Warburg effect in lung carcinoma, the logic?

Warburg effect in lung carcinoma, the logic?

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Outside of 'purposefully' removing mitochondria to diffuse cytochrome C's apoptotic threat, I would assume that lung carcinomas, and their proximity to high oxygen levels, would have the lowest rate of cells entering the Warburg effect. Would this be a correct assumption?


As far as I can tell, there are no data to support your hypothesis. Someone correct me if I'm wrong.

There's actually some data to support that aerobic glycolysis occurs in lung carcinomas despite their proximity to oxygen (ref). There's even an article on the prolifertative pathway behind lung microvascular endothelial cells (ref), alluding to this requirement for anaerobic glycolysis in rapid proliferation.

T cells exhibit a similar pathway, switching to aerobic glycolysis in the effector phase where T cells are rapidly expanding (see clonal expansion). The general trend is that the Warburg effect lends itself to the rapid accumulation of biomass, supporting rapid cell division, instead of energy production to support cell survival (ref). You could rationalize it like cancer is already a failure in cell death, and so the cancer cells need to worry more about carbon compounds that support rapid division. There was also a paper by Cai et al. in 2014 that described their hypothesis behind aerobic glycolysis in cancer, whereby the Warburg effect helps tumors resist anoikis and thereby promote metastasis (ref).

It may be that the Warburg effect is a means to a required end for cancer cells, but as far as I know there's is no correlation between proximity to oxygen and incidence of Warburg metabolism. There are papers out there that continue to ask: is the Warburg effect a cause or consequence of cancer (ref? My thought is that cancer cells will undergo the Warburg effect at a given stage regardless of histology or behavior because it's beneficial.

Warburg Effect

Meshach Asare-Werehene , . Benjamin K. Tsang , in The Ovary (Third Edition) , 2019

Hexokinase II and Chemoresistance

The Warburg effect is associated with increased glycolysis as a result of upregulation of several major glycolytic enzymes. The upregulation of hexokinase (HK) activity due to Warburg effect has been implicated in chemoresistance in many cancer types including OVCA [69] . The HK family has four main isoforms: HKI, II, III, and IV. However, the HKII is the major isoform expressed in cancers due to its high glycolytic phenotype, and its upregulation has been associated with tumor survival and chemoresistance [70] . HKII catalyzes the phosphorylation of glucose to glucose-6-phosphate in the committed step of glycolysis.

HKII competes for the binding sites of VDAC on the mitochondrial outer membrane and prevents the release of proapoptotic factors such as Bax and Bad. This antiapoptotic characteristic of HKII could contribute to tumor cell survival and chemoresistance in OVCA [71–73] . The immunohistochemical data from 110 epithelial OVCA patients revealed that HKII expression is significantly associated with chemoresistance and decreased progression-free survival (PFS) [74] . HKII expression is also an independent prognostic factor for early recurrence. Although HKII has been implicated in an array of cancers [75,76] , its mechanistic role in OVCA chemoresistance and CDDP-induced apoptosis is largely unknown. Understanding the underlying mechanism behind the exact role of HKII in OVCA chemoresistance will help in designing personalized drugs to target the gene.

New Cancer Paradigm and New Treatment: The Example of METABLOC

Hyperthermia has long been known to interfere with the tumor metabolism. The goal of this paper is to review the potential of metabolic therapy and to suggest that its combination with hyperthermia may be of interest.

1. Objective

In a land mark article, John Bailar published in the “New England Journal of Medicine” in 1997 “Are we losing the war on cancer?” We recently confirmed that this is, still the case. We obtained from the World Health Organization mortality time-series data of 20 countries over 45 years (1961–2005). During these 45 years the age standardised cancer death rate has varied little (−4%). There has been a slight decrease in breast cancer (−6.5%), lung cancer in men (−2.5%), and prostate cancer (−1.7%), but a sharp decrease in stomach cancer (−77%). These data confirm the preliminary results from Bailar and contradict the notion of a breakthrough in cancer prevention, early detection, and cancer treatment (Summa 2012). Today, as before, metastatic cancer to the notable exception of some childhood malignancies and of lymphoma remains almost universally fatal.

Today cancer is thought as an invasion by malignant cells which deserves to be killed either by surgery, radiation therapy, or chemotherapy. The screening of new drugs is done by assessing their efficacy in killing cancer cells. Modern drugs target one specific pathway in order to kill the malignant cell. But the logic is still the same: killing the cancer cell. None of these new drugs can be credited with having changed significantly the survival pattern. For example the overall response rate to Herceptin (a so called magic bullet) when administered alone is less than 5%.

In the meantime the cost of cancer drugs has increased exponentially. It is highly probable that we are witnessing a “bubble” based more on goodwill and hope than results.

There is an obvious need for change of paradigm.

Cancer is widely thought to be the consequence of genetic abnormalities such as oncogene activation or tumor suppressor inactivation. This is correct but only a partial view of the disease. For example, there is oncogene activation in normal cells or during development or benign inflammation.

There are alternative ways of understanding cancer. The most promising is considering cancer as a metabolic disease as a disease related to diabetes.

2. Metabolic Aspects of Cancer: Otto Warburg

Cancer is not only a genetic disease but also a disease of the metabolism. Since the work of Nobel Prize winner Otto Warburg, we know that the metabolism of cancerous cells clearly differs from that of normal cells [1], Cancerous cells consume higher amounts of glucose than they are able to fully degrade [2]. This is the actual basis for PET scan imagery, in which the intravenous injection of a radioactive substance similar to glucose is used to visualize the cancer and its metastases. This fact, which had long been forgotten, is starting to surface again. A considerable amount of recent work, including our own, shows that this metabolic disorder could be the source of the cancer development process [3].

Otto Warburg published his observations regarding a metabolic alteration frequently observed in the 1920s in cancer cells [1]. Warburg reported that the cancer cells he investigated metabolized glucose directly to lactic acid, as opposed to the pyruvate being converted to water and carbon dioxide in the mitochondria via the tricarboxylic acid (TCA) pathway. This metabolic property of cancer cells bears his name, that is, the Warburg effect. It is also referred to as aerobic glycolysis, as it takes place in cancer cells even under normoxic conditions.

Interest in the Warburg effect waned considerably for a long period of time. Part of the reason was the fact that Warburg was convinced that the altered glucose metabolism in cancer cells was actually the cause of cancer and that the most likely explanation for his observation was damage to the mitochondria [1]. Since then, modern molecular biology has demonstrated that cancer cannot originate without a change to a cell’s genome and that, at least in most cases, damage to the mitochondria is not the explanation for why many cancer cells adopt aerobic glycolysis as the principal pathway for glucose metabolism [4, 5]. However, during the last 15 years or so, there has been a considerable increase in interest regarding the Warburg effect and its role in cancer. As a result, some seminal publications have elucidated the role that the Warburg effect plays in cancer, and there are a number of recent excellent reviews as well [2, 6, 7]. Warburg understood that there is a prevalent defect in the anabolic pathway. He did not understand that the oxidative pathway (catabolism) is also flawed.

3. Change in Metabolism Explain Prominent Features of Cancer Such as Carcinogenesis and Response to Chemotherapy

There is a wide consensus today on the importance of metabolism in cancer [2, 6, 7]. Prominent features of cancer probably can be summarized by metabolic changes. For example, the oncogenes target the metabolic pathway (for review see Israël and Schwartz [3]: cancer as a dysmethylation syndrome). A retrovirus can capture a gene from a host cell and transmit it to a new host. Retroviral oncogenes disturb a major signaling pathway: the MAP kinases mitogenic pathways, while the different steps of PI3 kinase pathway are targets for DNA viruses. Oncogene can thus be seen as metabolic perturbators.

To confirm the role of metabolism in carcinogenesis, we exposed normal melanocytes (from adolescent’s foreskin) to high dose glucose and insulin. The proliferation increased (doubling time: 2.7 versus 5.6 days). After 3 weeks of exposure to glucose or after 3 weeks followed by 4-week culture in standard medium, melanocytes were able to grow in soft agar colonies, a feature of cancer cells [8]. Most anticancer drugs target the DNA. But their precise mechanism of action is debated. It is clear that even when the treatment is effective in patients with large metastatic disease, there are no signs of cell death. Minutes after the beginning of a small cardiac infarct, there is an increase in intracellular protein in the circulating blood. This is not the case after chemotherapy. However, the first sign of response of treatment is a decrease in glucose uptake as demonstrated by the PET scan.

Cancer drugs can kill cancer cells (it is what they are selected to do), but when a cell survives it stops to grow for days or weeks. This resting phase has been little studied because it is technically difficult (only a few cells survive) and time consuming. We were able to demonstrate that this growth arrest was because of a switch in metabolism [9].

4. Targeting Cancer Metabolism: Background

There is considerable logic in targeting metabolic changes as an approach to the development of pharmaceutical agents to treat cancer despite the fact that these changes are not causal in nature. A relatively recent publication has shown that the genes involved in glycolysis are over overexpressed expressed in at least 24 different types of cancers that correspond to approximately 70% of all cancers [10]. It has been hypothesized that this widespread prevalence is because aerobic glycolysis provides a competitive advantage to cancer cells, allowing the synthesis of compounds (ribonucleotides and lipids) required for proliferation [11–13].

A number of specific inhibitors of key enzymes involved in the aerobic glycolytic pathway have been evaluated as potential anticancer drugs (see reviews [14–16]). However, with rare exceptions none of these compounds has been used clinically. Michelakis reported that treatment of five patients with glioblastoma multiforme using dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, resulted in tumor regression in three individuals. Berkson treated four pancreatic cancer patients with a combination of lipoic acid and naltrexone with excellent results [17, 18]. The first patient treated was still alive and well 78 months after presentation. Somewhat coincidentally,

-lipoic acid is also known to be an inhibitor of pyruvate dehydrogenase kinase as is dichloroacetate. Naltrexone, on the other hand, is an opioid receptor antagonist and is primarily used for the treatment of alcohol and opioid dependence, although there are limited data suggesting a potential role in cancer inhibition.

This relative lack of success suggested to us that a single inhibitor of cancer cell metabolism might be insufficient to significantly inhibit cancer proliferation. Given the extreme plasticity of malignant tissue, it seemed logical to attempt to use at least two different compounds, each one targeted to interact with enzymes catalyzing different steps. We adopted a strategy to use compounds already proven to be nontoxic in humans.

5. Screening for a “Universal” Metabolic Combination

In 2004, we started collaborating with other scientists, among them Dr. Maurice Israël we focused their efforts on discovering a way to take advantage of one of the weaknesses of cancer: its poorly effective metabolism. Instead of targeting the mitotic process, they choose to target the metabolism of the cell [3].

In June 2007, the second phase of this work began with the selection of about a hundred molecules potentially active from the literature analysis. Focusing on the metabolic alterations of cancer cells, we identified molecules that have been described to act on enzymes whose activities are known to be affected by cancer. Our second selection criteria were the existence of data on human administration for these molecules. This approach allowed us to select 27 different molecules.

In the first animal study [19], a detailed literature analysis was conducted from which a first library of twenty-seven drugs that are known to target pathways potentially implicated in cancer was developed. We conducted in vitro tests on these molecules to determine their antiproliferative capacity on four cells lines at concentrations consistent with published human plasma levels. The data, summarized in Table 2, showed that 5 molecules were not effective 11 molecules were weakly effective, while 11 molecules were significantly effective.

Thus, this preliminary study (see [19] for details) suggested that a combination of ALA with HCA may have a high antitumoral potential. This efficacy was similar whatever the cell line tested.

Our group tested 15 combinations of two drugs based on the seven effective and least toxic molecules. Seven combinations showed a strong antiproliferative effect (< 20% of viable cells after 24 hours). They were acetazolamide and hydroxycitrate, lipoic acid and dichloroacetate, lipoic acid and hydroxycitrate, acetazolamide and miltefosine, albendazole and dichloroacetate, dichloroacetate and hydroxycitrate, and lipoic acid and miltefosine.

5.1. In Vivo Antitumoral Effect

We then proceeded to test these seven most effective combinations in vivo using mice bearing syngeneic MBT-2 bladder carcinoma. The majority of the combinations were not or only weakly effective (data not shown). The most effective treatment was hydroxycitrate and lipoic acid (designated as METABLOC) [19]. The efficacy of this combination was confirmed in B16-F10 melanoma and LL/2 Lewis lung carcinoma. This combination of both drugs slowed growth of the tumor and increased survival with an efficacy similar to conventional cytotoxic chemotherapy. This combination is effective whatever the tumor model, suggesting that these metabolic pathways are crucial for cancer survival.

The compositions were tested against different murine tumor models (MBT-2 bladder carcinoma, LLC Lewis lung carcinoma, and B16F10 melanoma implanted in syngeneic C3H mice (MBT-2 cells) or C57Bl6 (LLC and B16F10 cells)). Tumor cells were inoculated in the flank of the mice and tumor developed for few days before the beginning of the treatment. After randomization, the combinations were administered intraperitoneally, for 21 days. The change in tumor development was monitored by measuring the size of the tumours with a Vernier caliper and monitoring the survival of the animals during the experiment. The mice used in this study were treated in accordance with the ethical regulations in force. In the described results, the following doses and schedule of administration were used: alpha-lipoic acid 10 mg/kg, twice a day hydroxycitrate 250 mg/kg, twice a day.

The combination was used to treat mouse syngeneic cancer models: MBT-2 bladder transitional cell carcinoma, B16-F10 melanoma, and LL/2 Lewis lung carcinoma. The efficacy of this combination appears similar to conventional chemotherapy (cisplatin or 5-fluorouracil) as it resulted in significant tumor growth retardation and enhanced survival (Figure 1) (for details see article [19]).


A critical review of the role of M 2 PYK in the Warburg effect

It is becoming generally accepted in recent literature that the Warburg effect in cancer depends on inhibition of M2PYK, the pyruvate kinase isozyme most commonly expressed in tumors. We remain skeptical. There continues to be a general lack of solid experimental evidence for the underlying idea that a bottle neck in aerobic glycolysis at the level of M2PYK results in an expanded pool of glycolytic intermediates (which are thought to serve as building blocks necessary for proliferation and growth of cancer cells). If a bottle neck at M2PYK exists, then the remarkable increase in lactate production by cancer cells is a paradox, particularly since a high percentage of the carbons of lactate originate from glucose. The finding that pyruvate kinase activity is invariantly increased rather than decreased in cancer undermines the logic of the M2PYK bottle neck, but is consistent with high lactate production. The "inactive" state of M2PYK in cancer is often described as a dimer (with reduced substrate affinity) that has dissociated from an active tetramer of M2PYK. Although M2PYK clearly dissociates easier than other isozymes of pyruvate kinase, it is not clear that dissociation of the tetramer occurs in vivo when ligands are present that promote tetramer formation. Furthermore, it is also not clear whether the dissociated dimer retains any activity at all. A number of non-canonical functions for M2PYK have been proposed, all of which can be challenged by the finding that not all cancer cell types are dependent on M2PYK expression. Additional in-depth studies of the Warburg effect and specifically of the possible regulatory role of M2PYK in the Warburg effect are needed.

Copyright © 2019 Elsevier B.V. All rights reserved.


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Recent studies have demonstrated that many tumour-associated mutp53 proteins gain new oncogenic functions to promote tumour cell proliferation, anti-apoptosis, metastasis and lipid metabolism 4,5,6,7 . However, the mechanism of mutp53 GOF in tumorigenesis is not well understood. Results from this study clearly demonstrate a novel GOF of mutp53 in both cultured cells and mutp53 knockin mice, that is, mutp53 stimulates the Warburg effect. This function of mutp53 is contrary to the function of wtp53 in repressing the Warburg effect, which was confirmed by our results in this study (Figs 1 and 2). Mutp53 does not affect the expression of GLUT1, but promotes GLUT1 translocation to the PM. GLUT1 knockdown largely abolishes this stimulating effect of mutp53 on the Warburg effect in cells. Furthermore, mutp53 does not affect the expression or the PM translocation of GLUT2 or GLUT3. Knockdown of GLUT2 or GLUT3 clearly reduced the Warburg effect in cells, but did not clearly affect the stimulating effect of mutp53 on the Warburg effect. Currently, the mechanism by which mutp53 specifically regulates the translocation of GLUT1 in tumour cells remains unclear. These results strongly suggest that promoting the GLUT1 translocation to the PM is an important mechanism by which mutp53 stimulates the Warburg effect.

GLUTs, including GLUT1–4, mediate the transport of glucose across the PM, a critical step for glucose metabolism. Each of these GLUTs displays distinct expression patterns in cells and tissues. For example, GLUT4 is specifically and highly expressed in fat and muscle tissues, and is responsive to insulin-stimulated glucose uptake in these two tissues. In contrast, GLUT1 is ubiquitously expressed and is responsible for the basal glucose uptake in various types of cells. The regulation of GLUTs translocation is different among GLUTs and the mechanisms appear to be highly cell type and context dependent 25,32 . For example, although translocation of GLUT4 is dramatically stimulated by insulin in fat and muscle cells, the translocation of GLUT1 is not markedly regulated by insulin 25,32 . It has been reported that the RhoA/ROCK signalling is involved in the regulation of the insulin-stimulated translocation of GLUT4 and glucose uptake 25,32,33,40 . However, it is unclear whether RhoA/ROCK signalling can regulate GLUT1 translocation and the basal glucose uptake in cells, especially in cells other than fat and muscle. Furthermore, it is unclear whether RhoA/ROCK activation in cancer cells promotes the Warburg effect in cancer cells. Results from this study clearly show that RhoA/ROCK can promote the translocation of GLUT1 to the PM and promote the basal glucose uptake in various cells. Furthermore, the activation of RhoA/ROCK signalling by mutp53 mediates mutp53’s role in promoting GLUT1 translocation and the Warburg effect in cancer cells. Inhibition of RhoA/ROCK signalling by knocking down RhoA or ROCK1/2, or by the ROCK inhibitor Y27632, all largely abolishes the stimulating effect of mutp53 on the GLUT1 translocation to the PM and the Warburg effect in cells. Thus, our results reveal that promoting the GLUT1 translocation to the PM to stimulate the Warburg effect in cancer cells is a novel mechanism by which the activated RhoA/ROCK signalling promotes tumorigenesis, especially in cells containing mutp53.

The RhoA signalling is frequently activated in many types of cancer, which has a critical role in promoting tumour cell proliferation, invasion and metastasis 30,31 . In addition to our finding that mutp53 promotes the Warburg effect through RhoA activation, recent studies also showed that the activation of RhoA by mutp53 contributes to the GOF of mutp53 in tumour proliferation, invasion and metastasis 34,35,36,37 . These findings together indicate an important role of the RhoA signalling in mediating mutp53 GOF in cancer. However, it is still not well understood how mutp53 activates RhoA. As a small GTPase, RhoA activity is regulated by many positive and negative regulators, especially a group of Rho GEFs, Rho GAPs and Rho GDIs. Recently, it was reported that mutp53 activates RhoA through transcriptional upregulation of RhoGDI and Rho GEF-H1, two positive regulators for RhoA 34,35,36 . It will be of interest to examine whether mutp53 can regulate the expression of some other RhoA regulators to activate RhoA, including upregulation of additional positive regulators and/or downregulation of some negative regulators for RhoA. In addition to the transcription regulation, mutp53 can interact with other proteins to affect their functions, which contributes to the GOF of mutp53 in cancer 6,54 . It is possible that mutp53 interacts with some upstream regulators for RhoA to activate RhoA. Considering the important role of mutp53/RhoA signalling in tumorigenesis and potential therapeutic applications, future studies are needed to elucidate the molecular mechanism by which mutp53 activates RhoA.

In summary, our results clearly demonstrate that stimulating the Warburg effect in tumour cells is a novel GOF of tumour-associated mutp53. Our results also reveal that mutp53 acts as an important mediator for the Warburg effect in cancer cells, which provides a new mechanism for the Warburg effect. Emerging evidence has strongly suggested that as a hallmark of tumour cells, metabolic changes in tumours, such as the Warburg effect, could be targeted for tumour therapy. Our results strongly suggest that targeting altered glucose metabolism could be a feasible therapeutic strategy for tumour carrying mutp53.

Autophagy, Warburg, and Warburg Reverse Effects in Human Cancer

1 Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956 p5, 1113 Buenos Aires, Argentina

2 Department of Pharmacology, CEMIC University Institute, 1113 Buenos Aires, Argentina


Autophagy is a highly regulated-cell pathway for degrading long-lived proteins as well as for clearing cytoplasmic organelles. Autophagy is a key contributor to cellular homeostasis and metabolism. Warburg hypothesized that cancer growth is frequently associated with a deviation of a set of energy generation mechanisms to a nonoxidative breakdown of glucose. This cellular phenomenon seems to rely on a respiratory impairment, linked to mitochondrial dysfunction. This mitochondrial dysfunction results in a switch to anaerobic glycolysis. It has been recently suggested that epithelial cancer cells may induce the Warburg effect in neighboring stromal fibroblasts in which autophagy was activated. These series of observations drove to the proposal of a putative reverse Warburg effect of pathophysiological relevance for, at least, some tumor phenotypes. In this review we introduce the autophagy process and its regulation and its selective pathways and role in cancer cell metabolism. We define and describe the Warburg effect and the newly suggested “reverse” hypothesis. We also discuss the potential value of modulating autophagy with several pharmacological agents able to modify the Warburg effect. The association of the Warburg effect in cancer and stromal cells to tumor-related autophagy may be of relevance for further development of experimental therapeutics as well as for cancer prevention.

1. Introduction

Autophagy is an evolutionarily conserved and highly regulated lysosomal pathway involved in the degradation of macromolecules and cytoplasmic organelles [1–3]. Autophagy is a crucial contributor to maintain cellular homeostasis. The quality control of mitochondria structure and function, for instance, is important in the maintenance of cell energy and this process seems to involve autophagy.

By 1920, Otto Warburg hypothesized that tumor cells mainly generate energy by nonoxidative breakdown of glucose, making cancer growth feasible. This phenomenon is known as “Warburg effect.” This cellular event relies on mitochondrial dysfunction, characterized by respiratory impairment, resulting in a switch to glycolysis.

Originally, the Warburg effect was thought to occur only in cancer cells. Nevertheless, in 2008, Vincent et al. demonstrated that human skin keloid fibroblasts display similar bioenergetic changes as cancer cells in generating ATP mainly from glycolysis. The hypoxic microenvironment is a common fact in solid tumors and keloids, which may be the explanation for this thermodynamic phenomenon [4]. In line with these findings, Pavlides and col suggested, in 2009, a novel hypothesis for understanding the Warburg effect in tumors [5]: they proposed that epithelial cancer cells induce the Warburg effect in neighboring stromal fibroblasts.

A clear association among mitochondrial function, Warburg effect, the reverse Warburg effect, and autophagy can be established. The objective of this review is to discuss the autophagy process, its regulation, the selective pathways, and its role in cancer cell metabolism. We define Warburg effect and the “reverse” hypothesis and we discuss the potential value of modulating autophagy. The relevance of these interactions on cancer cell biology will be also discussed, as well as their potential impact on disease prevention and treatment.

2. Autophagy and Cancer

Autophagy is a highly regulated-cellular pathway for degrading long-lived proteins and is the only known pathway for clearing cytoplasmic organelles. This process is involved in the turnover of long-lived proteins and other cellular macromolecules, and, when normal, it might play a protective role in development, aging, cell death, and defense against intracellular pathogens [6, 7]. Autophagy has been associated with a variety of pathological processes such as degenerative diseases (diabetes, neurodegenerative processes, etc.) and carcinogenesis, with highlights of biomedical relevance [8, 9].

Autophagy consists of several sequential steps: induction, autophagosome formation, autophagosome-lysosome fusion, and degradation. Three major types of autophagy exist in eukaryotes: (1) chaperone mediated autophagy (CMA), (2) microautophagy, and (3) macroautophagy, hereafter referred to as autophagy [10]. CMA allows the direct lysosomal import of unfolded, soluble proteins that contain a particular pentapeptide motif. In microautophagy, cytoplasmic material is directly engulfed into the lysosome at the surface of the lysosome by membrane rearrangement. Finally, autophagy involves the sequestration of cytoplasm into a double-membrane cytosolic vesicle, referred to as an autophagosome that subsequently fuses with a lysosome to form an autolysosome for the degradation by lysosomal hydrolases [11].

Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes, which eventually acquire lysosomal-like features [11, 12].

Autophagy is mediated by a set of evolutionarily conserved gene products (termed the ATG proteins) originally discovered in yeast [13]. In mammalian cells, BECN1 [2, 14–16] promotes autophagosome formation when it functions as part of a complex with the class III phosphatidylinositol 3-kinase (PI3K) mediating the localization of other autophagic proteins to the autophagosomal membrane [17]. However, despite the advances in understanding autophagy, autophagosome formation in mammalian cells is a complex process, and neither the molecular mechanisms nor all the implicated genes involved in its formation are fully elucidated.

More than 30 highly conserved genes that are involved in autophagy have been identified so far [18]. A core molecular machinery has been defined and is composed of four subgroups: first, the ATG1/unc-51-like kinase (ULK) complex second, the class III phosphatidylinositol 3 kinase (PtdIns3K)/Vps34 complex I third, two ubiquitin-like protein (ATG12 and ATG8 (LC3) conjugation systems and four, two transmembrane proteins, ATG9/mATG9 (and associated proteins involved in its movement such as ATG18/WIPI-1) and VMP1 (whose expression triggers autophagy) [19–21]. Basal autophagy in unstressed cells is kept down by the action of the mammalian target of rapamycin complex 1 (mTORC1). Key upstream regulators of mTORC1 include the class I phosphoinositide 3-kinase (PI3K)-Akt pathway, which keeps mTORC1 active in cells with sufficient growth factors, and the AMP-activated protein kinase (AMPK) pathway that inhibits mTORC1 upon starvation and calcium signals [22, 23].

Autophagy is strongly induced in many types of cultured cells under stress conditions. These stress conditions include amino acid starvation. The effects of individual amino acids differ in their abilities to regulate autophagy. It has been suggested that amino acid starvation is followed by an activation of serine/threonine kinase mammalian target of rapamycin (mTOR) and the subsequent regulation of the class III PI3K. The mTOR is involved in the control of multiple cell processes in response to changes in nutrient conditions [24]. Particularly, mTOR complex 1 (mTORC1) requires Rag GTPase, Rheb, and Vps34 for its activation and the corresponding inhibition of autophagy in response to amino acids [25, 26]. AMP activated protein kinase (AMPK) senses energy levels and constitutes a key factor for cellular energy homeostasis. When energy levels are low, AMPK is activated. The activated AMPK then inactivates mTORC1 through TSC1/TSC2 and Rheb protein [27]. This inactivation of mTORC1 is an essential step for the induction of autophagy and plays a central role in autophagy. In addition to amino acid signaling, it has also been reported that other factors can regulate autophagy, such as hormones, growth factors, and many other factors, including bcl-2 [28], reactive oxygen species (ROS) [29], calcium [30], BNIP3 [31], p19ARF [32], DRAM [33], calpain [34], TRAIL [35], FADD [36], and myo-inositol-1,4,5-triphosphate (IP3) [37]. But it is important to point out that not all autophagy signals are transduced through mTOR signaling. A recent study showed that small-molecule enhancers of the cytostatic effects of rapamycin (called SMERs) induce autophagy independently of mTOR [38].

Depending on nutrient conditions, the activities of the ULK1 kinase complex can be regulated by mTOR. Under growing and high-nutrient conditions, the active mTORC1 interacts with the ULK1 kinase complex (ULK1-mATG13-FIP200-ATG101) and phosphorylates ULK1 and mATG13 and therefore inhibits the membrane targeting of the ULK1 kinase complex. On the other hand, during starvation condition, the inactivated mTORC1 dissociates from the ULK1 kinase complex. This dissociation results in the ULK1 kinase complex free to phosphorylate components, such as mATG13 and FIP200, in the ULK1 kinase complex, leading to autophagy induction [39].

The vacuole membrane protein 1 (VMP1), a pancreatitis-associated protein, is a transmembrane protein with no known homologues in yeast. Its expression induces autophagosome formation, even under nutrient-replete conditions while remaining an integrated autophagosomal membrane protein in mammalian cells [40]. Hyperstimulation of Gq-coupled CCK receptor in pancreatic acinar cells during acute pancreatitis [41] and mutated KRas in pancreatic cancer cells [42] induce VMP1 expression. In addition, VMP1 interacts with Beclin 1/ATG6 through its hydrophilic C-terminal region (VMP1-ATG domain), which is necessary for early steps of autophagosome formation [40, 43]. Besides, EPG-3/VMP1 is one of three essential autophagy genes conserved from worms to mammals. EPG-3/VMP1 regulates early steps of the autophagic pathway in Caenorhabditis elegans [44]. VMP1 along with ULK1 and ATG14 localizes in the endoplasmic reticulum-associated autophagosome formation sites in a PI3K activity-independent manner, confirming the key role of VMP1 in the formation of autophagosomes [19]. Interestingly, an accumulation of huge ubiquitin-positive protein aggregates containing the autophagy marker ATG8/LC3 was seen and p62 homolog [45] in Dictyostelium cells lacking Vmp1 gene showed. Moreover, the knockdown of VMP1 expression abolishes starvation and rapamycin-induced autophagosome formation [40]. It also abolishes autophagy induced by hyperstimulation of Gq-coupled CCK receptor in pancreatic acinar cells [41] or by chemotherapy in pancreatic tumor cells [46]. Furthermore, VMP1 is the only human disease-inducible ATG-protein described so far.

It has been shown that both downregulated and excessive autophagy have been implicated in the pathogenesis of diverse diseases. These diseases include a certain type of neuronal degeneration, diabetes and its complications, and cancer [47]. Autophagy has also been implicated in cell death called autophagic or type II programmed cell death, which was originally described on the basis of morphological studies detecting autophagic vesicles during tissue involution [48].

In general, cancer cells tend to undergo less autophagy than their normal counterparts, at least for some tumors [49, 50]. There is a monoallelic deletion of beclin1 autophagy gene in 40–74% of cases of human sporadic breast, ovarian, and prostate cancer [50]. Heterozygous disruption of beclin1 increases the frequency of spontaneous malignancies and accelerates the development of virus-induced premalignant lesions [50]. This suggests that defective regulation of autophagy promotes tumor genesis. It has been proposed that autophagy can suppress carcinogenesis by a cell-autonomous mechanism that involves the protection of genome integrity and stability and a nonautonomous mechanism that involves the suppression of inflammation and necrosis. On the other hand, autophagy may support the survival of rapidly growing cancer cells that have outgrown their vascular supply and are exposed to a hostile environment with an inadequate oxygen supply or metabolic stress. In contrast, excessive levels of autophagy promote cell death [51]. Accordingly, it has been proposed that autophagy can play an important role both in tumor progression and in promotion of cancer cell death [52]. For instance, in pancreatic ductal adenocarcinoma (PDAC), the cellular response to ROS initiates a survival or cell death pathway dependent on the severity of the oxidative damage [53]. ROS such as H2O2 can induce autophagy. The deregulation of the AGER ligand HMGB1 is expressed in many cancer cells including pancreatic cancer cells. ROS can increase the release of HMGB1 from necrotic cells and then activates Beclin-1-dependent autophagy by binding to AGER in pancreatic cancer cells [53, 54]. In addition, ROS can promote cytosolic translocation of HMGB1 to bind to Beclin-1 and then enhance autophagy [55]. Recent studies have demonstrated that oxidative stress increases the activity of NF-κB which upregulates the expression of AGER in pancreatic cancer [56]. This in turn promotes autophagy flux by upregulation of LC3-II levels and protects pancreatic cancer cells from oxidative injury. On the other hand, ascorbate leads to cell death in PDAC through a unique ROS-mediated caspase-independent autophagy pathway [57], and gemcitabine and cannabinoids combination induces ROS-mediated autophagic cell death in pancreatic tumor cells [58].

There are suggestions that autophagy may be a cancer cell survival response to tumor-associated hypoxia. Tumor hypoxia has been used as a marker of poor prognosis [59] in any case, how cancer cells become more malignant or survive with an extremely poor blood supply is poorly understood. When cancer cells are exposed to hypoxia, anaerobic glycolysis increases and provides energy for cell survival, but as the glucose supply is also insufficient due to the poor blood supply, there must be an alternative metabolic pathway that provides energy when both oxygen and glucose are depleted [60, 61]. In pancreatic cancer, there have been reports of hypoxia increasing the malignant potential of the tumor [59]. Proliferating cancer cells require more nutrients than surrounding noncancerous cells do. Nutrition of these proliferating cancer cells is supplied via functionally structurally immature neovessels. Autophagy may react to the cancer microenvironment to favor the survival of rapidly growing cancer cells. This is because autophagy-specific genes promote the survival of normal cells during nutrient starvation in all eukaryotic organisms. LC3 expression has been seen in surgically resected pancreatic cancer tissue that shows activated autophagy in the peripheral area, which included the invasive border and concomitantly exhibits enhanced expression of carbonic anhydrase [62]. This suggests that autophagy may promote cell viability in hypovascularized cancer tissue.

Another proposal is that autophagy is a survival cancer cell response to tumor-associated inflammation [63]. The promotion of carcinogenesis and resistance to therapy are two results of cancer-associated inflammation. Several phenotypic alterations observed in cancer cells are a result of inflammatory signals found within the tumor microenvironment [63]. The receptor for advanced glycation end products (RAGE) is an induced inflammatory receptor. It is constitutively expressed on many murine and human epithelial tumor cell lines [64, 65]. Murine and human pancreatic adenocarcinoma tumors have shown the highest levels of RAGE expression. Genotoxic and/or metabolic stress lead to modest but reproducible increases in overall expression of RAGE on epithelial cell lines. There is a direct correlation between RAGE expression and the ability of both murine and human pancreatic tumor cell lines to survive cytotoxic aggression. Targeted knockdown of RAGE significantly increases cell death, whereas forced overexpression promotes survival. It was recently reported that the enhanced sensitivity to cell death in the setting of RAGE knockdown is associated with increased apoptosis and decreased autophagy. In contrast, overexpression of RAGE is associated with an increased autophagy, but diminished apoptosis and enhances cancer cell viability. Knockdown of RAGE enhances mTOR phosphorylation in response to chemotherapy therefore, there is a prevention of an induction of a survival response. Inhibition of autophagy by means of silencing beclin1 expression in pancreatic cancer cells enhances apoptosis and cell death [66]. These findings suggest that RAGE expression in cancer cells has a role in tumor cell response to environmentally induced stress through the enhancement of autophagy. However, increased sensitivity to chemotherapeutic agents in RAGE-knockdown pancreatic cancer cells is dependent on ATG5 expression but independent of BECN1 expression [66]. These last findings suggested that the role of autophagy in the resistance to microenvironment insult or in the sensitivity to chemotherapeutic agent is the result of complex molecular pathways in the tumor cell.

On the other hand, inhibition of autophagy has been suggested as a tumor cell response to prolonged hypoxic conditions. Pancreatic cancer cell response to prolonged hypoxia may consist in inhibition of autophagic cell death. A member of the basic helix-loop-helix family of transcriptional regulators [67], the short isoform of single-minded 2 (SIM2s), is upregulated in pancreatic cancer. The procell death gene BNIP3 has been identified by microarray studies as a target of SIM2s repression. Prolonged hypoxia induces cell death via an autophagic pathway involving the HIF1alfa-mediated upregulation of BNIP3 [31, 68]. There is an association between the deregulation of both SIM2s and BNIP3 with poor prognostic outcomes [69]. Decreased BNIP3 levels and poor prognosis correlate with elevated SIM2s expression in pancreatic cancer. The loss of BNIP3, either by hypermethylation or by transcriptional repression, correlates with inhibition of cell death [70, 71]. On the contrary, upregulation of BNIP3 sensitizes pancreatic carcinoma cells to hypoxia-induced cell death [72]. SIM2s expression, concomitant with its repression of BNIP3, enhances tumor cell survival under prolonged hypoxic conditions. Recent data linked the increased SIM2s expression with enhanced cell survival during hypoxia-stress associated with BNIP3 repression and the attenuation of hypoxia-induced autophagic processes. Therefore, inhibition of autophagic cell death by BNIP3 repression enhances tumor cell survival under prolonged hypoxic conditions.

In some cancer cells a relation between a decreased autophagy and malignant stages of the disease has been found. Cancer cells present a general tendency to undergo less autophagy than their normal counterparts this supports the idea that defective autophagic cell death plays an important role in the tumor progression process. Pancreatic adenocarcinoma cells have lower autophagic capacity than premalignant cells. This has been proved by studies of carcinogen-induced pancreatic cancer in animal models [73]. The WIPI protein family, which includes ATG18, the WIPI-1 homolog in S. cerevisiae, was genetically identified as a gene contributing to autophagy [73]. Human WIPI-1a, a member of a highly conserved WD-repeats protein family, is linked to starvation-induced autophagic processes in the mammalian system. The deprivation of amino acids triggers an accumulation of endogenous hWIPI-1 protein. They are contained in large vesicular and cup-shaped structures where colocalize with LC3. The starvation-induced hWIPI-1 formation is blocked by wortmannin, a principal inhibitor of PI-3 kinase-induced autophagosome formation [74]. An interesting fact is that WIPI proteins are linked pathologically to cellular transformation. This is because all human WIPI genes are reported aberrantly expressed in a variety of matched human cancer samples. Strikingly, hWIPI-2 and hWIPI-4 mRNA expression is substantially decreased in 70% of matched kidney (10 patients) and 100% of pancreatic (seven patients) tumor samples. Most of these samples were derived from tumors in an advanced stage, such as pancreatic adenocarcinomas stages I–IV. Therefore, cancer-associated downregulation of hWIPI-2 and hWIPI-4 supports the possibility that decreased autophagic activity is necessary for the malignant stages of pancreatic cancer.

3. Warburg Effect and Cancer Cell Biology

Otto Warburg and colleagues performed studies measuring lactate production and oxygen consumption on liver rat carcinoma tissue and were able to propose that cancer cells display some very relevant differences when compared with normal tissues with regard to their glucose metabolism glycolysis is favored despite oxygen availability. The hypothesis of Warburg was that cancer growth is caused by the fact that tumor cells mainly generate energy (in the form of ATP) by nonoxidative breakdown of glucose. This view contrasts with the observation that normal cells produce ATP during oxidative phosphorylation obtaining “fuel” through the oxidative breakdown of glucose [75].

Glycolysis under anaerobic condition produces 2ATP per molecule of glucose. This yield of ATP is much lower than the production of ATP by means of a complete oxidation of glucose to CO2 under aerobic conditions (30 or 32 ATP per molecule of glucose) [76]. In other words, about a 15 times higher amount of glucose is consumed anaerobically when compared to the aerobic pathway to yield the same amount of ATP. As consequence, glucose uptake takes place about ten times faster in most solid tumors than in normal tissues [77]. Commonly, cancer cells depend on anaerobic glycolysis for their ATP production due to their exposure to a limited O2 supply (hypoxia).

The “Warburg effect” was the denomination given to this phenomenon of preferred aerobic glycolysis, which results in an increased lactate production even in presence of adequate pO2. It was suggested that this cellular behavior relies on mitochondrial dysfunction, characterized by respiratory impairment, resulting into a switch to glycolysis. It was also suggested that the high glycolytic rate might also result from a decreased mitochondrial mass in tumor cells [78].

This effect, first described in cancer tissues, was further identified in many other rapidly dividing normal cells [79]. Several mechanisms have been proposed to explain the Warburg effect in cancer tissues and they may be involved in transcriptional and posttranslational related metabolic changes.

A reduced expression of the tumor suppressor protein p53 in cancer cells might be linked to the Warburg effect. P53 is known to reduce the glycolysis rate by increasing the activity of a fructose-2,6-bisphosphatase. This mechanism is also involved in the regulatory pathways of apoptosis [80, 81]. In addition, this mechanism seems to increase the oxidative phosphorylation process. Other transcription regulators, such as the alpha estrogen-related receptor (of potential relevance in breast cancer) might be linked to the Warburg effect in the same way, an increased expression of oncogenes like MYC also seems to be associated with an increased glycolytic rate and might be involved in the pathophysiology of the metabolic modifications found in tumors [82]. Besides, glycolytic enzymes and glucose transmembrane transport are activated by MYC overexpression.

As mentioned before, the posttranslational regulation of the Warburg effect was also under scrutiny. As a relevant example, the activation of the PI3K/AKT downstream derives into an increased glucose influx and the phosphorylation of some enzymes like hexokinase and phosphofructokinase-2 with an upregulation of the glycolytic pathway [80]. Several posttranslational modifications of the M2 isoform pyruvate kinase result in a change in its activity, modulating the glycolytic pathway in several tissues. The K305 acetylation of this M2 isoform reduces its enzymatic activity and increases the enzyme degradation via chaperone-mediated autophagy [80]. The posttranscriptional modification of the M2 isoform of the pyruvate kinase has been shown to influence glycolysis at various models and experimental conditions, by oxidation, acetylation, phosphorylation, and so forth. A recent link was described among tumor overexpression of endogenous microRNA (miRNA), metabolic regulation of cancer cells, and the “Warburg effect” [80]. Even when attractive, the biological impact of this association remains to be clarified.

4. The Reverse Warburg Effect in Cancer: Pharmacological Modulation of Warburg and Reverse Warburg Effects

As stated before, it was thought that the Warburg effect only occurred in cancer cells. Recently, it has been shown that human skin keloid fibroblasts were able to generate energy (ATP) mainly from glycolysis this phenomenon was explained through the existence of similar hypoxic microenvironments in tumors and keloids [4, 5]. This observation led to suggest a new hypothesis where epithelial cancer cells are able to induce the Warburg effect in stromal fibroblasts. This process was termed “reverse Warburg effect” and it is based in studies performed in cocultures systems (e.g., stromal fibroblasts and human breast cancer cells) [4]. This hypothesis is consistent with the original view and is important to point out that in this situation the Warburg effect is not occurring in cancer cells but in the stroma. For a clearer understanding, the reverse Warburg effect can be explained as occurring in two steps.

As a first step, cancer-associated fibroblasts undergo myofibroblastic differentiation and secrete lactate and pyruvate through the glycolytic pathway. As stated before, this process is induced in cancer cells by a mechanism involving oxidative stress in association with loss of Caveolin-1, mitophagy, and/or mitochondrial dysfunction and increased production of NO [83].

Following these changes, epithelial cancer cells take up the energy-rich metabolites, which in turn enter in the tricarboxylic acid (TCA) pathway. This leads to production of ATP by oxidative phosphorylation. The mitochondrial mass of these cells expands to satisfy the increased metabolic demand. In addition, antioxidant enzymes are upregulated in order to cope with the oxidative stress generated and increase tumor aggressive behavior [84].

It is conceivable that different variants of similar types of cancer may differ with regard to their metabolic behavior. Breast cancer seems to be heterogeneous in its metabolic status, and therefore it can be classified into various metabolic phenotypes. “Warburg” and mixed variants had been identified, closely associated with the triple negative breast cancer phenotype, whereas the reverse Warburg and null types were frequently identified within the luminal type of breast tumors, suggesting a correlation between metabolic phenotype and the biology of breast cancer [85].

The Warburg effect might be modified and reversed by some pharmacological interventions. Even though several mechanisms for such actions were reported, in general, the clinical relevance of these findings is still on the way of being clarified.

One of the most studied agents in this area is a well-known antidiabetic agent, metformin. This drug has been proposed as a potential multifaceted agent for cancer prevention. Metformin acts as an indirect activator of AMPK and is able to reduce mitochondrial complex I activity. These have been proposed as mechanisms for reducing hepatic glucose output in patients with type 2 diabetes. Metformin treatment was associated with an increased cell death in P53-deficient cancer cells. In normal cells, there is an increase in glycolysis rates as an alternative ATP-producing mechanism that follows metformin treatment. In fact, one very rare but still possible adverse effect of metformin is lactic acidosis. It seems that P53-deficient cells experience problems in switching their metabolic pattern. This is followed by an enhanced cell death rate. Metformin diminishes ROS generation at mitochondria [86]. This is mainly achieved by reducing the activity of the respiratory chain complex I. Acknowledging this is important because the role of ROS in tumorigenesis and in cancer growth has been widely recognized. Metformin exhibits a mild to moderate antiangiogenic effect this is an effect that it shares with thrombospondin and endostatin. This effect on angiogenesis may be on the basis of its potential actions on cancer cells and/or its stroma [86].

In addition, as mentioned before, metformin activates the ATM/LKB1/AMPK axis. A very well-characterized tumor suppressor in the pathophysiology of melanoma and pancreatic and lung cancer, the tumor suppressor LKB1, might participate at the mechanism of action of metformin. It is thought that part of the preventive effects of metformin might be mediated by this suppressing factor. Metformin may inhibit the mTOR pathway by activating AMPK this effect has been proposed as an explanation for the potential antineoplastic effects of metformin in breast and renal tumors [87]. Metformin’s effects on the Warburg effect may be explained by many of the mentioned mechanisms. This drug has been suggested to reduce glycolysis and to increase mitochondrial respiration in tumors, and both effects have been associated with growth arrest [87]. It has been proposed that pyruvate kinase expression in fibroblast of tumoral stroma is linked to cancer growth. Cancer cells produce ROS that promote oxidative stress in fibroblasts. This results in the activation of HIF1 and NF-κB. NF-κB increases proinflammatory cytokines and HIF1 alpha promotes autophagy and anaerobic glycolysis. Pyruvate kinase activity results in an increase in ketones and lactate, and these nutrients are transferred to cancer cells where they are used for mitochondrial oxidative metabolism. As it has been said before, metformin reduces the mitochondrial chain activity by inhibiting complex I activity. In this manner, metformin may alter some of the mechanisms involved into the reverse Warburg effect [88]. It may also affect cell reprogramming by modifying the lipogenic enzymes acetyl-Co A carboxylase and fatty acid synthase [89]. These changes may also affect the metabolic behavior of both stroma and tumor cells. As mentioned before, the clinical impact of these modifications is still uncertain.

There are other drugs that exhibit potential for the modification of Warburg effect and authophagy rates. Mild autophagy induction by hypoxia or starvation seems to protect the cells, but rapamycin or sulforaphane leads to its elimination [90]. In contrast, an excessive autophagy rate may induce cell death. Elimination of highly aggressive pancreatic adenocarcinoma cells can be achieved by inhibition of autophagy by monensin or 3-methyladenine [90]. This is possible because these drugs may totally block continuous recycling of cellular components necessary for new synthesis and survival. This information suggests that both inhibitors and activators of autophagy may have utility in the treatment of patients with pancreatic ductal adenocarcinoma, since strong overactivation as well as strong inhibition of autophagy induces death in highly aggressive adenocarcinoma cells and sensitizes them to hypoxia-starvation [90]. Both autophagy activating (e.g., rapamycin—derivates sirolimus and temsirolimus or sulforaphane-a naturally occurring dietary substance enriched in broccoli) or inhibiting drugs (e.g., antibiotic monensin, antimalarial drug chloroquine) are available and generally tolerated well by patients.

Autophagy may be necessary for the maintenance of the tumor in advanced cancer. This is why multiple clinical trials are on their way to test this as a therapeutic approach in human patients using hydroxychloroquine (HCQ) [91, 92].

Autophagy can be affected in different manner and several ways by standard cancer chemotherapies. Gemcitabine monotherapy or its combination with other agents has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Gemcitabine is a relatively effective chemotherapeutic agent acting by competition with dCTP for the incorporation into DNA causing chain termination. On the other hand, gemcitabine serves as an inhibitory alternative substrate for ribonucleotide reductase and leads to a reduction of deoxynucleotide pools [93, 94]. This molecule inhibits cells that are insensitive to classic anticancer drugs, including other nucleoside analogs with similar structures. It has been recently suggested that gemcitabine also induces autophagy in pancreatic cancer cells [46] even though gemcitabine seems to exert its toxicity at least in part by activation of apoptosis [93]. It has been proposed that the early induction of autophagy with gemcitabine may be mediated by an increased expression of VMP1 [46]. Capecitabine, which is a pyrimidine analog, induces apoptosis in several cancer lines and shows a modest efficacy in locally advanced pancreatic ductal adenocarcinoma when associated with limited field radiotherapy [95]. It has been proposed that capecitabine modulates autophagy by displaying a Src kinase modulatory effect [96], but the results on this area are still contradictory. Irinotecan is a topoisomerase I inhibitor which prevents DNA from unwinding. In a phase III trial, the combination of 5-fluouracil, leucovorin, oxaliplatin, and irinotecan resulted in better responses, progression-free survival, and overall survival when compared with the standard single drug therapy with gemcitabine for metastatic pancreatic ductal adenocarcinoma [97]. In small-cell prostatic carcinoma, irinotecan promoted an increase in autophagy of treated tumors as indicated by an increase in LC3B expression [98, 99]. Nevertheless, authors of this research state that the role of autophagy is complex. This can be said because there is evidence that autophagy supports both promotion and suppression of cancer growth. In general, as mentioned before, a considerable amount of caution should be exercised for the interpretation of the consequences of cancer chemotherapy on autophagy. Other chemotherapeutic agents like the glycoside oleandrin, some platinum compounds, the multikinase inhibitor sorafenib, and some histone-deacetylase inhibitors have demonstrated effects on the autophagy rate in pancreatic carcinoma cell lines [98, 99]. As proposed, autophagy may be involved in carcinogenesis, tumor progression, and dissemination and may be associated at least in part with the actions of some chemotherapy for pancreatic ductal adenocarcinoma as well. All these modifications may alter Warburg and reverse Warburg effects, but it is important to remember that the real contribution of these metabolic changes to tumor cell survival and clinical prognosis remains unclear.

5. Conclusions

Autophagy regulation involves a set of key processes needed for a normal cell survival and turnover. The association between abnormal or defective autophagy and cancer development has been strongly suggested by several authors. This association is currently under an intense scrutiny aimed to contribute to a better understanding of the tumor cell biology. Figure 1 summarizes the link between abnormalities in autophagy and the Warburg and the reverse Warburg effects, critical to understand several tumor adaptive behaviors. A better knowledge of these metabolic interactions may be of importance in the development of new therapeutic agents in oncology, as well as for the development of more efficient preventive strategies for some cancer phenotypes.

The Clinical Biochemistry of Neoplasia


Glucose intolerance in association with malignancy was first described in 1885. 3 Endocrine tumours secreting ectopic or eutopic hormones normally associated with blood glucose elevation (Cortisol by pituitary adenomas and adrenocortical carcinoma growth hormone by pituitary adenomas and lung carcinoma glucagon by pancreatic tumours catecholamines by phaeochromocytoma and neuroblastoma) may be expected to manifest themselves with hyperglycaemia, but these are rare. The presence of glucose intolerance, defined by the oral glucose tolerance test, has a prevalence of 36.7% in patients with all malignant tumours as compared to 9.3% in benign tumours. 4 The mechanism for this effect is not absolutely clear. There is an increased rate of endogenous glucose production and turnover in cancer that is apparently influenced by tumour stage, type and is associated with cachexia. There are four possible mechanisms involved.

The Warburg effect and the Cori cycle. Otto Warburg in 1930 noted an excess lactate production by tumour cells due to anaerobic glycolysis in spite of an apparently adequate oxygen availability. 5 Excess lactate production by tumour is taken up by the liver and used to produce glucose, which is then passed back into the circulation and may be reused for glycolysis— the Cori cycle 6 ( Fig. 9.1 ). This is an energy expending or ‘futile’ cycle and its flux is increased in both disseminated and localized tumours. 7, 8

Figure 9.1 . The Cori cycle: lactate and glucose cycling between neoplastic tissue ana the liver results in a net loss of four molecules of ATP per molecule of glucose cycled and is therefore an energy-consuming process.

Insulin resistance. Marks and Bishop in 1956 9 noted that the fall in glucose in response to intravenous insulin was significantly smaller in patients with carcinoma, leukaemia and lymphoma. These findings have been confirmed and there is no apparent defect in insulin receptors, but it is not clear whether this a specific effect of cancer rather than the effect of associated weight loss or sepsis.

Impaired insulin secretion. There is a decreased insulin response to glucagon and the oral glucose tolerance test in cancer but again this is only seen inassociation with weight loss malnutrition per se is also associated with insulin resistance.

Increased gluconeogenesis. The gluconeogenic hormones, insulin, Cortisol and growth hormone show consistent increases in cancer patients but there is evidence that gluconeogenesis in cancer is substrate-led, presumably from lactate (due to the Warburg effect) and alanine rather than glycerol. 10–12

On balance the excessive Cori cycle activity combined with a defect in insulin response or action would seem to be the most likely explanation for glucose intolerance in cancer. The contribution of factors such as weight loss, sepsis and bedrest associated with cancer is difficult to assess but when present such factors probably contribute significantly.

Glucose intolerance secondary to destruction ofβ-cells in the pancreas by primary or secondary tumours occurs, but this also appears to be unusual in spite of a well-recognized association between carcinoma of the pancreas and glucose intolerance or diabetes (an incidence of 81% in one series). 13

Treatment of malignant disease by high doses of steroids and oestrogens, asparaginase and cyclophosphamide, and pancreatic resection can also induce glucose intolerance and diabetes.

UChicago scientists unveil the secret of cancer-associated Warburg effect

A macrophage (in white) engulfs a cancer cell. When these immune cells respond to infection or tumors, they produce a substance call lactate, which can actually fuel cancer growth.

A new study, led by researchers at the University of Chicago, provides an answer to why cancer cells consume and use nutrients differently than their healthy counterparts and how that difference contributes to their survival and growth.

All cells need to generate energy to keep living, but cancer cells have an increased demand for energy in order to grow and multiply quickly. Understanding how different types of cells fuel themselves, or metabolize, is an attractive area of study because new drugs could be developed to interrupt and exploit the process. Metabolism also plays a role in the responsiveness of immune cells that protect against harmful pathogens, such as viruses, bacteria and the body&rsquos own cells that have changed, such as cancerous cells. Until recently, the intricacies surrounding how cellular metabolism affects the cell&rsquos function have eluded biologists for decades.

The study, published in the October 23 issue of Nature, shows that lactate, an end product of metabolism, changes the function of an immune cell known as a macrophage, thereby rewiring it to behave differently.

Almost 90 years ago, German physiologist and physician Otto Warburg first posed the question about why some cells consume nutrients differently. He knew that normal cells use oxygen to turn food into energy through a process called oxidative phosphorylation. But when he observed cancer cells, he saw that they preferred to fuel their growth through glycolysis, a process that involves consuming and breaking down glucose for energy. The phenomenon was coined &ldquothe Warburg effect."

Lactate, the end-product of the Warburg effect, has long been considered a metabolic waste product. More recent studies showed that lactate can regulate the functions of many cell types, such as immune cells and stem cells. Thus, lactate is not simply a waste product, but may be a key regulator of cell functions in Warburg-associated diseases. Despite this progress, the mechanisms by which lactate controls cellular functions remains unknown, representing a fundamental and long-standing question in the field. And, because the Warburg effect occurs in virtually all cancers, unraveling its mechanisms presents a rare opportunity to develop new targeted therapies that could have broad implications for many types of cancer.

&ldquoWhat makes the Warburg effect so interesting to study is that it&rsquos an important and common cancer phenomenon, but no one ever understood if this process has regulatory functions on diverse types of cells in a tumor, and how,&rdquo said Yingming Zhao, PhD, professor in the Ben May Department for Cancer Research at the University of Chicago and the lead author of the study. &ldquoAs a technologist and biochemist, I enjoy figuring out how we can answer exciting questions like this and figure out details.&rdquo

Zhao and Lev Becker, PhD, an associate professor at UChicago, used a laboratory technique called mass spectrometry to analyze the mechanisms driving the Warburg effect. They noticed that lactate, a compound generated during this process, also plays a non-metabolic role. Lactate is the source and stimulator of a new type of histone modification, which they termed histone lactylation.

Histones are a group of proteins found in eukaryotic cell nuclei that organize DNA into structural units and control which genes are expressed. In turn, those particular genes determine cell type and function. The researchers demonstrated that histone lactylation alters these structural units to change the combination of genes expressed and functions of macrophages, white blood cells that play an important role in infections and cancer.

Lactate production by macrophages is triggered by bacterial infection or by lack of adequate oxygen supply (hypoxia) in tumors, both of which stimulate glycolysis. Using bacterially-exposed macrophages as a model system, the researchers found that histone lactylation alters the cells from a pro-inflammatory and anti-bacterial state (known as M1) to an anti-inflammatory and reparative state (known as M2).

In response to bacterial infection, macrophages must react rapidly with a substantial pro-inflammatory burst to help kill bacteria and recruit additional immune cells to the infection site. During this process, macrophages switch to aerobic glycolysis, which is thought to support generation of pro-inflammatory immune substances called cytokines. However, the researchers show that over time, this metabolic switch also increases lactate, which stimulates histone lactylation to express stabilizing genes that may repair collateral damage to the host incurred during infection.

Although this reparative M2 macrophage phenotype may help control damage during infection, its presence in tumors is known to promote growth, metastasis and immune suppression in cancer. Interestingly, the researchers also detected histone lactylation in macrophages isolated from mouse melanoma and lung tumors, and observed positive correlations between histone lactylation and cancer-promoting genes made by reparative M2 macrophages. These findings suggest that high lactate and histone lactylation levels in macrophages may contribute to the formation of tumors and their progression.

&ldquoThat a single metabolite can have such a powerful effect on immune cell function is both remarkable and surprising,&rdquo Becker said. &ldquoOur discovery of histone lactylation and its impact on macrophage biology serves as a blueprint to understand how lactate alters other cell types and unravel the mysteries of the Warburg effect and its impact on human disease.&rdquo

The authors said studying these effects on macrophages is just the beginning. They speculate that cancer cells and other immunological cells, such as T cells, could be regulated by this mechanism. In addition to cancer, the Warburg effect is also observed in other diseases, including sepsis, autoimmune diseases, atherosclerosis, diabetes and aging. More research is needed on the role and regulation of this new histone modification, but the discovery draws an exciting link between cellular metabolism and gene regulation that was previously unknown and could have promising implications for human health.

The study, &ldquoMetabolic Regulation of Gene Expression by Histone Lactylation,&rdquo was published in Nature and supported by the University of Chicago, Nancy and Leonard Florsheim Family Fund and the National Institutes of Health.

Additional authors include Di Zhang, Guolin Zhou, Chang Cui, Yejing Weng, Wenchao Liu, Mathew Perez-Neut, Jun Ding, Daniel Czyz and Howard A. Shuman from the University of Chicago Zhanyun Tang and Robert G. Roeder from the Rockefeller University He Huang from the University of Chicago, Chinese Academy of Sciences Sunjoo Kim and Sangkyu Lee from Kyungpook National University Rong Hu, Zhen Ye and Bing Ren from the University of California, San Diego School of Medicine Maomao He and Y. George Zheng from the University of Georgia and Lunzhi Dai from the University of Chicago, Sichuan University and Collaborative Innovation Center of Biotherapy.

The Warburg Effect and cancer

The Warburg Effect refers to the fact that cancer cells, somewhat counter intuitively, prefers fermentation as a source of energy rather than the more efficient mitochondrial pathway of oxidative phosphorylation (OxPhos). We discussed this in our previous post.

In normal tissues, cells may either use OxPhos which generates 36 ATP or anaerobic glycolysis which gives you 2 ATP. Anaerobic means ‘without oxygen’ and glycolysis means ‘burning of glucose’. For the same 1 glucose molecule, you can get 18 times more energy using oxygen in the mitochondrion compared to anaerobic glycolysis. Normal tissues only use this less efficient pathway in the absence of oxygen – eg. muscles during sprinting. This creates lactic acid which causes the ‘muscle burn’.

However, cancer is different. Even in the presence of oxygen (hence aerobic as opposed to anaerobic), it uses a less efficient method of energy generation (glycolysis, not phosphorylation). This is found in virtually all tumors, but why? Since oxygen is plentiful, it seems inefficient, because it could get way more ATP using OxPhos. But it can’t be that stupid, because it happens in virtually every single cancer cell in history. This is such as striking finding that it has become one of the emerging ‘Hallmarks of Cancer’ as detailed previously. But why? When something seems counterintuitive, but happens anyways, it’s usually that we simply do not understand. So we need to try to understand it rather than dismissing it as a freak of nature.

For single-celled organisms like bacteria, there is evolutionary pressure to reproduce and grow as long as nutrients are available. Think of a yeast cell on a piece of bread. Grows like crazy. Yeast on a dry surface like a countertop stays dormant. There are two very important determinants of growth. You need not only the energy to grow, but also the raw building blocks. Think of a brink house. You need construction workers, but also bricks. Similarly, cells need the basic building blocks (nutrients) to grow.

For multi-celled organisms, there is generally plenty of nutrients floating around. The liver cell, for example, finds lots of nutrients all over the place. The liver does not grow because it only takes up these nutrients when stimulated by growth factors. In our house analogy, there are plenty of bricks, but the foreman has told the construction workers not to build. So nothing is built.

One theory is that perhaps the cancer cell is using the Warburg Effect to not just generate energy, but also the substrate needed to grow. For a cancer cell to divide, it needs lots of cellular components, which requires building blocks like Acetyl-Co-A, which can be made into other tissues like amino acids and lipids.

For example, palmitate, a major constituent of the cell wall requires 7 ATP of energy, but also 16 carbons that can come from 8 Acetyl-CoA. OxPhos provides lots of ATP, but not much Acetyl-CoA because it is all burned to energy. So, if you burn all the glucose to energy, there are no building blocks with which to build new cells. For the palmitate, 1 glucose molecule will provide 5 times the energy needed, but will need 7 glucose to generate the building blocks. So, for a proliferating cancer cell, generating pure energy is not great for growth. Instead, aerobic glycolysis, which produces both energy and substrate will maximize the rates of growth and proliferate the fastest.

This may be important in an isolated environment, but cancer does not arise in a petri dish. Instead nutrients are rarely a limiting factor in the human body – there is plenty of glucose and amino acids everywhere. There’s lots of available energy and building blocks so there is no selective pressure to maximize ATP yield. Cancer cells perhaps use some glucose for energy and some for biomass to support expansion. In an isolated system, it may make sense to use some resources for bricks and some for construction workers. However, the body is not such a system. The burgeoning breast cancer cell, for example, with access to the blood stream, which has both glucose for energy and amino acids and fat for building cells.

It also does not make any sense of the link with obesity, where there are plenty of building blocks around. In this situation, cancer should maximize glucose for energy, since it can easily obtain building blocks. Thus, it is debatable whether this explanation of the Warburg Effect plays any role in cancer’s origin.

There is an interesting corollary, however. What if nutrient stores were significantly depleted? That is, if we are able to activate our nutrient sensors to signal ‘low energy’ then the cell would face selective pressure to maximize energy production (ATP) moving away from cancer’s preferred aerobic glycolysis. If we lower insulin and mTOR, while increasing AMPK. There is a simple dietary manipulation that does this – fasting. Ketogenic diets, while lowering insulin, will still activate the other nutrient sensors mTOR and AMPK.


Another misconception of the Warburg Effect is that cancer cells can only use glucose. This is not true. There are two main molecules that can be catabolized by mammalian cell – glucose, but also the protein glutamine. Glucose metabolism is deranged in cancer, but so is glutamine metabolism. Glutamine is the most common amino acid in the blood and many cancers seem to be ‘addicted’ to glutamine for survival and profileration. The effect is most easily seen in the Positron Emission Tomography (PET) scan. PET scans are a form of imaging used heavily in oncology. A tracer is injected into the body. The classic PET scan used fluorine-18 fluorodeoxyglucose (FDG) which is a variant of regular glucose which is tagged with a radioactive tracer so it can be detected by the PET scanner.

Most cells take up glucose at a relatively low basal rate. However, cancer cells drink up the glucose like a camel drinks up water after a desert trek. These tagged glucose cells accumulate in the cancerous tissue and can be seen as active sites of cancer growth.

In this example of lung cancer, there is a large area in the lung that is drinking up the glucose like crazy. This demonstrates that cancer cells are far, far more glucose avid than regular tissues. However, there is another way to do the PET scan, and that is to use the radioactively tagged amino acid glutamine. What this demonstrates is that some cancer are just as avid for glutamine. Indeed, some cancers cannot survive without glutamine and seem ‘addicted’ to it.

Where Warburg made his seminal observations about cancer cells and perverted glucose metabolism in the 1930s, it was not until 1955 that Harry Eagle noted that some cells in culture consumed glutamine by over 10 times that of other amino acids. Later studies in the 1970’s showed that this was true for many cancer cell lines also. Further studies showed that the glutamine was being converted to lactate, which seems rather wasteful. Instead of burning it as energy, the glutamine was being changed to lactate, seemingly a waste product. This was the same ‘wasteful’ process seen in the glucose. Cancer was changing glucose to lactate and not getting the full energy bonanza from each molecule. Glucose provides the mitochondria with a source of acetyl-CoA and glutamine provides a pool of oxaloacetate (see diagram). This supplies the carbon needed to maintain citrate production in the first step of the TCA cycle.

Certain cancers seem to have exquisite sensitivity to glutamine starvation. In vitro, pancreatic cancer, glioblastoma multiform, acute myelogenous leukaemia for example often die off in the absence of glutamine. The simplistic notion that a ketogenic diet may ‘starve’ the cancer of glucose does not hold up to the facts. Indeed, in certain cancers, glutamine is the more important component.

What’s so special about glutamine? One of the important observations is that mTOR complex 1, mTORC1 a master regulator of protein production is responsive to glutamine levels. In the presence of sufficient amino acids, growth factor signaling occurs through the insulin-like growth factor (IGF)-PI3K-Akt pathway.

This PI3K signalling pathway is critical for both growth control and glucose metabolism, underscoring once again the close relationship between growth and nutrient/ energy availability. Cells do not want to grow unless nutrients are available.

We see this in the study of oncogenes, most of which control for enzymes called tyrosine kinases. One common feature of tyrosine kinase signaling associated with cell proliferation is regulation of glucose metabolism. This does not happen in normal cells that are not proliferating. The common MYC oncogene is particularly sensitive to glutamine withdrawal.

So, here’s what we know. Cancer cells:

  1. Switch over from the more efficient energy generating OxPhos to a less efficient process, even though oxygen is freely available.
  2. Need glucose, but also need glutamine.

But the million-dollar question still remains. Why? It is too universal to be just a fluke. It’s also not simply a dietary disease, since many things, including viruses, ionizing radiation and chemical carcinogens (smoking, asbestos) cause cancer. If it is not simply a dietary disease, then a purely dietary solution does not exist. The hypothesis that makes the most sense to me is this. The cancer cell does not use the more efficient pathway, because it can’t.

If the mitochondrion are damaged or senescent (old), then cells will naturally look for other pathways. This drives cells to adopt a phylogenetically ancient pathway of aerobic glycolysis in order to survive. Now, we come to the atavistic theories of cancer.


This work has been supported in part by the China National 973 project (2013CB911303), China National 863 project (2007AA02Z143), China Natural Sciences Foundation projects (81272456) and the Fundamental Research Funds for the Central Universities, National Ministry of Education, China, to X Hu. We thank Prof. M.X. Guan, Zhejiang University College of Life Sciences, for allowing us to use the Seahorse XF96 extracellular analyzer for metabolic flux measurements. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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