control proliferation, apoptosis, transformation and differentiation along with migration. In response to many types of stress such as radiation and hypoxia, this pathway signals for mitochondrialdependent apoptosis (Sanchez-Prieto et al., 2000). Thus, the SAPK/JNK signaling cascade is a protective mechanism for cells. However, any mutations or aberrant signaling could also lead to further glioma progression. Up-regulation of proteins involved in these pathways is a good indicator of cellular stress.

Additionally, other markers of general stress have also been found. The MDR1 (multi-drug resistance 1) gene, which encodes the P-glycoprotein responsible for reducing drug accumulation in cancer cells, is actually induced by stresses like acidity, drug treatment, and radiation (Szabo et al., 2000). Thus, cancer cells under stress have created multiple mechanisms to evade cell death. The original intent for non-transformed, normal cells was for them to be able to pump out toxins encountered in their environment for survival purposes. Transformed cancer cells have adapted those responses to their own needs.

Some indicators of specific stresses have also been revealed. An example of a marker for specific stress can be found in hypoxia. The transcription factor HIF-1 (hypoxia-inducting factor-1) is a major regulator of the cellular hypoxia response, which binds to hypoxiaresponsive elements (HREs) leading to the transcription of genes involved in cell survival, metabolism, angiogenesis and invasion. It can increase expression of glycolysis genes and VEGF protein (Jiang et al., 1996). The expression of HIF-1 is increased in glioma usually through induction by EGFR signaling the PI3 kinase pathway and loss of the tumor suppressors p53 and PTEN. HIF-1 expression, and thus hypoxic stress, in tumors can be determined by immunohistochemical staining. Another indicator of stress linked to HIF-1 is NF-κB induction. NF-κB activation leads to the rapid transcription of important genes involved with the stress response. Because it is a transcription factor, it is often thought of as a first line of defense, especially against activators of the immune system (Garg & Aggarwal, 2002). NF-κB is able to regulate many proteins involved in proliferation and survival, including HIF-1. Another isoform, HIF-2α, appears to be a specific marker for pH imbalance as it is increased with exposure to acidic stress (Hjelmeland et al., 2010).

Overall, there are numerous markers of stress in glioma cells. Many are the result of a general response to stress, but as research continues, better markers for specific stress, like HIF-1 in the case of hypoxia, will be developed as our understanding continues to grow. These markers may eventually help clinicians to positively identify the stresses the tumor is under, which will inevitably lead to more effective glioma treatment.

#### **2.3 Mitigation of metabolic and therapeutic stress by autophagy**

Glioma cells are able to employ several ways to overcome stress and the resulting energy depletion. Autophagy, the catabolic recycling of the cell's own components, takes advantage of this idea as a survival mechanism. The autophagic process allows the tumor cells to reallocate amino acids, fatty acids and other macromolecules for energy and go into a hibernation-like state until the surrounding environment is more favorable. Glioma therapies, like temozolimide and etoposide, have been shown to induce autophagy as have various metabolic stresses. While autophagy is also viewed as a cell death mechanism, new research indicates that autophagy can actually be responsible for glioma cell survival and resistance to therapies and stresses. Molecules like beclin-1 and elongation factor-2 kinase (EF-2K) have been shown to play a critical role in the autophagic response in glioma.

#### **2.3.1 Role of autophagy in stress response**

32 Advances in the Biology, Imaging and Therapies for Glioblastoma

receptors (Fas), and antigen receptors. SAPK/JNK pathways can control proliferation, apoptosis, transformation and differentiation along with migration. In response to many types of stress such as radiation and hypoxia, this pathway signals for mitochondrialdependent apoptosis (Sanchez-Prieto et al., 2000). Thus, the SAPK/JNK signaling cascade is a protective mechanism for cells. However, any mutations or aberrant signaling could also lead to further glioma progression. Up-regulation of proteins involved in these pathways is

Additionally, other markers of general stress have also been found. The MDR1 (multi-drug resistance 1) gene, which encodes the P-glycoprotein responsible for reducing drug accumulation in cancer cells, is actually induced by stresses like acidity, drug treatment, and radiation (Szabo et al., 2000). Thus, cancer cells under stress have created multiple mechanisms to evade cell death. The original intent for non-transformed, normal cells was for them to be able to pump out toxins encountered in their environment for survival purposes. Transformed cancer cells have adapted those responses to their own needs. Some indicators of specific stresses have also been revealed. An example of a marker for specific stress can be found in hypoxia. The transcription factor HIF-1 (hypoxia-inducting factor-1) is a major regulator of the cellular hypoxia response, which binds to hypoxiaresponsive elements (HREs) leading to the transcription of genes involved in cell survival, metabolism, angiogenesis and invasion. It can increase expression of glycolysis genes and VEGF protein (Jiang et al., 1996). The expression of HIF-1 is increased in glioma usually through induction by EGFR signaling the PI3 kinase pathway and loss of the tumor suppressors p53 and PTEN. HIF-1 expression, and thus hypoxic stress, in tumors can be determined by immunohistochemical staining. Another indicator of stress linked to HIF-1 is NF-κB induction. NF-κB activation leads to the rapid transcription of important genes involved with the stress response. Because it is a transcription factor, it is often thought of as a first line of defense, especially against activators of the immune system (Garg & Aggarwal, 2002). NF-κB is able to regulate many proteins involved in proliferation and survival, including HIF-1. Another isoform, HIF-2α, appears to be a specific marker for pH imbalance

as it is increased with exposure to acidic stress (Hjelmeland et al., 2010).

under, which will inevitably lead to more effective glioma treatment.

**2.3 Mitigation of metabolic and therapeutic stress by autophagy**

Overall, there are numerous markers of stress in glioma cells. Many are the result of a general response to stress, but as research continues, better markers for specific stress, like HIF-1 in the case of hypoxia, will be developed as our understanding continues to grow. These markers may eventually help clinicians to positively identify the stresses the tumor is

Glioma cells are able to employ several ways to overcome stress and the resulting energy depletion. Autophagy, the catabolic recycling of the cell's own components, takes advantage of this idea as a survival mechanism. The autophagic process allows the tumor cells to reallocate amino acids, fatty acids and other macromolecules for energy and go into a hibernation-like state until the surrounding environment is more favorable. Glioma therapies, like temozolimide and etoposide, have been shown to induce autophagy as have various metabolic stresses. While autophagy is also viewed as a cell death mechanism, new research indicates that autophagy can actually be responsible for glioma cell survival and resistance to therapies and stresses. Molecules like beclin-1 and elongation factor-2 kinase

(EF-2K) have been shown to play a critical role in the autophagic response in glioma.

a good indicator of cellular stress.

Autophagy was first discovered as a Type II programmed cell death (PCD) mechanism with distinct differences from apoptosis in yeast. Most initial research was done in yeast, and later, it was found that many of the proteins involved are conserved in higher eukaryotes with human homologues. During autophagy, double membrane autophagosomes or autophagic vacuoles engulf cytoplasm along with long-lived proteins and damaged organelles through a process of self-digesting. The enveloped contents of the autophagosome are degraded by fusing with the lysosome. Their constituents of fatty acids and amino acids are recycled into new molecules or shunted into the synthesis of ATP to meet energy needs. If left unchecked, autophagy does indeed lead to cellular destruction and canabolitic cell death. Because autophagy occurs in many cells immediately preceding cell death, it was initially seen as a cell death mechanism (Levine & Yuan, 2005). However, cells can also utilize autophagy as a means to go into a cellular "hibernation" state where they have decreased energy needs as a strategy of survival (Kuma et al., 2004). Thus, autophagy can also be seen as a temporary protective response of cells.

Autophagy is known to occur in the brain during neurodegenerative processes such as Alzheimer, Parkinson, and Huntington disease. However, its role as a protector or cause of disease is debated (Nixon, 2006). As for its role in glioma, cancer cells were shown to have decreased levels of autophagy compared to non-malignant cells, while nutrient deprivation upregulated its autophagic activity in cancer cells (Wu et al., 2006). This could be due to the early stages of tumorigenesis needing increased protein synthesis and proliferation, where autophagy would impede the growth of the cells. Also, since autophagy removes damaged organelles, it can decrease the mutation rate of the cancer cells, leaving them at a disadvantage during early stages of the disease. Later stages of glioma progression see an increase in autophagy to protect against the numerous cellular stresses present at this stage like nutrient deficiency.

An important mechanism of regulation of autophagy is through the PI3K-Akt-mTOR pathway, which is activated in many cancers. Suppression of autophagy occurs through class 1 PI3K, while class III PI3K promotes autophagosome development (Lum et al., 2005). This is because class III PI3K binds to a molecule known as beclin-1 (BECN1). Beclin-1, the homologue of yeast Atg6, is part of an early-autophagy complex that participates in the autophagosome formation (Liang et al., 1999). It is also known to interact with the antiapoptotic protein Bcl-2, binding the molecule and preventing cell death (Erlich et al., 2007). Bcl-2 may regulate the balance between autophagy and apoptosis, since it plays a role in both. Down-regulation of Bcl-2 or up-regulation of its binding protein, BNIP3, induces autophagy. Beclin-1 expression has been shown to be aberrant in cancers, including glioma (Liang et al., 2006). When beclin-1 binds class III PI3 kinase, the complex can activate autophagy. This complex is located in the cell cytoplasm and trans-Golgi network where it can sort the necessary autophagosome components. It, in turn, can be regulated through miRNA miR-30a which is able to down-regulate beclin-1 and thus regulate autophagy. miR-30a expression in glioma was initially implicated in a miRNA screen for differential expression during conditions that induce autophagy and was found to be decreased during autophagy. Further studies with the miRNA showed that expression of miR-30a in glioma cells decreased beclin-1 expression and decreased autophagy (Zhu et al., 2009). This was the first report of miRNA regulating autophagy, so it is certainly possible that other miRNA's may play a role in this process.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 35

Many neoplastic cells, including glioma cells, survive metabolic stress through autophagy. Autophagy can originally act as a tumor suppressor due to the inactivation of apoptosis and subsequent immune reaction. In fact, autophagic markers localize i*n vivo* to areas of tumors that are undergoing metabolic stress (White, 2007). Autophagy supports metabolic functions during periods of starvation by cannibalizing and recycling need elements, in short, providing alternative energy sources. Initially, autophagy in non-malignant cells can help prevent tumorigenesis through the removal of damaged organelles and defective proteins, since accumulation of these particulars can lead to oxidative stress. In fact, research shows that it even protects the genomic stability (White, 2007). However, as genetic mutations from other sources accumulate, autophagy allows compromised cells to survive. Defective autophagy can lead to cell death through apoptosis and necrosis, which further stresses neighboring cells through the recruitment of inflammatory molecules. Damage can occur to cellular DNA, creating further genomic instability. Increased mutation rate can further

Stresses known to induce autophagy include starvation, ER stress, mitochondrial damage, protein aggregation, radiation, hypoxia, and pathogens stimulation. Failure of autophagy has been reported to be the mechanism behind cell damage accumulation and aging.

Fig. 2. Autophagy machinery and its regulation by EF-2K.

tumor progression at the expense of some cells.

The formation of the autophagosome is mediated by a series of autophagy specific genes (ATGs) originally identified in yeast like Beclin-1. Other important human counterparts of regulators of autophagy have been found, such as LC3, a mammalian homologue of yeast gene Atg8, which is one of the primary markers of autophagy. It is a ubiquitin-like protein which cooperates with Atg4 protease (Mizushima, 2004). Other autophagy proteins include another system of an Atg12-Atg5-Atg16 complex in addition to gene products like Atg5, Atg7, and Atg10, which also play roles in activating autophagy.

Another regulator of autophagy is the protein synthesis inhibitor, elongation factor-2 kinase (EF-2K). EF-2K, a Ca2+/calmodulin kinase, halts translation by phosphorylating elongation factor-2 (EF-2), a protein responsible for moving the peptide strand along the ribosome during elongation. It does so through hydrolyzing GTP to GDP, which provides the energy for elongation. Phosphorylation of EF-2 negatively reduces its affinity for the ribosome (Ryazanov et al., 1991). Regulating this energy-consuming step in translation is important to cell survival during periods of stress and reduced nutrients, so it is unsurprising that EF-2K protein and activity levels are found to be increased in glioma and other cancers.

The first link of EF-2K to cellular stress was found in hibernating squirrels. During hibernation, decreased respiration and blood flow along with abstinence from food intake, greatly reduces the amount of oxygen and nutrients that are available to cells. In tissues with high metabolic rate, both p-EF-2 and EF-2K levels were found to be increased(Chen et al., 2001). Further studies in cells and mouse models showed increases in EF-2K during nutrient deprivation, hypoxia, radiation exposure, and drug treatment. As EF-2K is a calmodulin kinase known to be activated by calcium flux, ER stress was also found to be dependent on EF-2K status (Py et al., 2009). Unsurprisingly, it was later discovered that EF-2K regulates autophagy. Down-regulation of EF-2K reduces autophagy and increases cell death during stress in glioma (Wu et al., 2006).

EF-2K is regulated through the PI3K/mTOR/S6 kinase pathway by nutrient and growth factor availability, which links together cellular stress and the autophagic response, as they are regulated by the same pathway (Hait et al., 2006). In fact, disruption of the PI3K/mTOR/S6K pathway is known to induce autophagy, probably through EF-2K activation (Fig. 2). Nutrients and growth factors activate mTOR that in turn activates S6 kinase. Both mTOR and S6 kinase negatively regulate EF-2K by phosphorylating it on Ser 78 and Ser366, respectively (Browne & Proud, 2004). This inhibits EF-2K activity and its induction of autophagy, since nutrients and cellular building blocks are plentiful. Nutrient deprivation not only inhibits mTOR signaling and regulation of EF-2K, but it also increases AMP kinase activity due to the depletion of ATP. AMP kinase can positively regulate EF-2K activity by phosphorylating it on a different site, Ser 398, leading to its activation and induction of autophagy, as measured by autophagic markers, like LC3 and acidic vacuole organelle staining (Browne et al., 2004).

It is through the mTOR/S6 kinase pathway that cellular stress can cause the induction of autophagy. Some stresses, dependent on severity and duration, can instantaneously cause both apoptosis and autophagy, while the same stress under different conditions make cause one or another. While it is not yet clear why one pathway is chosen over another, new studies into the induction of autophagy have helped to determine the conditions under which it is stimulated. Disruption of the PI3K/Akt pathway has been associated with autophagy induction as well as stimulation of the AMP kinase pathway. mTOR inhibits autophagy through activating one of its downstream targets, S6 kinase (Abeliovich, 2003).

The formation of the autophagosome is mediated by a series of autophagy specific genes (ATGs) originally identified in yeast like Beclin-1. Other important human counterparts of regulators of autophagy have been found, such as LC3, a mammalian homologue of yeast gene Atg8, which is one of the primary markers of autophagy. It is a ubiquitin-like protein which cooperates with Atg4 protease (Mizushima, 2004). Other autophagy proteins include another system of an Atg12-Atg5-Atg16 complex in addition to gene products like Atg5,

Another regulator of autophagy is the protein synthesis inhibitor, elongation factor-2 kinase (EF-2K). EF-2K, a Ca2+/calmodulin kinase, halts translation by phosphorylating elongation factor-2 (EF-2), a protein responsible for moving the peptide strand along the ribosome during elongation. It does so through hydrolyzing GTP to GDP, which provides the energy for elongation. Phosphorylation of EF-2 negatively reduces its affinity for the ribosome (Ryazanov et al., 1991). Regulating this energy-consuming step in translation is important to cell survival during periods of stress and reduced nutrients, so it is unsurprising that EF-2K

The first link of EF-2K to cellular stress was found in hibernating squirrels. During hibernation, decreased respiration and blood flow along with abstinence from food intake, greatly reduces the amount of oxygen and nutrients that are available to cells. In tissues with high metabolic rate, both p-EF-2 and EF-2K levels were found to be increased(Chen et al., 2001). Further studies in cells and mouse models showed increases in EF-2K during nutrient deprivation, hypoxia, radiation exposure, and drug treatment. As EF-2K is a calmodulin kinase known to be activated by calcium flux, ER stress was also found to be dependent on EF-2K status (Py et al., 2009). Unsurprisingly, it was later discovered that EF-2K regulates autophagy. Down-regulation of EF-2K reduces autophagy and increases cell

EF-2K is regulated through the PI3K/mTOR/S6 kinase pathway by nutrient and growth factor availability, which links together cellular stress and the autophagic response, as they are regulated by the same pathway (Hait et al., 2006). In fact, disruption of the PI3K/mTOR/S6K pathway is known to induce autophagy, probably through EF-2K activation (Fig. 2). Nutrients and growth factors activate mTOR that in turn activates S6 kinase. Both mTOR and S6 kinase negatively regulate EF-2K by phosphorylating it on Ser 78 and Ser366, respectively (Browne & Proud, 2004). This inhibits EF-2K activity and its induction of autophagy, since nutrients and cellular building blocks are plentiful. Nutrient deprivation not only inhibits mTOR signaling and regulation of EF-2K, but it also increases AMP kinase activity due to the depletion of ATP. AMP kinase can positively regulate EF-2K activity by phosphorylating it on a different site, Ser 398, leading to its activation and induction of autophagy, as measured by autophagic markers, like LC3 and acidic vacuole

It is through the mTOR/S6 kinase pathway that cellular stress can cause the induction of autophagy. Some stresses, dependent on severity and duration, can instantaneously cause both apoptosis and autophagy, while the same stress under different conditions make cause one or another. While it is not yet clear why one pathway is chosen over another, new studies into the induction of autophagy have helped to determine the conditions under which it is stimulated. Disruption of the PI3K/Akt pathway has been associated with autophagy induction as well as stimulation of the AMP kinase pathway. mTOR inhibits autophagy through activating one of its downstream targets, S6 kinase (Abeliovich, 2003).

protein and activity levels are found to be increased in glioma and other cancers.

Atg7, and Atg10, which also play roles in activating autophagy.

death during stress in glioma (Wu et al., 2006).

organelle staining (Browne et al., 2004).

Fig. 2. Autophagy machinery and its regulation by EF-2K.

Many neoplastic cells, including glioma cells, survive metabolic stress through autophagy. Autophagy can originally act as a tumor suppressor due to the inactivation of apoptosis and subsequent immune reaction. In fact, autophagic markers localize i*n vivo* to areas of tumors that are undergoing metabolic stress (White, 2007). Autophagy supports metabolic functions during periods of starvation by cannibalizing and recycling need elements, in short, providing alternative energy sources. Initially, autophagy in non-malignant cells can help prevent tumorigenesis through the removal of damaged organelles and defective proteins, since accumulation of these particulars can lead to oxidative stress. In fact, research shows that it even protects the genomic stability (White, 2007). However, as genetic mutations from other sources accumulate, autophagy allows compromised cells to survive. Defective autophagy can lead to cell death through apoptosis and necrosis, which further stresses neighboring cells through the recruitment of inflammatory molecules. Damage can occur to cellular DNA, creating further genomic instability. Increased mutation rate can further tumor progression at the expense of some cells.

Stresses known to induce autophagy include starvation, ER stress, mitochondrial damage, protein aggregation, radiation, hypoxia, and pathogens stimulation. Failure of autophagy has been reported to be the mechanism behind cell damage accumulation and aging.

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 37

mispaired with thymine instead of cytosine during the next cycle of DNA synthesis. Initial research into the mechanism of cell death by the drug indicated that TMZ causes cell death through autophagy not through apoptosis (Kanzawa et al., 2004). . However, not much cell death occurred, and it was eventually discovered that glioma cells were using autophagy initially as a protective mechanism as glioma cells were able to start proliferating again after a week of TMZ treatment. This could be due to the previously stated idea that inhibiting autophagy at different stages of induction leads to different outcomes. Before the recruitment of LC3, cells can be rescued from autophagy by treatment with 3-MA (inhibitor of PI3K) (Kanzawa et al., 2004). Bafilomycin A1, an inhibitor of lysosomal ATPase and atuophagy, in combination with TMZ induces

While TMZ induces autophagy and not apoptosis in glioma cell lines, another alkylating agent cisplatin induces both apoptosis and autophagy. Apoptosis is further activated if autophagic inhibitors were added to cisplatin-treated glioma cells due to the release of Bcl-2 from beclin-1. Thus, cisplatin also utlizes autophagy as a protective mechanism (Harhaji-Trajkovic, 2009). Additional studies were done on TMZ and etopside showing that autophagy clearly protects cells from the multimicronucleation and cell death normally associated with TMZ and etoposide treatment. This was discovered through the detection of a concomitant ATP-surge that occurred with treatment. The associated unsustained ATP surge was not through glycolysis but through a brief period of oxidative phosphorylation. This was due to an induction of autophagy that increased catabolic metabolism to increase ATP levels (Katayama et al., 2007). Many chemotherapeutic agents have also been shown to cause hyperthermia in areas of tumors due to increased inflammation and stress in glioma cells. Hyperthermia itself is also known to induce autophagy, adding another mechanism by which glioma chemotherapies are able to activate autophagic response (Sanchez-Prieto et

Growth factor inhibitors are in the experimental stage of glioma therapy development. Platelet-derived growth factor receptor (PDGFR) antagonists, such as imatinib, and epidermal growth factor receptor (EGFR) antagonists, like erlotinib, have been developed to inhibit the growth signals transmitted through these pathways. Autocrine signaling of growth factors can occur, with glioma cells over-expressing both the growth factor and its receptor together to signal through PI3K pathway. Inhibition of PDGF and EGF signaling induced autophagy but not apoptosis (Takeuchi et al., 2004). This result could be due to inhibitory effect of class I PI3K and/or stimulatory effect of class III PI3K. Downstream targets are also available for inhibition of growth factor pathways. Rapamycin, the inhibitor of mTOR, was able to induce autophagy along with suppressing proliferation. Since mTOR regulates both cell proliferation and autophagy, this could be a good target for future combined therapies. Combining rapamycin with an Akt or PI3K inhibitor increased glioma cell death, and future studies will look at the combination of rapamycin with growth factor

Another category of experimental therapies, glycolytic inhibitors, work similarly to nutrient deprivation as they have preferential uptake by glioma cells that are normally dependent on high levels of glucose to satisfy their rapid glycolysis needs. 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that blocks the effects of glucose on metabolic pathways. It had previously been shown to inhibit growth of cancer cells and enhance the efficacy of other glioma treatments. 2-DG causes oxidative stress in glioma cells which lead to the discovery

apoptosis.

al., 2000).

inhibitors (Takeuchi et al., 2005).

Starvation is readily linked to autophagy through activation the mTOR pathway. Other stresses regulate autophagy in different manners. During hypoxia, autophagic clearing of damaged mitochondria is advantageous to cells. The source of pro-apoptotic signals and ROSs is removed, and thus the cancer cell is able to survive even under oxygen deprived conditions. Bcl-2 family protein, BNIP3, is known to induce autophagy during hypoxia, while others believe it induces apoptosis especially after the cellular environment becomes too acidic from hypoxia (Azad et al., 2008). Metabolic stress activates the p53 pathway, which can normally induce apoptosis through proteins like Puma and Noxa, but metabolic stress increases Bim instead, which signals through Bax and Bak (Vousden & Lane, 2007). Therefore, autophagy can suppress apoptosis during both hypoxia and metabolic stress in glioma.

In short, autophagy plays a critical role in glioma cell survival during various stresses. While autophagy can lead to cell death if not properly regulated, neoplastic cells can also use it as a protective mechanism. The autophagic response is activated by the typical hazards encountered by glioma cells during tumorigenesis, helping the cells to survive periods of limited oxygen and nutrients. Thus, exploiting autophagy as a therapeutic intervention is a subject that has been actively explored.

#### **2.3.2 Glioma treatments and autophagy**

Since autophagy can serve as a cell survival mechanism, it is unsurprising that cancer cells would adapt to use it to their advantage. Not only can neoplastic cells survive intrinsic stresses through autophagy, but they can use it to evade therapeutic interventions. Autophagy-associated therapy resistance is gaining recognition as a key resistance mechanism. Glioma cells tend to undergo autophagy rather than apoptosis, perhaps due to their advanced nature created by genomic instability. Autophagy has been shown to be induced in a wide range of glioma therapies.

Radiation was the first therapy shown to cause glioma cells to undergo autophagy. As stated previously, the main mechanism of damage caused by radiation is through DNA double-strand breaks (DBSs), which can lead to translocation, misrepair, and even loss of chromosomes. Gamma radiation is known to induce autophagy in human glioma cells, but there is some controversy as to whether it causes cell death or if it protects cells (Paglin et al., 2001). This could be due to autophagy playing different roles at different times, acting as a cell death mechanism during early stages and acting as a protective mechanism later in tumor development after the accumulation of more advantageous mutations. Autophagy itself is also regulated on many levels and at different stages of induction, producing differing effects in glioma cells. Inhibiting autophagy is sometimes protective and other times destructive, indicating that autophagy is a sensitive modulator of cell survival. Even with glioma cells using autophagy as a way of survival, prolonged radiation may eventually switch the autophagic program from cell survival to cell death as too many damaged proteins accumulate. Glioma cells treated with autophagic inhibitors were radiosensitized, and radiation was able to create more DBSs (Ito et al., 2005).

Many chemotherapies cause autophagy in glioma. Mainstay treatment, temozolomide (TMZ), is used for high-grade gliomas (late stage). It is a small, lipophilic agent that easily passes through the BBB. Although referred to as an alkylating agent, TMZ does not actually cause cross-linking but instead adds a methyl group to a guanine which gets

Starvation is readily linked to autophagy through activation the mTOR pathway. Other stresses regulate autophagy in different manners. During hypoxia, autophagic clearing of damaged mitochondria is advantageous to cells. The source of pro-apoptotic signals and ROSs is removed, and thus the cancer cell is able to survive even under oxygen deprived conditions. Bcl-2 family protein, BNIP3, is known to induce autophagy during hypoxia, while others believe it induces apoptosis especially after the cellular environment becomes too acidic from hypoxia (Azad et al., 2008). Metabolic stress activates the p53 pathway, which can normally induce apoptosis through proteins like Puma and Noxa, but metabolic stress increases Bim instead, which signals through Bax and Bak (Vousden & Lane, 2007). Therefore, autophagy can suppress apoptosis during both hypoxia and metabolic stress in

In short, autophagy plays a critical role in glioma cell survival during various stresses. While autophagy can lead to cell death if not properly regulated, neoplastic cells can also use it as a protective mechanism. The autophagic response is activated by the typical hazards encountered by glioma cells during tumorigenesis, helping the cells to survive periods of limited oxygen and nutrients. Thus, exploiting autophagy as a therapeutic

Since autophagy can serve as a cell survival mechanism, it is unsurprising that cancer cells would adapt to use it to their advantage. Not only can neoplastic cells survive intrinsic stresses through autophagy, but they can use it to evade therapeutic interventions. Autophagy-associated therapy resistance is gaining recognition as a key resistance mechanism. Glioma cells tend to undergo autophagy rather than apoptosis, perhaps due to their advanced nature created by genomic instability. Autophagy has been shown to be

Radiation was the first therapy shown to cause glioma cells to undergo autophagy. As stated previously, the main mechanism of damage caused by radiation is through DNA double-strand breaks (DBSs), which can lead to translocation, misrepair, and even loss of chromosomes. Gamma radiation is known to induce autophagy in human glioma cells, but there is some controversy as to whether it causes cell death or if it protects cells (Paglin et al., 2001). This could be due to autophagy playing different roles at different times, acting as a cell death mechanism during early stages and acting as a protective mechanism later in tumor development after the accumulation of more advantageous mutations. Autophagy itself is also regulated on many levels and at different stages of induction, producing differing effects in glioma cells. Inhibiting autophagy is sometimes protective and other times destructive, indicating that autophagy is a sensitive modulator of cell survival. Even with glioma cells using autophagy as a way of survival, prolonged radiation may eventually switch the autophagic program from cell survival to cell death as too many damaged proteins accumulate. Glioma cells treated with autophagic inhibitors were radiosensitized,

Many chemotherapies cause autophagy in glioma. Mainstay treatment, temozolomide (TMZ), is used for high-grade gliomas (late stage). It is a small, lipophilic agent that easily passes through the BBB. Although referred to as an alkylating agent, TMZ does not actually cause cross-linking but instead adds a methyl group to a guanine which gets

intervention is a subject that has been actively explored.

and radiation was able to create more DBSs (Ito et al., 2005).

**2.3.2 Glioma treatments and autophagy** 

induced in a wide range of glioma therapies.

glioma.

mispaired with thymine instead of cytosine during the next cycle of DNA synthesis. Initial research into the mechanism of cell death by the drug indicated that TMZ causes cell death through autophagy not through apoptosis (Kanzawa et al., 2004). . However, not much cell death occurred, and it was eventually discovered that glioma cells were using autophagy initially as a protective mechanism as glioma cells were able to start proliferating again after a week of TMZ treatment. This could be due to the previously stated idea that inhibiting autophagy at different stages of induction leads to different outcomes. Before the recruitment of LC3, cells can be rescued from autophagy by treatment with 3-MA (inhibitor of PI3K) (Kanzawa et al., 2004). Bafilomycin A1, an inhibitor of lysosomal ATPase and atuophagy, in combination with TMZ induces apoptosis.

While TMZ induces autophagy and not apoptosis in glioma cell lines, another alkylating agent cisplatin induces both apoptosis and autophagy. Apoptosis is further activated if autophagic inhibitors were added to cisplatin-treated glioma cells due to the release of Bcl-2 from beclin-1. Thus, cisplatin also utlizes autophagy as a protective mechanism (Harhaji-Trajkovic, 2009). Additional studies were done on TMZ and etopside showing that autophagy clearly protects cells from the multimicronucleation and cell death normally associated with TMZ and etoposide treatment. This was discovered through the detection of a concomitant ATP-surge that occurred with treatment. The associated unsustained ATP surge was not through glycolysis but through a brief period of oxidative phosphorylation. This was due to an induction of autophagy that increased catabolic metabolism to increase ATP levels (Katayama et al., 2007). Many chemotherapeutic agents have also been shown to cause hyperthermia in areas of tumors due to increased inflammation and stress in glioma cells. Hyperthermia itself is also known to induce autophagy, adding another mechanism by which glioma chemotherapies are able to activate autophagic response (Sanchez-Prieto et al., 2000).

Growth factor inhibitors are in the experimental stage of glioma therapy development. Platelet-derived growth factor receptor (PDGFR) antagonists, such as imatinib, and epidermal growth factor receptor (EGFR) antagonists, like erlotinib, have been developed to inhibit the growth signals transmitted through these pathways. Autocrine signaling of growth factors can occur, with glioma cells over-expressing both the growth factor and its receptor together to signal through PI3K pathway. Inhibition of PDGF and EGF signaling induced autophagy but not apoptosis (Takeuchi et al., 2004). This result could be due to inhibitory effect of class I PI3K and/or stimulatory effect of class III PI3K. Downstream targets are also available for inhibition of growth factor pathways. Rapamycin, the inhibitor of mTOR, was able to induce autophagy along with suppressing proliferation. Since mTOR regulates both cell proliferation and autophagy, this could be a good target for future combined therapies. Combining rapamycin with an Akt or PI3K inhibitor increased glioma cell death, and future studies will look at the combination of rapamycin with growth factor inhibitors (Takeuchi et al., 2005).

Another category of experimental therapies, glycolytic inhibitors, work similarly to nutrient deprivation as they have preferential uptake by glioma cells that are normally dependent on high levels of glucose to satisfy their rapid glycolysis needs. 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that blocks the effects of glucose on metabolic pathways. It had previously been shown to inhibit growth of cancer cells and enhance the efficacy of other glioma treatments. 2-DG causes oxidative stress in glioma cells which lead to the discovery

Impact of Metabolic and Therapeutic Stresses on Glioma Progression and Therapy 39

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

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

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

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

there was probably a cancer stem cell population in gliomas.

glioma cells.

according to nestin status.

GCSC population.

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

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 currently used treatments, providing a way around tumor resistance.
