Hypoxia, Angiogenesis and Mechanisms for Invasion of Malignant Gliomas

Malignant gliomas are not only the most frequent primary brain tumor in the adult pop‐ ulation, but also the most aggressive. Despite recent therapeutic advances, they remain associated with high morbidity and mortality. determinant of key biological features of these tumors, like their response, resistance, and patterns of recurrence when challenged by standard and new therapeutic options. Glioblastoma multiforme (GBM), the most ag‐ gressive of the astrocytic neoplasms, is characterized by both necrosis as well as high rates of endothelial proliferation and neo-vascularization. that generates a high consump‐ tion of oxygen and nutrients, thus leading to local hypoxia. The latter activates a cascade of signaling pathways leading to: 1) angiogenesis, 2) enhanced motility/invasion, 3) changes in metabolism, and 4) the ability to survive oxidative stress. This is a dynamic and multifactorial process whose outcome, either death or survival, is dependent on the timing and the rates of each of these processes, as well as on the molecular characteris‐ tics of the tumor. Therefore, hypoxia-induced enhancement of motility/invasion may de‐ termine the outcome and response to therapy of a particular GBM. We seek a better understanding of the molecular and phenotypic effects of local hypoxia in GBM. Here, we review the definition of hypoxia and its molecular and phenotypic effects on cancer cells in general and then turn our attention to its impact on malignant glioma cells. We also examine the interplay between anti-angiogenesis, survival, and enhanced motility, and explore potential therapeutic implications. We close with unresolved questions and potential future directions.


Introduction
Malignant gliomas are not only the most frequent primary brain tumor in the adult population, but also the most aggressive. Despite recent therapeutic advances, they remain associated with high morbidity and mortality. determinant of key biological features of these tumors, like their response, resistance, and patterns of recurrence when challenged by standard and new therapeutic options. Glioblastoma multiforme (GBM), the most aggressive of the astrocytic neoplasms, is characterized by both necrosis as well as high rates of endothelial proliferation and neo-vascularization. that generates a high consumption of oxygen and nutrients, thus leading to local hypoxia. The latter activates a cascade of signaling pathways leading to: 1) angiogenesis, 2) enhanced motility/invasion, 3) changes in metabolism, and 4) the ability to survive oxidative stress. This is a dynamic and multifactorial process whose outcome, either death or survival, is dependent on the timing and the rates of each of these processes, as well as on the molecular characteristics of the tumor. Therefore, hypoxia-induced enhancement of motility/invasion may determine the outcome and response to therapy of a particular GBM. We seek a better understanding of the molecular and phenotypic effects of local hypoxia in GBM. Here, we review the definition of hypoxia and its molecular and phenotypic effects on cancer cells in general and then turn our attention to its impact on malignant glioma cells. We also examine the interplay between anti-angiogenesis, survival, and enhanced motility, and explore potential therapeutic implications. We close with unresolved questions and potential future directions. meet consumption. For example, a local environment including a large number of metabolically active cancer cells may be hypoxic when the oxygen supply is not enhanced; to prevent hypoxia, supply must be increased. Factors that contribute to hypoxia include low partial pressure of oxygen in the blood, reduced ability of blood to carry oxygen, reduced tissue perfusion, increased diffusion distances, or inability of cells to use oxygen (Hockel and Vaupel 2001). Hypoxia is threatening to the normal cellular environment and a series of highly regulated and coordinated response-mechanisms are necessary in order for cells to survive in hypoxic environments (Wheaton and Chandel 2011).

Hypoxia and tumor cells
One of the characteristics of cancer cells is deregulated, high cellular proliferation that ultimately results in structurally and functionally abnormal blood vessels that are unable to provide an adequate amount of oxygen to meet the increased metabolic demands of proliferation, which ultimately results in hypoxia (Vaupel, Kallinowski et al. 1989;Harris 2002;. Although indirect evidence for hypoxia in human tumors was first reported in the 1950s, Peter Vaupel and colleagues were among the first researchers to demonstrate direct evidence of hypoxia in human cancers, as well as linking hypoxia with increased metastasis and poor prognosis in patients with squamous tumors of the head and neck, cervical cancers, and breast cancers (Thomlinson and Gray 1955;Hockel, Schlenger et al. 1999;Hockel and Vaupel 2001;Harris 2002).
Tumor cells generally respond to hypoxia in 1 of 2 ways: either cellular proliferation is restricted and apoptosis and necrosis may occur or the tumor cells adapt to the stress of the hypoxic environment and become more aggressive (Vaupel 2008). This adaptation towards a more aggressive phenotype is accomplished by regulating the expression of various genes that have critical roles in cellular events, which include cellular proliferation, differentiation, tumor glycolysis, angiogenesis, and metastasis and invasion (Vaupel 2004; Lu and Kang 2010; Semenza 2012). Additionally, tumor cells not only acquire more invasive and metastatic properties in response to hypoxia, but also become more resistant to standard treatments, such as chemotherapies and radiation therapies ). Hypoxia-associated resistance to radiation therapy can occur when the partial pressure of oxygen in a tumor is less than 25-30 mmHg. The mechanism of this resistance is thought to be multi-factorial, however some proposed possibilities include decreased oxygen concentrations, higher levels of heat shock proteins, and the presence of cells with decreased apoptotic potential (Hockel and Vaupel 2001). Hypoxia-induced resistance to chemotherapy is likely affected by inhibition of cellular proliferation, decreased effectiveness of some agents in the setting of hypoxia, tissue acidosis, and/or the loss of apoptotic potential of cells (Hockel and Vaupel 2001).

Molecular signals of hypoxia
Hypoxia-inducible factor (HIF) is a transcription factor that plays a critical a central role in mediating the ability to adapt to low-oxygen concentrations (Wang, Jiang et al. 1995;Semen-za 1998). One of the primary cellular events in response to the initial exposure to hypoxia is activation of hypoxia-inducible factor 1 (HIF-1), a hetero-dimeric basic helix-loop-helix protein, composed of 2 subunits: HIF-1α, which is up-regulated in an oxygen-dependent manner, and HIF-1β, which is constitutively expressed (Semenza 2000;Lee, Bae et al. 2004;Zhu, Zhou et al. 2011). Over-expression of HIF-1α is seen in many cancer types associated with a poor prognosis, like malignancies of the brain, oropharynx, breast, cervix, ovary, and uterus (Semenza 2003; Lu and Kang 2010).
Hypoxia-inducible factor-2 alpha (HIF-2α) is another mammalian protein that appears to play a role in the cellular response to hypoxia (Hu, Wang et al. 2003;. HIF-2α is structurally similar to HIF-1α, sharing 48 percent of the overall amino acid identity (Hu, Wang et al. 2003;Ke and Costa 2006). Unlike HIF-1α, which is ubiquitously expressed, HIF-2α is mainly expressed in the lung, endothelium, and carotid body

Mechanisms of hypoxia-induced signaling
Under normal oxygen conditions, HIF-1α is hydroxylated by prolyl hydroxylase (PHD). This hydroxylation leads to the recognition and binding of von Hippel-Lindau (VHL), which is part of an E3 ubiquitin ligase complex that targets HIF-1α for degradation through a proteasomal pathway (Wheaton and Chandel 2011; Semenza 2012). hypoxic conditions, the hydroxylation of the HIF-1α subunit by PHD is inhibited and, therefore, HIF-1α is stabilized, resulting in its translocation to the cell nucleus where it dimerizes with the HIF-1β subunit and forms the active HIF-1. Activation of HIF-1 subsequently results in the recruitment of transcriptional co-activators and this complex then binds to consensus hypoxia-responsive elements (HREs) within the promoter regions of target genes and initiates the transcription of a variety of hypoxia-responsive genes (Vaupel 2004). These genes can be grouped into 4 functional categories (see Figure), namely, survival/proliferation, metabolism, angiogenesis, and invasion/metastasis (Lu and Kang 2010).

Effects on cell proliferation and survival
Increased rates of cell proliferation and decreased rates of cell death are 2 of the defining differences between neoplastic cells and normal cells. HIF-1 plays an important role in cellular proliferation. Iyer et al. demonstrated that cells deficient in HIF-1α have reduced rates of cellular proliferation as compared to wild-type cells under both normoxic and hypoxic conditions, with the degree of reduction worsened in hypoxia (Iyer, Kotch et al. 1998;Feldser, Agani et al. 1999). Additionally, several growth factors that contribute to increased cell proliferation are also HIF-1 target genes. insulin-like growth factor-2 (IGF2) and transforming-growth factor-alpha (TGF-α) (Feldser, Agani et al. 1999;Krishnamachary, Berg-Dixon et al. 2003). Binding of these factors to their receptors, namely insulin-like growth factor receptor-1 (IGFR1) and epidermal growth-factor receptor (EGFR), respectively, leads to autocrine signaling, resulting in both increased cell proliferation and survival as well as increased expression of HIF-1 (Semenza 2003;Ke and Costa 2006). These autocrine signaling pathways are necessary for cancer progression (Semenza 2003).

Effects on metabolism
The uptake of glucose by metastatic cancer cells is markedly increased as compared to noncancer cells and under hypoxic conditions cells switch from the oxygen-dependent metabolic pathway, the tricarboxylic acid (TCA) cycle, to the oxygen-independent pathway, glycolysis (Dang and Semenza 1999; Ke and Costa 2006). HIF-1 plays an active role in this metabolic response to hypoxia by up-regulating the expression of glycolytic enzymes, such as aldolase A, phosphoglycerate kinase 1, and lactate dehydrogenase, that convert glucose to lactate (Vaupel 2004). HIF-1 also induces the over-expression of glucose transporters, namely GLUT1 and GLUT3, that facilitate the uptake of glucose by the cells ( found that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1α and HIF-2α and that an increase in ROS links this complex to HIF-alpha stabilization (Guzy, Hoyos et al. 2005). This important study linked an oxygen-dependent pathway to the stabilization of HIF-1. Thus, while HIF-1 controls glycolysis and shuts-down OXPHOS, functional mitochondria play a role in the regulation of HIF-1.

Effects on angiogenesis
Lee et. al demonstrated that inhibition of HIF-1 activity by either anthracycline chemotherapy or acriflavine prevents tumor vascularization in mouse models, thus lending support to the idea that HIF-1 activity is critical in tumor vascularization (Lee, Qian et al. 2009; Lee, Zhang et al. 2009;. Angiogenesis is a complex process by which the vascular system is formed through growth of new capillaries from pre-existing vessels (Wang, Fei et al. 2004). In addition, although there is great diversity in the factors and signals that contribute to angiogenesis, the chemical signal that appears to play the most critical role in the process is Vascular Endothelial Growth Factor, or VEGF. VEGF is a pro-angiogenic growth factor that is secreted by many cells, including mesenchymal, stromal, and especially tumor cells. VEGF induces the migration of the endothelial precursor cells to sites of angiogenesis and is also responsible for the proliferation and differentiation of these cells (

Effects of hypoxia on tumor invasion and motility
Metastasis is a complex process that consists of a series of highly regulated, rate-limiting steps in which tumor cells gain more invasive properties. Metastasis begins with a change in tumor plasticity in a process called epithelial-mesenchymal transition (EMT). During this process there is a loss of epithelial cell characteristics and a gain of mesenchymal gene expression. In short, this process is characterized by the down-regulation of epithelial (E)-cadherin, involved in adherence junctions and endothelial stabilization, and the up-regulation of mesenchymal (N)-cadherin, which allows cells to become more motile ( Motility is also an important concept in the process of metastasis. Once the cell-cell junctions have been broken down by the down-regulation of (E)-cadherin, the cells are quickly able to reassemble the actin cytoskeleton into protrusive and invasive structures, such as lamellipodia and filopodia, that help them to migrate through the degraded extracellular matrix (ECM), into the blood vessels, and ultimately to their target location (Fathallah-Shaykh 2005). Actin-Related Protein 2/3 (Arp2/3), which is composed of 2 actin related proteins and 5 structural subunits, is controlled by the Scar/WAVE complex, also known as the WANP complex, and is essential to the rapid assembly of actin networks that make up lamellipodia (Machesky 2008). Additionally, the up-regulation of the capping protein increases the protrusion activity of invasive tumor cells (Yamaguchi, Wyckoff et al. 2005). The formation of filopodia is controlled by proteins such as fascin, diaphanous, and Mena/VASP (Machesky 2008).

HIF in gliomas
In addition to decreased oxygen tension in brain and GBM tissues, the expression of HIF in gliomas can be affected by the activation of oncogenes, namely epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), and/or the loss of tumor suppressor function through p53 and phosphatase and tensin homolog gene

Hypoxia, angiogenesis, and GBM
GBM are highly cellular astrocytic neoplasms; they are histologically characterized by an area of central necrosis surrounded by a highly cellular rim of viable tumor (

Hypoxia and invasiveness of GBM
As discussed earlier, cancer cell migration and invasion consists of a series of highly coordinated steps including dissociation of cellular adhesions, remodeling of the actin cytoskeleton, proteolytic degradation of the ECM, and formation of new adhesions. Brain invasion is a characteristic feature of malignant gliomas. In vivo studies of glioblastoma have shown that tumor hypoxia results in increased cell migration (Plasswilm, Tannapfel et al. 2000). Once again, the pseudopalisading cells found around the necrotic cores in GBM, which upregulate HIF-1, appear to be hypoxic and migrating away from the necrotic core (Elstner, Holtkamp et al. 2007;Onishi, Ichikawa et al. 2011). discovery has revealed that the protein MINK, which inactivates ADF/cofilin by phosphorylation, is up-regulated in glioma cells as compared to normal brain cells (Fathallah-Shaykh 2005). Additionally, under hypoxic conditions, glioblastoma induce the expression of c-Met, which stimulates gliomas cells to secrete urokinase plasminogen activator (uPA), which converts circulating plasminogen into plasmin. The latter helps promote invasion by degrading ECM proteins as well as by activating

Hypoxia and cell survival in GBM
Microarray analysis of cultured glioma cells revealed over-expression of molecules that protect them from reactive oxygen species (ROS) and endoplasmic reticulum (ER)-induced apoptosis (Fathallah-Shaykh 2005). ROS, generated by the rapid multiplication of cancer cells, cause a release of cytochrome c leading to apoptosis (Filomeni, Aquilano et al. 2003;Petrosillo, Ruggiero et al. 2003;. Glioma cells appear to acquire protective mechanisms against ROS-induced apoptosis. First, by up-regulating protective enzymes such as AKR1A1 and AKR1C1, which belong to the aldo-keto reductase enzyme super-family ( The ER, one of the cells largest organelles, is responsible for folding, glysolating, and sorting proteins to their correct destinations, as well as for synthesizing lipids and cholesterol components of the cell membrane. When homeostasis is disrupted in the ER, mis-folded and un-folded proteins can accumulate with the ER lumen, resulting in ER stress, which shuts off protein synthesis leading to apoptosis. This response is beneficial to the cell as a safeguard against carcinogenesis because mis-folded proteins may acquire aberrant functions. On the other hand, bypassing this response is an important milestone in the development of cancer. Glioma cells appear to acquire pathways that help them recover from ER stress and avoid apoptosis. A normal ER stress-induced response ultimately leads to inactivation of the eukaryotic initiation factor 2 (eIF-2α), which shuts off protein synthesis. By up-regulating activating transcription factor 4 (ATF4), malignant glioma cells appear to bypass the ER stress-induced protein synthesis inhibition. ATF4 induces the growth arrest and DNA damage-inducible protein (GADD34), which dephosphorylates eIF2α, causing protein synthesis recovery (

Hypoxia, invasion, and angiogenesis in malignant gliomas
Hypoxia, invasion, and angiogenesis are interrelated through a variety of molecular pathways and feedback loops. divide and increase in number, they often out-grow their diffusion-limited oxygen supplies and hypoxia results. Once the tumor micro-environment becomes hypoxic, cellular and molecular signaling pathways are initiated in order to promote overall tumor survival. These adaptive responses include: 1) increasing cellular resistance to hypoxia, 2) favoring metabolic pathways that are optimal in hypoxia, 3) increasing motility and invasive properties in order to seek new oxygen supplies, and 4) initiating angiogenesis in order to create a new vascular supply, or a combination of these responses. (Shimizu, Eguchi et al. 1995). Therefore, there is evidence that local oxygen concentrations modulate a switch between proliferative phenotype and an invasive in GBM cells; this is called the "Go or Grow" mechanism (Giese, Kluwe et al. 1996;Giese, Loo et al. 1996;Hatzikirou, Basanta et al. 2012).
When considering angiogenesis as the adaptive mechanism in glial cells, it is necessary to evaluate the relationship between the rate of angiogenesis and the rate of cellular division. If cellular division proceeds at a rate faster than angiogenesis, then oxygen supply cannot meet metabolic demands causing not only local necrosis, but also dissemination of tumor cells by enhanced brain invasion. On the other hand, if angiogenesis proceeds at a rate faster than cellular division then oxygen supplies are sufficient to meet metabolic demands and no necrosis occurs, which is characteristic of anaplastic astrocytoma.

Anti-angiogenic agents as treatment for malignant gliomas
Both prospective and retrospective clinical trials have shown that Bevacizumab, an anti-angiogenic agent, is effective as a single-agent against recurrent glioblastoma as compared to historical controls of salvage chemotherapy (Friedman, Prados et al. 2009

Implications for treatment of malignant gliomas
Treatment with Bevacizumab also has important implications for how tumor progression is defined. Traditionally, tumor progression has been measured according to the MacDonald criteria which define progression by increase in contrast enhancement. However, as a result of blood-brain barrier stabilization secondary to anti-angiogenic agents, contrast enhancement is no longer a reliable measure of progression. Therefore, the Response Assessment in Neuro-Oncology (RANO) Working Group developed the RANO criteria as a means to address potential progression by non-enhancing T2/FLAIR (fluid attenuated inversion recovery) lesions as well as by increases in contrast enhancement in malignant gliomas (Province, Han et al. 2011). The use of anti-angiogenic agents, namely Bevacizumab, has also raised many questions about patterns of recurrence in malignant gliomas. Additionally, although there is some conflicting data on this subject secondary to poor study design and low study power, there is evidence that patients who are treated with Bevacizumab exhibit more diffuse or distant recurrence patterns as compared to those who do not receive Bevacizumab. Once again, the possibility of more diffuse and distant recurrence patterns may be explained by the "Go or Grow" mechanism, as explained above, with further supporting evidence derived from the glioblastoma xenograft model, also detailed above ( Hypoxia-mediated enhancement of brain invasion has profound biological and clinical implications on GBM, in particular in the context of treatment with anti-angiogenic drugs. Recall that necrosis is a hallmark of GBM; thus, one can safely assume that, by the time of the initial clinical presentation, GBM cells have already invaded the surrounding brain parenchyma because of local hypoxia. This feature of GBM may limit the effectiveness of local therapeutic options, like radiation therapy and surgical resection. Furthermore, one could argue that, though anti-angiogenic drugs prolong progression free survival times, they may create a more aggressive and invasive tumor by hypoxia-mediated enhancement of motility, which delays clinical progression. These patients may experience a socalled "honeymoon period," in which they may appear stable; however, once they recur, the rate of progression of Bevacizumab-treated tumors may be very acute secondary to the large volume of invaded brain. This rationale may explain the limited effects of Bevacizumab on overall survival times.

Future directions
The Figure depicts the effects of HIF-1α on key cellular functions. Though some of the signaling molecules are known, we seem to be only looking at the tip of the iceberg. More research is needed to fit the pieces of the puzzle. What other molecules are activated by hypoxia to influence the 4 phenotypes shown in the Figure? How do they relate to HIF-1? Are there feedback loops between the phenotypes? That is, does motility affect metabolism and vice versa? We have presented a succinct view of the molecular basis of the four phenotypes; however, a detailed analysis is not within the scope of this chapter.
Although preclinical studies have revealed a molecular link between hypoxia, angiogenesis, and invasion, the clinical implications of these relationships are still not well known and future studies are needed. Drawing from what has already been established regarding the effects of anti-angiogenic agents on increased migration and invasion in malignant gliomas, a plausible direction for future therapeutic development would include blocking not only angiogenesis but also invasion and migration. motility agents in malignant GBM will result in significant improvement in PFS as well as overall survival times by blocking two of the primary mechanisms for their survival.

Conclusion
From this chapter, it is our hope that the reader has gained an understanding of the extensive role of hypoxia in brain tumors, with specific regard to its role in angiogenesis, invasion, metabolism, and overall survival of the tumor. Furthermore, although the use of antiangiogenic agents as therapeutic options for malignant gliomas has provided some promising results, it has also revealed the need for further molecular research into the mechanisms for invasion of malignant gliomas, especially as it relates to the hypoxic environments created by anti-angiogenic drugs. Hopefully, this combination of efforts to decrease both angiogenesis and invasion will result in the development of more promising therapeutic agents to treat this disease. Under normal oxygen supply, one of the two proline residues (Pro-402, Pto-564) in HIF-1α is hydroxylated by prolyl hydroxylase. Once hydroxylated HIF-1α is recognized by VHL, an E3 ubiquitin (Ub) ligase, which targets HIF-1α for degradation through the proteasomal pathway. In hypoxic conditions, hydroxylation is inhibited causing the accumulation of HIF-1α, which translocates to nucleus where it associates with HIF-1β then binds to the HIF responsive Element (HRE) of target genes activating their transcription. The HIF targeting genes modulate cell survival, metabolism, angiogenesis, and tumor invasion. PGK: phosphoglycerate kinase, LDH: lactate dehydrogenase.

Author details
Paula Province, Corinne E. Griguer, Xiaosi Han, Nabors Louis B. and Hassan Fathallah Shaykh The University of Alabama at Birmingham, Birmingham, Alabama, United States of America