Metabolic therapy: a new paradigm for managing malignant brain cancer.

Cancer Letters xxx (2014) xxx–xxx

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Mini-review

Metabolic therapy: A new paradigm for managing malignant brain cancer Thomas N. Seyfried a,⇑, Roberto Flores a, Angela M. Poff b, Dominic P. D’Agostino b, Purna Mukherjee a a b

Biology Department, Chestnut Hill, MA, USA Department of Molecular Pharmacology and Physiology, University of South Florida, 33612 Tampa, FL, USA

a r t i c l e

i n f o

Article history: Received 10 February 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online xxxx Keywords: Caloric restriction Brain tumor Energy metabolism Glioma Ketogenic diet

a b s t r a c t Little progress has been made in the long-term management of glioblastoma multiforme (GBM), considered among the most lethal of brain cancers. Cytotoxic chemotherapy, steroids, and high-dose radiation are generally used as the standard of care for GBM. These procedures can create a tumor microenvironment rich in glucose and glutamine. Glucose and glutamine are suggested to facilitate tumor progression. Recent evidence suggests that many GBMs are infected with cytomegalovirus, which could further enhance glucose and glutamine metabolism in the tumor cells. Emerging evidence also suggests that neoplastic macrophages/microglia, arising through possible fusion hybridization, can comprise an invasive cell subpopulation within GBM. Glucose and glutamine are major fuels for myeloid cells, as well as for the more rapidly proliferating cancer stem cells. Therapies that increase inflammation and energy metabolites in the GBM microenvironment can enhance tumor progression. In contrast to current GBM therapies, metabolic therapy is designed to target the metabolic malady common to all tumor cells (aerobic fermentation), while enhancing the health and vitality of normal brain cells and the entire body. The calorie restricted ketogenic diet (KD-R) is an anti-angiogenic, anti-inflammatory and pro-apoptotic metabolic therapy that also reduces fermentable fuels in the tumor microenvironment. Metabolic therapy, as an alternative to the standard of care, has the potential to improve outcome for patients with GBM and other malignant brain cancers. Ó 2014 Published by Elsevier Ireland Ltd.

Glioblastoma multiforme (GBM) GBM is the most malignant of the primary brain cancers with only about 12% of patients surviving beyond 36 months (long-term survivors) [1–3]. Most glioblastomas are heterogeneous in cellular composition consisting of tumor stem cells, mesenchymal cells, and host stromal cells [4–9]. Primary GBM can arise de novo, whereas secondary GBM is thought to arise from lower-grade gliomas [6,10,11]. The timing and incidence of malignant progression from low-grade glioma to GBM is variable and unpredictable [12]. In addition to the neoplastic cell populations, tumor-associated macrophages/monocytes (TAM) also comprise a significant cell population in GBM sometimes equaling the number of tumor cells [13–18]. TAM can contribute to tumor progression through release

Abbreviations: KD-R, calorically restricted ketogenic diet; OxPhos, oxidative phosphorylation; HCMV, human cytomegalovirus. ⇑ Corresponding author. Address: Biology Department, Boston College, Chestnut Hill, MA 0246, USA. Tel.: +1 617 552 3563; fax: +1 617 552 2011. E-mail address: [email protected] (T.N. Seyfried).

of pro-inflammatory and pro-angiogenic factors [14,16,18,19]. Moreover, many cells appearing as TAM might actually be neoplastic macrophages/microglia. Recent studies show that some neoplastic cells within GBM stain positive for both markers of astrocytes (GFAP) and macrophages (CD68, CD163) consistent with the fusion hybrid hypothesis for origin of invasive/metastatic tumor cells [20–25]. Although systemic metastasis is not common for GBM, GBM cells can be metastatic if given access to extraneural sites [26–30]. Using the secondary structures of Scherer, the neoplastic cells in GBM invade through the neural parenchyma well beyond the main tumor mass making complete surgical resections exceedingly rare [1,31–34]. Despite extensive analysis from the cancer genome projects, no mutation is known that is unique to GBM [35–37]. Although many GBMs contain mutations in genes thought to provoke progression (epidermal growth factor receptor, tumor promoter p53, PTEN, etc.) these mutations were not found in all GBM evaluated [37]. These observations are consistent with evidence from other tumors demonstrating the extensive genetic heterogeneity seen in most cells of natural tumors [38]. Recent evidence also suggests that

http://dx.doi.org/10.1016/j.canlet.2014.07.015 0304-3835/Ó 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: T.N. Seyfried et al., Metabolic therapy: A new paradigm for managing malignant brain cancer, Cancer Lett. (2014), http:// dx.doi.org/10.1016/j.canlet.2014.07.015

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T.N. Seyfried et al. / Cancer Letters xxx (2014) xxx–xxx

the somatic mutations seen in cancer cells can arise as downstream secondary effects of disturbed energy metabolism and are unlikely to provide useful information for therapeutic treatment strategies for the majority of GBM patients [11,39,40]. The retrograde or mitochondrial stress response can lead to genomic instability as the result of protracted disruption of oxidative phosphorylation with generation of reactive oxygen species [41–48]. However, the correlation between IDH1 gene mutation status and survival for patients with recurrent glioma is interesting [37,49]. IDH1 mutations have been shown to alter the enzymatic activity of the encoded protein, resulting in up-regulation of hypoxia inducible factor-1a (HIF-1a) [49,50]. HIF-1a plays an important role in the process of angiogenesis while also supporting tumor cell survival and proliferation [49,51]. Although the properties of the IDH1 mutation would promote tumor growth, IDH1 expression in glioma patients is associated with increased survival [37,49]. It remains unclear how the IDH mutations confer a survival advantage. It is also unclear if the IDH1 mutation acts in gliomas as a tumor promoting oncogene, a tumor inhibiting suppressor gene, or can act as both an oncogene and suppressor gene [52]. This conundrum should not be surprising, however, as Soto and Sonnenschein have indicated that when it comes to the somatic mutation theory of cancer; something can be anything and it opposite [53]. We and others suggest that targeting the metabolic malady common to most neoplastic cells in the tumor will likely be more effective for management than in targeting genetic differences that are not expressed in all cells of the tumor [39,54].

Standard of care for malignant glioma The current standard of care for GBM and many malignant brain cancers includes maximum surgical resection, radiation therapy, and chemotherapy [2,55–57]. The toxic alkylating agent, temozolomide (TMZ), is the most common chemotherapy used for treating GBM. The most common adverse events of TMZ exposure besides hematological toxicities are alopecia, nausea, vomiting, anorexia, headache, and constipation. These adverse effects have been described on the NCI web site (http://www.cancer.gov/cancertopics/druginfo/fda-temozolomide), and in original research articles [58–60]. Recent evidence also indicates that TMZ and most other chemotherapies currently approved by the FDA for cancer management can potentially facilitate tumor recurrence through an effect on the Janus kinase–signal transducers and activators of transcription (JAK–STAT) pathway [61]. Moreover, Johnson et al. recently reported that 6 of 10 patients treated with TMZ followed an alternative evolutionary path to high-grade glioma [11]. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt–mTOR pathways that bore the signature of TMZ-induced mutagenesis. These observations are concerning and suggest that better approaches to GBM management are needed. Although high-dose perioperative corticosteroids (dexamethasone) are not recommended in all cases [55], most GBM patients receive corticosteroids as part of the standard of care, which is often extended throughout the course of the disease [16,62,63]. The antiangiogenic drug, bevacizumab (Avastin) can also be given to GBM patients despite the Food and Drug Administration’s removal of Avastin for breast cancer due to toxicity and lack of efficacy [64,65]. Bevacizumab treatment increases progression free survival in GBM patients, but does not increase overall patient survival [66]. There have been no major advances in GBM management for over 50 years, though use of temozolomide has produced marginal improvement in survival over radiation therapy alone [2,67]. Despite conventional treatments, prognosis remains poor for most patients with high-grade brain tumors [1,3,56,67,68]. (Fig. 1 Stupp

Fig. 1. Kaplan–Meier estimates of overall survival of patients with glioblastoma multiforme by treatment group. The two patient groups included radiotherapy alone (n = 278) and radiotherapy with temolozomide (n = 254). Overall patient survival has remained largely unchanged form the study published in 2005 [58]. Reprinted with permission from [2].

Fig. 2). Is it possible that the limitations of standard therapy produce untoward effects that undermine efficacy by increasing aggressiveness of surviving tumor cells? Mitochondrial abnormalities in malignant brain tumors Substantial evidence collected from numerous investigators over many years indicates that abnormalities in mitochondrial structure and function are the hallmark of most cancers including brain cancer [69–74]. These mitochondrial abnormalities will reduce energy production through oxidative phosphorylation (OxPhos) [69–72,75–86]. Although pathological mitochondrial DNA (mtDNA) mutations were not found in a broad range of mouse brain tumors [87], recent studies identified potential pathological mutations in mtDNA from human GBM that were correlated with tumor origin [88]. The work of several investigators showed that the ultra structure of mitochondria in malignant brain tumors differs markedly from the ultra structure of normal tissue mitochondria [78,79,89–92]. In contrast to normal mitochondria, which contain numerous cristae, mitochondria from GBM tissue samples showed swelling with partial or total cristolysis (Fig. 2). Cristae contain the proteins of the respiratory complexes, and play an essential structural role in facilitating energy production through OxPhos [93]. Many of the mitochondrial defects seen in excised tumor tissue, however, are not often seen in the cell lines derived from the tumor tissue. This is thought to arise from the abnormal in vitro growth environment that selects for cells with some level of mitochondrial function [39]. The structural defects in human glioma mitochondria seen in tissue sections are consistent with lipid biochemical defects in murine gliomas. We showed that cardiolipin was abnormal in five independently derived mouse brain tumors [94,95]. These tumors arose spontaneously in the brains of inbred VM mice or were induced in C57/BL6 mice with 20-methylcholanthrene implantation into the brain as we previously described [96,97]. Cardiolipin is the signature lipid of the inner mitochondrial membrane and controls the efficiency of OxPhos. Any alterations in the content or fatty acid composition of cardiolipin will reduce cellular respiration [98–100]. No tumors have been found to our knowledge that contain a normal content or composition of cardiolipin. In addition to these findings, Poupon and colleagues also indicated that the high glycolytic activity seen in malignant gliomas could arise from mitochondrial structural abnormalities [101]. Despite abnormalities in mitochondrial structure and function, TCA cycle activity



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cristolysis Fig. 2. Typical ultrastructure of a normal mitochondrion and a mitochondrion from GBM tissue. Normal mitochondria contain elaborate cristae, which are extensions of the inner membrane and contain the protein complexes of the electron transport chain necessary for producing ATP through OxPhos. The mitochondrion from the GBM (m) is enlarged and shows a near total breakdown of cristae (cristolysis) and an electron-lucent matrix. The absence of cristae in GBM mitochondria indicates that OxPhos would be deficient. The arrow indicates an inner membrane fold. Bar: 0.33 lm. Method of staining: uranyl acetate/lead citrate. The GBM mitochondrion was reprinted with permission from Journal of Electron Microscopy [79]. The normal mitochondria and diagram were from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/mito.htm. This figure was previously cited [65].

can be robust in the mitochondria of some GBM [102]. The elevated lactate production in these cells, however, would be indicative of insufficient respiration despite having robust TCA activity [39]. Hence, substantial morphological and biochemical evidence exists showing that respiratory capacity is defective to some degree in most gliomas. Based on numerous findings in human glioma cell lines and tissues, several research groups suggested that the majority of malignant brain tumors are incapable of producing adequate amounts of energy through oxidative phosphorylation [78,79,91,92,101,103– 105]. Besides these ultrastructure findings, Renner and co-workers showed that tumor cells isolated from human GBM could produce ATP in the presence of potassium cyanide [106]. Cyanide blocks cytochrome c oxidase and kills normal control cells, which obtain energy through OxPhos. Mitochondrial energy production in the presence of cyanide suggests that OxPhos is not likely the origin of the energy produced in these GBM cells. These and other studies suggest that OxPhos is deficient in malignant gliomas and that energy through oxidative metabolism alone would be incapable of maintaining viability in glioma cells [107]. The Warburg effect in malignant brain tumors Otto Warburg first proposed that all cancers arise from irreversible damage to cellular respiration. As a result, cancer cells increase their capacity to ferment lactate even in the presence of oxygen in order to compensate for their insufficient respiration [108,109]. Although confusion has surrounded Warburg’s hypothesis on the origin of cancer cells [39,110,111], his hypothesis has never been formally disproved and remains a credible explanation for the origin of tumor cells [39,84,112–114]. Consequently, Warburg’s explanation for the origin of cancer can no longer be viewed as a hypothesis, but can now be viewed as a theory [39,40]. The key points of Warburg’s theory are; (1) insufficient respiration initiates tumorigenesis and ultimately cancer, (2) energy through glycolysis gradually compensates for insufficient energy through respiration, (3) cancer cells continue to ferment lactate in the presence of oxygen, and (4) respiratory insufficiency eventually becomes irreversible [108,109,115–117]. Warburg referred to the phenomenon of enhanced glycolysis in cancer cells as ‘‘aerobic fermentation’’ to highlight the abnormal production of lactate in

the presence of oxygen [108,109,115–117]. The ‘‘Warburg effect’’ refers to the aerobic fermentation of cancer cells [113,114]. Substantial evidence exists showing that gliomas avidly consume glucose and produce lactate [74,102,107,118]. This would be expected for any tumor cell with quantitative or qualitative or abnormalities in mitochondria. Although the abnormal energy metabolism and mitochondrial abnormalities seen in most cancers including glioma could arise in part through oncogenic modulation of metabolism [111], the data from the nuclear and mitochondrial transfer experiments seriously challenge this possibility [39,119]. The nuclear cytoplasmic transfer experiments date back to 1969 when McKinnell et al. showed that normal frogs could be cloned from the nucleus of a frog kidney tumor cell implanted into a fertilized egg [120,121]. The Mintz group showed that mice could be cloned from the nuclei of teratomas, while Morgan and colleagues showed that differentiated post-implantation mouse embryos could be cloned from the nuclei of medulloblastoma [122–124]. The work of Jaenisch and colleagues showed that nuclei from numerous tumor types could support early development of mouse embryos without signs of abnormal cell proliferation [125]. These studies were supported from numerous other studies in mouse and human cybrid cells showing that normal cytoplasm could suppress tumorigenesis when combined with tumor nuclei [119]. On the other hand, tumors could arise when a normal nucleus was place into an enucleated tumor cytoplasm [126,127]. The study of Howell and Sager recognized that these phenomena supported Warburg’s original hypothesis that tumor cells arise from abnormalities in the mitochondria rather than from abnormalities in the nucleus [128]. The distinguished British geneticist Darlington also presented evidence showing that tumor cells arose from defects in the cytoplasm and not the nucleus [129]. These observations indicate that nuclear gene mutations are not the drivers of tumorigenesis and that normal mitochondria can suppress tumorigenesis [130]. A comprehensive discussion of these findings in relationship to the origin of cancer has appeared [119]. It is anticipated that the cancer field will move forward in new directions once the implications of these findings become more widely recognized. As the result of insufficient respiration, cancer cells must rely on non-oxidative energy metabolism to maintain energy balance and viability. Consequently, aerobic fermentation plays a role in


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producing energy through substrate level phosphorylation in the cytoplasm (glycolysis) [84,85,116,131]. Besides aerobic fermentation in the cytoplasm, TCA cycle substrate level phosphorylation might also produce ATP through non-oxidative metabolism. [132–135]. Amino acid fermentation can generate energy through TCA substrate level phosphorylation under hypoxic conditions [134–137]. Hochachka showed that succinate is a waste product of amino acid fermentation [138,139]. Succinate is expressed in tumor cells and is thought to inhibit a family of prolyl hydroxylases, which facilitate Hif-1a degradation through the von Hippel–Lindau (VHL) gene product [140]. Hif-1a stabilization inhibits mitochondrial respiration and facilitates aerobic fermentation (Warburg effect) through its action on several glycolytic pathways [51,141,142]. It can be difficult to determine, however, the degree to which mitochondrial ATP production arises from coupled respiration or from TCA cycle substrate level phosphorylation. Emerging evidence also indicates that the function of DNA repair enzymes and the integrity of the nuclear genome are dependent to a large extent on the energy derived from normal respiration [41,42,143–149]. Previous studies in yeast and mammalian cells show that disruption of mitochondrial function can cause mutations (loss of heterozygosity, chromosome instability, and epigenetic modifications) in the nuclear genome [149–151]. These studies suggest that genomic instability could arise from a protracted reliance on non-oxidative energy. The process by which substrate level phosphorylation could be linked to the hallmarks of cancer was discussed [39,84,85,152]. It remains speculative whether respiratory insufficiency is irreversible, as Warburg suggested, or might be reversed through metabolic therapy. It is difficult to imagine, however, how respiration or the mitochondria cristolysis seen in GBM could be easily reversed (Fig. 2).

of extracellular glutamate, which is then metabolized to glutamine for delivery back to neurons. Neurons metabolize the glutamine to glutamate, which is then repackaged into synaptic vesicles for future release [162]. The glutamate–glutamine cycle maintains low extracellular levels of both glutamate and glutamine in normal neural parenchyma. Disruption of the glutamate–glutamine cycle can provide neoplastic GBM cells access to glutamine as we recently described [16]. Cytomegalovirus: an onco-modulator of brain tumor energy metabolism Many cancers including GBM are infected with human cytomegalovirus (HCMV), which acts as an oncomodulator of tumor progression [164–168]. Onco-modulation differs from initiation in facilitating progression after tumor initiation. The infection appears to be localized to the tumor cells and not to normal cells [167]. Products of the virus can damage mitochondria in the infected tumor cells thus contributing to a further dependence on glucose and glutamine for energy metabolism [169–172]. The virus often infects cells of monocyte/macrophage origin, which are considered the origin of many metastatic cancers [25,173–175]. Indeed, we proposed that neoplastic microglia/macrophages were the most invasive cells within GBM [24]. GBM malignancy is correlated with the titer of HCMV infection. The higher is the viral titer, the greater is the malignancy [168]. We suggest that HCMV infection in these neoplastic cells will contribute to the progression of GBM through an effect on tumor cell energy metabolism. Does the current standard of care accelerate GBM recurrence and progression through effects on energy metabolism?

Role of glucose and glutamine in brain tumor progression Glucose is the predominant fuel of the brain, but also fuels tumor cell glycolysis as well as serving as a precursor for glutamate synthesis [107,109,153,154]. Using linear regression analysis, we showed that the growth rate of the CT-2A experimental mouse astrocytoma was directly dependent on blood glucose levels [153]. The higher the blood glucose levels, the faster the tumors grew. As glucose levels fall, tumor size and growth rate falls. Hyperglycemia not only contributes to rapid tumor cell growth, but also enhances white matter damage in patients receiving radiation therapy [155]. Hyperglycemia was also directly linked to poor prognosis in humans with malignant brain cancer [156,157]. In other words, our findings in mice were corroborated with similar findings in humans. Moreover, we found that the expression of insulin-like growth factor 1 (IGF-1) was also dependent in part on circulating glucose levels [51,153]. IGF-1 is a cell surface receptor linked to rapid tumor growth through the PI3K/Akt signaling pathway [51]. The association of plasma IGF-1 levels with tumor growth rate is due in part to elevated levels of blood glucose. These findings in animal models and in brain cancer patients indicate that tumor growth rate and prognosis is dependent to a significant extent on circulating glucose levels. Glucose is the prime fuel for glycolysis, which drives growth of most brain cancers [101,104,107]. As long as circulating glucose levels remain elevated, brain tumor growth will be difficult to manage. In addition to glucose, glutamine is also suggested to play an role in tumor energy metabolism [158–161]. In contrast to extracranial tissues where glutamine is the most available amino acid, glutamine is tightly regulated in the brain through its involvement in the glutamate–glutamine cycle of neurotransmission [154,162]. Glutamate is a major excitatory neurotransmitter that must be cleared rapidly following synaptic release in order to prevent excitotoxic damage to neurons [162,163]. Glial cells possess transporters for the clearance

It is our view that the current standard of care for managing GBM and other malignant brain cancers will contribute to tumor recurrence and progression. This prediction comes from information describing how the standard of care can enhance the availability of glucose and glutamine within the tumor microenvironment [16,130,176]. It is well documented that neurotoxicity from mechanical trauma (surgery), radiotherapy, and chemotherapy, can increase tissue inflammation and glutamate levels [32,177– 179]. Local astrocytes rapidly clear extracellular glutamate metabolizing it to glutamine for release to neurons. In the presence of dead or dying neurons, however, surviving tumor cells and the tumorassociated macrophages (TAMs) will use astrocyte-derived glutamine for their energy and growth. TAMs also release pro-angiogenic and growth factors, which further stimulate tumor progression [16,18,180]. Radiation damage to tumor cell mitochondria will hasten a dependence on glucose and glutamine for growth and survival [84,109]. Both TAM and neoplastic microglia/macrophages could possibly ferment glutamine leading to the formation of succinate, a waste product of glutamine fermentation that contribute to local inflammation [132,181]. The HCMV infection in the neoplastic GBM cells will further accelerate tumor cell growth through increased metabolism of glucose and glutamine [169]. Tumor radiation will exacerbate the HCMV infection while also up-regulating the PI3K/ Akt signaling pathway, which will drive glioma glycolysis and chemotherapeutic drug resistance [84,165,182–185]. In contrast to normal glia that metabolize glutamate to glutamine, neoplastic glioma cells secrete glutamate [163]. It is not clear if the secreted glutamate is derived from glutamine in the necrotic microenvironment or is synthesized from glucose. Glioma glutamate secretion is thought to contribute in part to neuronal excitotoxicity and tumor expansion [163]. These observations indicate that the current standard of care creates a metabolic environment that would rescue neoplastic cells and facilitate GBM progression.



In addition to the enhancing effects of radiation and HCMV on GBM energy metabolism, most GBM patients are also given highdose glucocorticoids (dexamethasone) [63]. Although dexamethasone is given to reduce radiation-associated brain swelling and tumor edema, dexamethasone significantly elevates blood glucose levels [184,186–188]. Glucose fuels tumor cell glycolysis as well as serving as a precursor for glutamate synthesis [107,109,153,154]. Many GBM patients are also treated with the anti-angiogenic drug bevacizumab (Avastin). Bevacizumab targets leaky blood vessels thus enhancing hypoxia and radiation-induced necrosis in the tumor microenvironment. Increased hypoxia will further enhance tumor cell glycolysis and select for neoplastic microglia/macrophages with greatest invasive properties [24,189–191]. As macrophages/microglia evolved to survive in hypoxic environments, therapies that enhance hypoxia, like bevacizumab and radiation, will contribute to tumor progression. Bevacizumab also exacerbates radiation-induced necrosis, which will create a more favorable environment for tumor recurrence [192]. Viewed collectively, these findings illustrate how the current standard of care will create a microenvironment that facilitates the energy needs of tumor cells and the inevitable recurrence of the tumor. It is not clear if those who administer the standard of care to glioma patients are aware of these issues, as the standard of care has remained largely unchanged for decades [1]. The key processes are illustrated in Fig. 3. Although the existing standard of care for malignant brain cancer will increase patient survival over the short term (months) compared to the ‘‘no therapy’’ option, it is clear how this therapeutic strategy could accelerate the energy metabolism of surviving tumor cells. Moreover, the malignant phenotype of brain tumor cells that survive radiotherapy is often greater than that of the cells from the original tumor [165,184]. Treatments that increase tumor energy metabolism will facilitate tumor cell growth and survival, thus decreasing overall patient survival. In a randomized phase III study, none (0/278) of the patients receiving radiation alone

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survived, whereas only about 2% (6/254) of patients receiving radiation and TMZ became long-term survivors [2] (Fig. 1). While GBM is certainly a deadly disease, it remains to be determined if the current standard of care contributes to an irreversible progression of the disease [1,2,165]. These findings address the general inadequacy of current therapies in providing long-term management of GBM. The slight improvement of patient survival with TMZ is interesting in light of findings showing that TMZ enhances the number of driver mutations in the tumor tissue [11]. Driver mutations are thought to provoke tumor progression by conferring a growth advantage to the most neoplastic cells [36,37,193–195]. These findings question the role of driver mutations and tumor progression and also question the gene theory of cancer [130]. Is it possible that the toxic effects of TMZ (fatigue, nausea, diarrhea, etc.) cause an indirect calorie restriction, which thus improves survival?[196]. Further studies would be needed to determine if TMZ improves patient survival through indirect effects from calorie restriction. As long as brain cancer is viewed as something other than a metabolic disease, there will be little progress in improving progression free survival in our opinion [130,180]. The current standard of care for GBM offers little hope of long-term patient survival. This situation is even more disturbing, as the standard of care is not considered curative, but only palliative [197]. If GBM becomes viewed as a metabolic disease, however, we might anticipate major advances in treatment and substantial enhancement of progression free survival. Exploiting mitochondrial dysfunction for the metabolic management of GBM GBM, like most cancers, can be considered primarily a disease of energy metabolism [40,65,198]. Rational strategies for GBM management should therefore be found in therapies that specifically target tumor cell energy metabolism. As glucose is the major fuel for tumor energy metabolism through lactate fermentation, the restriction of glucose becomes a prime target for GBM manage-

Fig. 3. How the standard of care can accelerate brain tumor growth and recurrence. GBM and other high-grade brain tumors consist of multiple neoplastic cell types as well as TAMs, which release proinflammatory and pro-angiogenic factors. All these cells will use glucose and glutamine (Gln) as major metabolic fuels for their growth and survival. Recent evidence suggests that nearly all GBM are infected with human cytomegalovirus, which enhances glucose and glutamine metabolism in the tumor cells. Increased glutamate (Glu) concentrations will arise after radiation/drug-induced necrosis. Reactive astrocytes (RA) take up and metabolize glutamate to glutamine, whereas hyperglycemia will arise after corticosteroid (dexamethasone) therapy. Together, these standard treatments will provide a microenvironment that facilitates tumor cell growth, survival, and the likelihood of tumor recurrence [16]. With permission from Lancet Oncology.


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ment. However, most normal cells of the brain also need glycolytic pathway products, such as pyruvate, for energy production through OxPhos. It therefore becomes important to protect normal brain cells from drugs or therapies that disrupt glycolytic pathways or cause systemic reduction of glucose [130]. It is well known that ketones can replace glucose as an energy metabolite and can protect the brain from severe hypoglycemia [199–201]. Hence, the shift in energy metabolism associated with a low carbohydrate, high-fat ketogenic diet administered in restricted amounts (KDR) can protect normal brain cells from glycolytic inhibition and the brain from hypoglycemia. When systemic glucose availability becomes limiting, most normal cells of the body will transition their energy metabolism to fats and ketone bodies. Ketone bodies are generated almost exclusively in liver hepatocytes largely from fatty acids of triglyceride origin during periods of fasting [199,202]. The brain is exceptionally capable of transitioning from the metabolism of glucose to the metabolism of ketone bodies (b-hydroxybutyrate and acetoacetate) during periods of prolonged fasting [203–206]. The metabolism of ketones is an evolutionary adaptation that allows the body and brain to function at a high state of efficiency when food is unavailable [203]. A restriction of total caloric intake will facilitate a reduction in blood glucose and insulin levels and an elevation in ketone bodies. As long as the body is in a state of physiological ketosis (2–7 mM ketones in blood) blood glucose levels can be reduced to very low levels (2–3 mM) without producing the adverse effects of hypoglycemia [207]. Recent studies indicate that b-OHB is a histone deacetylase inhibitor that can reduce oxidative stress in normal cells through effects on the FOX3A and MT2 transcription factors [208]. Many tumors including gliomas, however, have reduced activity of succinyl-CoA: 3-ketoacid CoA transferase, the rate-controlling step for utilizing b-OHB as a respiratory fuel [209–214]. Defects in this enzyme will limit the ability of tumor cells to utilize ketone bodies as an alternative fuel to glucose. Metabolic stress following the gradual replacement of glucose with ketone bodies will be therefore greater in tumor cells than in normal cells [215]. Our group and others showed that ketone bodies could also be toxic to some cancer cells [39,209,216,217]. Nutritional ketosis induces metabolic stress on tumor tissue that is selectively vulnerable to glucose deprivation [65]. Hence, metabolic stress will be greater in tumor cells than in normal cells when the whole body is transitioned away from glucose and to ketone bodies for energy. The metabolic shift from glucose metabolism to ketone body metabolism creates an anti-angiogenic, anti-inflammatory, and pro-apoptotic environment within the tumor microenvironment [153,196,218–221]. Calorie restriction and ketogenic diets lower glucose and elevate ketones, which can account in part for the therapeutic benefit of these approaches [153,176,210,222]. The general concept of a survival advantage of tumor cells over normal cells occurs when fermentable fuels are abundant, but not when they become limiting [223]. Fig. 4 illustrates the theoretical changes in whole body levels of blood glucose and ketone bodies (b-hydroxybutyrate) that will metabolically stress tumor cells while enhancing the metabolic efficiency of normal cells. The efficacy of this therapeutic strategy was illustrated previously in cancer patients and in preclinical models [210,224–228].

The calorie restricted ketogenic diet as a non-toxic metabolic therapy for brain cancer Emerging evidence suggests that metabolic therapies using ketogenic diets that lower glucose levels can help retard GBM growth in younger and older patients [227–229]. Restricted diets are those that deliver fewer total calories in order to lower circulat-

Fig. 4. Relationship of circulating levels of glucose and ketones (b-hydroxybutyrate, b-OHB) to tumor management. The glucose and ketone values are within normal physiological ranges under fasting conditions in humans and will produce antiangiogenic, anti-inflammatory, and pro-apoptotic effects. We refer to this state as the zone of metabolic management. Metabolic stress will be greater in tumor cells than in normal cells when the whole body enters the metabolic zone. The values for blood glucose in mg/dl can be estimated by multiplying the mM values by 18. The glucose and ketone levels predicted for tumor management in human cancer patients are 3.1–3.8 mM (55–65 mg/dl) and 2.5–7.0 mM, respectively. These ketone levels are well below the levels associated with ketoacidosis (blood ketone values greater than 15 mmol). Elevated ketones will sustain metabolic pressure on tumor cells, buffer daily fluctuations in blood glucose levels, and protect the brain from hypoglycemia. Modified from a previous version [258].

ing glucose and insulin levels. We found that the KD reduced brain cancer growth and angiogenesis in mice only when administered in restricted amounts [65,153,210]. The importance of this point cannot be overemphasized, as unrestricted KD administration was largely without effect on the growth of the CT-2A astrocytoma [153,210]. We also showed that CR alone could reduce inter-hemispheric invasion through the ‘‘Secondary Structures of Scherer’’ in a natural mouse model of GBM [230]. In contrast to restricted administration, blood glucose levels remain high and ketones are largely excreted in the urine when the KD is fed to mice in unrestricted amounts. Blood ketone levels are also higher when the KD is administered in restricted than in unrestricted amounts. Harik et al. also showed that unrestricted feeding of the KD had no significant effect on brain glucose metabolism in rats [231]. However, Scheck and colleagues reported growth inhibition of the mouse GL261 cells from an unrestricted KD suggesting that some tumors might be susceptible to KD growth inhibition without calorie restriction or reduction [225]. On the other hand, glucose was reduced in mice receiving an unrestricted KD administered together with radiation therapy [222]. In contrast, we found that unrestricted consumption of the KD was ineffective in reducing tumor growth or angiogenesis in the syngeneic CT-2A glioma [153,210]. The unrestricted feeding of the KD prevented glucose and ketones from reaching the therapeutic levels necessary for blocking angiogenesis and enhancing tumor cell apoptosis [153,210]. Dyslipidemia and insulin insensitivity can also occur in association with the unrestricted feeding of the KD in mice [232]. We documented the importance of calorie restriction for management of mouse and human astrocytoma growth using either a standard high carbohydrate diet or a KD [210,219]. Due to differences in basal metabolic rate between man and mouse, water-only therapeutic fasting in man would be equivalent to a 40% CR in mice [233]. As the KD-R produces metabolic effects similar to that of water-only fasting, it is our opinion that it will be easier for people to implement and comply with the KD-R than with water-only fasting. To avoid the possibility of dyslipidemia and insulin insensitivity, we believe that the therapeutic benefits of the KD will also be best in humans if consumed in restricted



amounts, but in sufficient quantities to maintain energy levels and lean body mass. Nebeling and co-workers first showed that the restricted KD was an effective non-toxic management for advanced stage astrocytoma in children [228]. The malignant brain tumors in the two children of this study were largely unresponsive to the standard of care, which caused significant toxicity to both children. The standard of care was discontinued in one child (#1) and reduced in the other (#2), as the children were administered a medium chain triglyceride KD that lowered blood glucose and elevated blood ketone bodies [228]. Ketone bodies (b-hydroxybutyrate and acetoacetate) become an alternative fuel for brain energy metabolism when glucose levels are reduced [199,205,234,235]. Ketone bodies have known neuroprotective and anti-inflammatory action against a number of neurological and neurodegenerative diseases [236]. Ketone body metabolism increases the redox span of the Co-Q couple thereby enhancing the proton motive gradient and reducing the production of ROS [201,235]. Ketone body metabolism reduces ROS while enhancing metabolic efficiency of normal cells [225,235–238]. In addition to providing an alternative fuel to glucose, b-hydroxybutyrate also acts as a histone deacetylase inhibitor, which could reduce glucose and glutamine metabolism [208,237]. It is also important to recognize that circulating ketone levels will rarely exceed 7–9 mmol in most non-diabetic patients since excess ketones will be excreted in the urine [235]. Hence, ketones are considered ‘‘good medicine’ for several neurological and neurodegenerative diseases [199,205,239]. The health status of both children in the Nebeling et al. study improved after initiation of the ketogenic diet and overall survival was longer than initially predicted [228]. The quality of life improved significantly for the adult GBM patient in the Zuccoli et al. study while she remained on a calorie restricted KD [227]. Although this patient experienced radiological resolution while on the KD, the tumor recurred after the diet was discontinued [227]. The KD administered to the GBM patients in the study of Champ et al., appeared to improve response to the standard of care [229]. These observations are consistent with those from Scheck et al. in showing that survival was greater in mice with GBM that received radiotherapy with the KD than in mice that received radiotherapy alone [222,240]. The results from these case reports and preclinical studies suggest that the KD can be effective in improving overall survival and quality of life in children and adults with malignant brain cancer. In addition to lowering glucose availability to the tumor microenvironment, the KD could potentially lower brain glutamine levels thus restricting availability of this energy metabolite for tumor growth [237,241,242]. The KD-R could be even more therapeutic if combined with non-toxic drugs that also target glycolysis, e.g., 2deoxyglucose or 3-bromopyruvate, and dichloroacetate (DCA) [243–245]. The KD-R might also further enhance GBM patient survival when combined with the anti-human cytomegalovirus (HCMV) drug, valganciclovir. HCMV infection of tumor cells contributes to elevated metabolism of glucose and glutamine [169,246]. Recent evidence indicates that valganciclovir can enhance survival of GBM patients [247]. Poff et al. also recently showed a synergistic interaction between the KD and hyperbaric oxygen therapy (HBO2T) [248]. The KD reduces glucose for glycolytic energy, while also reducing NADPH levels for anti-oxidant potential through the pentose-phosphate-pathway. HBO2T will increase ROS in the tumor cells while the ketones protect normal cells against ROS damage and from potential oxygen toxicity [201,235]. Glucose deprivation will enhance oxidative stress in tumor cells, while increased oxygen can reduce tumor cell proliferation [249,250]. HBO2T has been used as a radiosensitizer for glioma patients with notable improvement over radiation alone [251]. In contrast to radiation therapy,

7

which also kills tumor cells through ROS production [252], the KD + HBO2T will kill tumor cells without causing toxic collateral damage to normal cells. Some ketogenic diets might also enhance the therapeutic action of radiation therapy against brain and lung tumors [222,253]. It is not yet known if therapeutic efficacy of the KD-R will be greater when combined with HBO2T than when combined with radiation therapy. It will therefore be important to compare and contrast the therapeutic efficacy of conventional radiation therapy with HBO2T when used with the KD-R. According to the evidence presented here, the KD-R can be a viable non-toxic option to the current standard of care for managing malignant brain cancer. The KD-R can target tumor cells globally without harming normal neurons and glia. The blood brain barrier is less of an issue with the KD-R therapy than with conventional therapies. We showed that the KD-R could enhance drug delivery to the brain [254]. Although radiotherapy was superior to best supportive care in older patients (>70 years of age) with high-grade glioma, the definition of ‘‘best supportive care’’ is ambiguous at best [255]. Would the KD-R + HBO2T be a better therapeutic option than radiotherapy or ‘‘best supportive care? Which brain tumor organizations might be interested in testing these options? Although metabolic therapy could be a more rational approach to malignant brain cancer management than is the current standard of cancer, the KD-R is not without some shortcomings. Compliance can be a major obstacle in attempting to implement the KD-R [65]. Some people can have difficulty in maintaining blood glucose and ketones in the ranges needed to target angiogenesis and to control tumor growth. Considerable patient discipline and motivation is required for implementing the KD-R as a therapy [198]. However, administration of ketone esters could possibly enable patients to circumvent the dietary restriction generally required for sustained nutritional ketosis. Ketone ester-induced ketosis would make sustained hypoglycemia more tolerable and thus assist in metabolic management of cancer [39,256,257]. It would be helpful if more neurooncologists could become familiar with the principles and concepts about how metabolic therapy controls tumor growth and how this therapy can be used as an alternative to the standard of care. This type of information is not generally covered as part of the training in the field. Consequently, some glioma patients might be discouraged from using the KD-R. Nevertheless, we remain hopeful that the metabolic approach to brain cancer management using the KD-R together with synergistic drugs, and possibly HBO2T could offer a better chance for improved quality of life and longer-term survival for younger and older GBM patients. Will brain tumor organizations step forward to test our predictions? Conflict of interest None of the authors have a conflict of interest. Acknowledgements This work was supported in part from NIH - United States Grants (HD-39722, NS-1080 55195, and CA-102135), a Grant from the American Institute of Cancer Research, and the Boston College Expense Fund. We thank Dr. Giulio Zuccoli and Jeremy Marsh for helpful comments, which were included in our similar review published in ‘‘Oncology & Hematology Reviews’’ and cited as reference 176 in the current review. References [1] P.G. Fisher, P.A. Buffler, Malignant gliomas in 2005: where to GO from here?, JAMA 293 (2005) (2005) 615–617


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