Warburg,me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers’ most common phenotypes,the “Warburg Effect”, i.e., elevated glycolysis in the presence of oxygen

As a new faculty member at The Johns Hopkins University, School of Medicine, the author began research on cancer in 1969 because this frequently fatal disease touched many whom he knew. He was intrigued with its viscous nature, the failure of all who studied it to find a cure, and also fascinated by the pioneering work of Otto Warburg, a biochemical legend and Nobel laureate. Warburg who died 1 year later in 1970 had shown in the 1920s that the most striking biochemical phenotype of cancers is their aberrant energy metabolism. Unlike normal tissues that derive most of their energy (ATP) by metabolizing the sugar glucose to carbon dioxide and water, a process that involves oxygen-dependent organelles called “mitochondria”, Warburg showed that cancers frequently rely less on mitochondria and obtain as much as 50% of their ATP by metabolizing glucose directly to lactic acid, even in the presence of oxygen. This frequent phenotype of cancers became known as the “Warburg effect”, and the author of this review strongly believed its understanding would facilitate the discovery of a cure. Following in the final footsteps of Warburg and caught in the midst of an unpleasant anti-Warburg, anti-metabolic era, the author and his students/collaborators began quietly to identify the key molecular events involved in the “Warburg effect”. Here, the author describes via a series of sequential discoveries touching five decades how despite some impairment in the respiratory capacity of malignant tumors, that hexokinase 2 (HK-2), its mitochondrial receptor (VDAC), and the gene that encodes HK-2 (HK-2 gene) play the most pivotal and direct roles in the “Warburg effect”. They discovered also that like a “Trojan horse” the simple lactic acid analog 3-bromopyruvate selectively enters the cells of cancerous animal tumors that exhibit the “Warburg effect” and quickly dissipates their energy (ATP) production factories (i.e., glycolysis and mitochondria) resulting in tumor destruction without harm to the animals.     

乳酸代谢——肿瘤治疗新靶点

恶性肿瘤严重危害人类健康,其治疗目前主要有手术、放疗和化疗三种方式,但疗效尚无法达到令人满意的程度,因此寻找肿瘤治疗新靶点、实现肿瘤的靶向治疗非常迫切.Warburg效应普遍存在于多种肿瘤中,其重要特征是在氧气充足的条件下,癌细胞的能量代谢仍以糖酵解为主.Warburg效应是糖酵解的典型过程,葡萄糖被大量吸收并通过糖酵解转化为乳酸.糖酵解产物乳酸可以激活癌细胞中许多重要的信号通路,促进癌细胞的存活、侵袭、免疫逃逸、转移和血管生成.因此,靶向乳酸代谢过程及其关键酶可能为肿瘤治疗提供新的靶点.本文对肿瘤细胞代谢方式的改变,乳酸对肿瘤细胞免疫逃逸、肿瘤转移、肿瘤血管生成的影响,以及以乳酸为靶点的肿瘤治疗等方面进行综述.     

The cancer cell’s “power plants” as promising therapeutic targets: An overview

This introductory article to the review series entitled “The Cancer Cell’s Power Plants as Promising Therapeutic Targets” is written while more than 20 million people suffer from cancer. It summarizes strategies to destroy or prevent cancers by targeting their energy production factories, i.e., “power plants.” All nucleated animal/human cells have two types of power plants, i.e., systems that make the “high energy” compound ATP from ADP and P _i . One type is “glycolysis,” the other the “mitochondria.” In contrast to most normal cells where the mitochondria are the major ATP producers (>90%) in fueling growth, human cancers detected via Positron Emission Tomography (PET) rely on both types of power plants. In such cancers, glycolysis may contribute nearly half the ATP even in the presence of oxygen (“Warburg effect”). Based solely on cell energetics, this presents a challenge to identify curative agents that destroy only cancer cells as they must destroy both of their power plants causing “necrotic cell death” and leave normal cells alone. One such agent, 3-bromopyruvate (3-BrPA), a lactic acid analog, has been shown to inhibit both glycolytic and mitochondrial ATP production in rapidly growing cancers (Ko et al., Cancer Letts., 173, 83–91, 2001), leave normal cells alone, and eradicate advanced cancers (19 of 19) in a rodent model (Ko et al., Biochem. Biophys. Res. Commun., 324, 269–275, 2004). A second approach is to induce only cancer cells to undergo “apoptotic cell death.” Here, mitochondria release cell death inducing factors (e.g., cytochrome c). In a third approach, cancer cells are induced to die by both apoptotic and necrotic events. In summary, much effort is being focused on identifying agents that induce “necrotic,” “apoptotic” or apoptotic plus necrotic cell death only in cancer cells. Regardless how death is inflicted, every cancer cell must die, be it fast or slow.     

Specific Phage-Displayed Peptides Binding to Tumor Vasculature

Tumor growth, progression and metastasis are critically dependent on blood supply, which has received increased attention as a potential target of new anticancer therapies. Antiangiogenic therapy to limit and even reverse the growth of tumors is under investigation and showing promise. Moreover, tumor vascular express specific surface proteins (“vascular zip codes”), not present in resting blood vessels of normal tissues, are suitable for targeting purposes. The specific “vascular zip codes” can be identified by screening phage-displayed peptide library in vivo. This technology is simple but powerful, providing the advantages of selectivity of action plus improved accessibility and efficacy. To date, a variety of tumor-homing peptides have been isolated in this method, and most of the peptides have been used for targeting devices to concentrate drugs and other therapeutic materials to tumors. Such a targeting strategy can decrease toxicity and increase the therapeutic efficacy of the drug.Dr.Yu Han and Dr. Liu Hong contributed equally to this study.     

Energy dependant plant stress acclimation

Plants may live and grow under suboptimal environmental conditions having certain biochemical and metabolic adaptations that facilitate their survival. Plant “metabolic flexibility” consists of the accomplishment of the same step in a metabolic pathway in a variety of different ways. Pyrophosphate which works as an energy donor when cellular ATP pools become diminished during stresses, alternative glycolytic reactions that bypass ATP-requiring steps, additional pathways for electron transport in plant mithocondria and the salvage pathways are some of the aspects related to “energetic flexibility”. This key feature that plays an important role in plant acclimation to stress can be an important target for engineering enhanced stress tolerance in crop plants.     

Cancer cell bioenergetics and pH regulation influence breast cancer cell resistance to paclitaxel and doxorubicin

The multidrug resistance (MDR) phenotype, frequently observed during cancer treatment, is often associated with drug efflux pump activity. However, many other factors are also known to be involved. Cancer cells often rely on aerobic glycolysis for energy production; this is known as the “Warburg effect” and is used as a survival mechanism. Associated to this event, a reverse pH gradient across the cell membrane occurs, leading to cytosol alkalinization and extracellular acidification. In the present study, we investigated the role of different mechanisms involved in MDR, such as altered tumor microenvironment and energetic metabolism. The breast cancer cell line MCF-7, used as model, was exposed to two widely used antitumor drugs, paclitaxel (antimitotic agent) and doxorubicin (alkylating agent). Cancer pH regulation was shown to be crucial for malignant characteristics such as cell migration and drug resistance. Our results showed that a lower extracellular pH induced a higher migratory capacity and higher resistance to the studied chemotherapeutical compounds in MCF-7 cells. Besides the influence of the extracellular pH, the role of the tumor metabolism in the MDR phenotype was also investigated. Pre-treatment with different bioenergetic modulators led to cell ATP depletion and altered lactic acid production and glucose consumption, resulting in increased sensitivity to paclitaxel and doxorubicin. Overall, this study supports the potential use of compounds targeting cell metabolism and tumor microenvironment factors such as pH, as co-adjuvants in conventional chemotherapy.     

Orchestrated downregulation of genes involved in oxidative metabolic pathways in obese vs. lean high-fat young male consumers

There are major variations in the susceptibility to weight gain among individuals under similar external influences (decreased physical activity and excessive calorie intake), depending on the genetic background. In the present study, we performed a microarray analysis and real-time PCR validations in order to find out differential gene expression in subcutaneous abdominal adipose tissue from two groups of subjects that despite living in similar environmental conditions such as a habitual high-fat dietary intake (energy as fat >40%) and similar moderate physical activity, some of them were successfully “resistant” (lean) to weight gain, while others were “susceptible” to fat deposition (obese). The classification of up- and downregulated genes into different categories, together with the analysis of the altered biochemical pathways, revealed a coordinated downregulation of catabolic pathways operating in the mitochondria: fatty acid β oxidation (P = 0.008), tricarboxylic acid cycle (P = 0.001), and electron transport chain (P = 0.012). At the same time, glucose metabolism (P = 0.010) and fatty acid biosynthesis (P = 0.011) pathways were also downregulated in obese compared to lean subjects. In conclusion, our data showed an orchestrated downregulation of nuclear-encoded mitochondrial gene expression. These genes are involved in cellular respiration and oxidative metabolic pathways and could play a role in the susceptibility to weight gain in some individuals.     

Identification of cancer stem cells-associated “side population” by flow cytometry with a violet laser

The possibility of identification of a “side population” of cancer stem cells in solid tumors by a flow cytometer equipped with a 405 nm violet laser has been investigated. Ovarian cancer (Skov-3) and colorectal cancer (Colo 320) cell lines formed the “side population” after vital staining with Hoechst 33342. Analysis of cells isolated from the tumor tissue of malignant melanoma and colorectal cancer, also revealed the “side population” characterized by increased fluorescent dye exclusion. The percentage of melanoma cells included in the “side population” was the same as that of cells co-expressing the cancer stem cells markers CD34 and CD44. However, the colon cancer “side population” was significantly smaller than the minor populations of colon cancer cells identified by either CD133 expression or exclusion of Rhodamine 123 exclusion.     

13C-pyruvate imaging reveals alterations in glycolysis that precede c-Myc-induced tumor formation and regression

Tumor cells have an altered metabolic phenotype characterized by increased glycolysis and diminished oxidative phosphorylation. Despite the suspected importance of glycolysis in tumorigenesis, the temporal relationship between oncogene signaling, in?vivo tumor formation, and glycolytic pathway activity is poorly understood. Moreover, how glycolytic pathways are altered as tumors regress remains unknown. Here, we use a switchable model of Myc-driven liver cancer, along with hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) to visualize glycolysis in de novo tumor formation and regression. LDHA abundance and activity in tumors is tightly correlated to?in?vivo pyruvate conversion to lactate and is rapidly inhibited as tumors begin to regress, as are numerous glycolysis pathway genes. Conversion of pyruvate to alanine predominates in precancerous tissues prior to observable morphologic or histological changes. These results demonstrate that metabolic changes precede tumor formation and regression and are directly linked to the activity of a single oncogene.     

Targeting amino acid metabolism in cancer growth and anti-tumor immune response

Recent advances in amino acid metabolism have revealed that targeting amino acid metabolic enzymes in cancer therapy is a promising strategy for the development of novel therapeutic agents. There are currently several drugs in clinical trials that specifically target amino acid metabolic pathways in tumor cells. In the context of the tumor microenvironment,however,tumor cells form metabolic relationships with immune cells,and they oftencompete for common nutrients. Many tumors evolved to escape immune surveillance by taking advantage of their metabolic flexibility and redirecting nutrients for their own advantage. This review outlines the most recent advances in targeting amino acid metabolic pathways in cancer therapy while giving consideration to the impact these pathways may have on the anti-tumor immune response.     

Preniche as missing link of the metastatic niche concept explaining organ-preferential metastasis of malignant tumors and the type of metastatic disease

Here we attempt to supplement the metastatic niche concept with a stage of “preniche” that determines the site of development of a premetastatic niche and of a subsequent metastasis. The “preniche” includes all cellular and molecular events in the site of a prospective metastasis preceding the entrance of myeloid progenitor cells. The “preniche” integrates an activation of vascular endothelium of the microcirculatory vessels of target organs in the site of a future metastasis under conditions of chronic persistent productive inflammation that can be induced by cytokines from the primary tumor and independently of it. The endothelium activation is responsible for adhesion and clustering of the recruited myeloid progenitor cells and also for the retention of cells of malignant tumors. The preniche easily arises in organs enriched with organspecific macrophages (lungs, liver, brain, etc.) where the endothelium is predisposed for intensive recruiting of myeloid progenitor cells of macrophages, especially under conditions of inflammation. Introduction of the “preniche” concept allows us to avoid difficulties associated with the development of the metastatic niche concept, especially concerning the problem of organ-preferential localization of metastases, and to make some predictions for experimental verification and potential approaches for preventing metastasizing in some oncologic patients.     

3-bromopyruvate (3BP) a fast acting, promising, powerful, specific, and effective “small molecule” anti-cancer agent taken from labside to bedside: introduction to a special issue

Although the “Warburg effect”, i.e., elevated glucose metabolism to lactic acid (glycolysis) even in the presence of oxygen, has been recognized as the most common biochemical phenotype of cancer for over 80 years, its biochemical and genetic basis remained unknown for over 50 years. Work focused on elucidating the underlying mechanism(s) of the “Warburg effect” commenced in the author’s laboratory in 1969. By 1985 among the novel findings made two related most directly to the basis of the “Warburg effect”, the first that the mitochondrial content of tumors exhibiting this phenotype is markedly decreased relative to the tissue of origin, and the second that such mitochondria have markedly elevated amounts of the enzyme hexokinase-2 (HK2) bound to their outer membrane. HK2 is the first of a number of enzymes in cancer cells involved in metabolizing the sugar glucose to lactic acid. At its mitochondrial location HK2 binds at/near the protein VDAC (voltage dependent anion channel), escapes inhibition by its product glucose-6-phosphate, and gains access to mitochondrial produced ATP. As shown by others, it also helps immortalize cancer cells, i.e., prevents cell death. Based on these studies, the author’s laboratory commenced experiments to elucidate the gene basis for the overexpression of HK2 in cancer. These studies led to both the discovery of a unique HK2 promoter region markedly activated by both hypoxic conditions and moderately activated by several metabolites (e.g., glucose), Also discovered was the promoter’s regulation by epigenetic events (i.e., methylation, demethylation). Finally, the author’s laboratory turned to the most important objective. Could they selectively and completely destroy cancerous tumors in animals? This led to the discovery in an experiment conceived, designed, and conducted by Young Ko that the small molecule 3-bromopyruvate (3BP), the subject of this mini-review series, is an incredibly powerful and swift acting anticancer agent. Significantly, in subsequent experiments with rodents (19 animals with advanced cancer) Ko led a project in which 3BP was shown in a short treatment period to eradicate all (100%). Ko’s and co-author’s findings once published attracted global attention leading world-wide to many other studies and publications related to 3BP and its potent anti-cancer effect. This Issue of the Journal of Bioenergetics and Biomembranes (JOBB 44-1) captures only a sampling of research conducted to date on 3BP as an anticancer agent, and includes also a Case Report on the first human patient known to the author to be treated with specially formulated 3BP. Suffice it to say in this bottom line, “3BP, a small molecule, results in a remarkable therapeutic effect when it comes to treating cancers exhibiting a “Warburg effect”. This includes most cancer types.     

Tumor mechanics and metabolic dysfunction

Desmosplasia is a characteristic of most solid tumors and leads to fibrosis through abnormal extracellular matrix (ECM) deposition, remodeling, and posttranslational modifications. The resulting stiff tumor stroma not only compromises vascular integrity to induce hypoxia and impede drug delivery, but also promotes aggressiveness by potentiating the activity of key growth, invasion, and survival pathways. Intriguingly, many of the protumorigenic signaling pathways that are mechanically activated by ECM stiffness also promote glucose uptake and aerobic glycolysis, and an altered metabolism is a recognized hallmark of cancer. Indeed, emerging evidence suggests that metabolic alterations and an abnormal ECM may cooperatively drive cancer cell aggression and treatment resistance. Accordingly, improved methods to monitor tissue mechanics and metabolism promise to improve diagnostics and treatments to ameliorate ECM stiffening and elevated mechanosignaling may improve patient outcome. Here we discuss the interplay between ECM mechanics and metabolism in tumor biology and suggest that monitoring these processes and targeting their regulatory pathways may improve diagnostics, therapy, and the prevention of malignant transformation.     

On the analysis of substrate cycles in large metabolic systems

The simultaneous operation of paired, opposing reactions (substrate cycles) or parallel reactions (dual pathways) with seeming wastage of ATP is widespread in cellular metabolism. Analysis of such “futile” pathways has hitherto been limited to loci with only two or three interconnecting fluxes. We introduce here a method that allows straightforward analysis of more complex systems. The method involves the linear superposition of “fundamental” modes, one or more of which may be energetically wasteful. Decomposition of a flux pattern into such modes allows computation of the amount of free energy “wasted” at any locus. Appropriate normalizations of energy wastage yield a number of indices useful for assessing the energetic impact of futile pathways on the cell and for comparing the degree of regulation of substrate cycles or dual pathways under different metabolic conditions. This approach is applied to steady-state flux data obtained in the protozoanTetrahymena pyriformis and in isolated rat hepatocytes under a variety of conditions.     

Evaluation of endogenous acidic metabolic products associated with carbohydrate metabolism in tumor cells

Tumor cells have a high tolerance for acidic and hypoxic microenvironments, also producing abundant lactic acid through accelerated glycolysis in the presence or absence of O₂. While the accumulation of lactate is thought to be a major contributor to the reduction of pH-circumscribing aggressive tumors, it is not known if other endogenous metabolic products contribute this acidity. Furthermore, anaerobic metabolism in cancer cells bears similarity to homo-fermentative lactic acid bacteria, however very little is known about an alternative pathway that may drive adenosine triphosphate (ATP) production independent of glycolysis. In this study, we quantify over 40 end-products (amines, acids, alcohols, aldehydes, or ketones) produced by malignant neuroblastoma under accelerated glycolysis (+glucose (GLU) supply 1–10 mM) ± mitochondrial toxin; 1-methyl-4-phenylpyridinium (MPP⁺) to abate aerobic respiration to delineate differences between anaerobic vs. aerobic cell required metabolic pathways. The data show that an acceleration of anaerobic glycolysis prompts an expected reduction in extracellular pH (pH_ex) from neutral to 6.7 ± 0.006. Diverse metabolic acids associated with this drop in acidity were quantified by ionic exchange liquid chromatography (LC), showing concomitant rise in lactate (Ctrls 7.5 ± 0.5 mM; +GLU 12.35 ± 1.3 mM; +GLU + MPP 18.1 ± 1.8 mM), acetate (Ctrl 0.84 ± 0.13 mM: +GLU 1.3 ± 0.15 mM; +GLU + MPP 2.7 ± 0.4 mM), fumarate, and a-ketoglutarate (<10 μM) while a range of other metabolic organic acids remained undetected. Amino acids quantified by o-phthalaldehyde precolumn derivatization/electrochemical detection–LC show accumulation of l-alanine (1.6 ± .052 mM), l-glutamate (285 ± 9.7 μM), l-asparagine (202 ± 2.1 μM), and l-aspartate (84.2 ± 4.9 μM) produced during routine metabolism, while other amino acids remain undetected. In contrast, the data show no evidence for accumulation of acetaldehyde, aldehydes, or ketones (Purpald/2,4-dinitrophenylhydrazine—Brady's reagent), acetoin (Voges–Proskauer test), or alcohols (NAD⁺-linked alcohol dehydrogenase). In conclusion, these results provide preliminary evidence to suggest the existence of an active pyruvate–alanine transaminase or phosphotransacetylase/acetyl-CoA synthetase pathway to be involved with anaerobic energy metabolism of cancer cells.     

Small Azurin Derived Peptide Targets Ephrin Receptors for Radiotherapy

Many lung cancer treatment regimens include radiotherapy. We sought to improve the efficacy of such treatment by invoking the targeted delivery of a model radiosensitizer (nicotinamide) to malignant tissues. Ephrin receptors (Eph), which are often overexpressed in lung cancers, were selected as the target of our delivery system. Molecular targeting was achieved utilizing a small peptide derived from the C-terminal portion of azurin, a copper-containing redox protein (“cupredoxin”) that is capable of binding to ephrin receptors. We prepared and screened a sub-library of peptides derived from the C-terminal region of azurin and found several small analogues that bound ephrin receptors EphA2, EphB2, and EphB4. One such peptide, termed AzV36, was selected for conjugation with nicotinic acid via an amide bond to form AzV36-NicL. The resulting linear peptide contains 15 residues (including unusual and d-amino acids), is very stable in human serum, and can be easily manufactured. AzV36-NicL conjugate was tested in vivo for its ability to radiosensitize Lewis lung carcinoma (LCC) in artificial metastasis and solid tumor engraftment models. The compound increased the efficacy of radiotherapy with tumor colonies being ~2–13 fold lower than with radiation alone depending on experimental schedule. In contrast, equimolar amounts of unconjugated peptide (AzV36-L) or nicotinamide alone only marginally improved radiation efficacy. The targeted delivery of radiosensitizer(s) to ephrin receptors may enhance the efficacy of radiation treatment of lung cancer and of other cancers that overexpress ephrin receptor(s).     

Global Metabolic Profiling of Infection by an Oncogenic Virus: KSHV Induces and Requires Lipogenesis for Survival of Latent Infection

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi''s Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi''s Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.