Archive for the ‘Hexokinase’ Category

Pancreatic Cancer Targeted Treatment?

Curator: Larry H. Bernstein, MD, FCAP



MGH study identifies potential treatment target for pancreatic cancer

Molecular signature found in 30 percent of PDAC tumors, associated with more aggressive cancer


Massachusetts General Hospital (MGH) investigators have identified the first potential molecular treatment target for the most common form of pancreatic cancer, which kills more than 90 percent of patients. Along with finding that the tumor suppressor protein SIRT6 is inactive in around 30 percent of cases of pancreatic ductal adenocarcinoma (PDAC), the team identified the precise pathway by which SIRT6 suppresses PDAC development, a mechanism different from the way it suppresses colorectal cancer. The paper will appear in the June 2 issue of Cell and have been published online.

“With the advance of cancer genomics, it has become evident that alterations in epigenetic factors – those that control whether and when other genes are expressed – represent some of the most frequent alterations in cancer,” says Raul Mostoslavsky, MD, PhD, of the MGH Cancer Center, senior author of the report.  “Yet, not many of those factors have been described before, and those that have been identified have not been linked to specific downstream targets.  Not only did more than a third of analyzed PDAC patient samples exhibit the molecular signature we identified, those patients also turned out to have very poor prognoses.”

Among its other functions, SIRT6 is known to control how cells process glucose, and a 2012 study by Mostoslavsky’s team found that its ability to suppress colorectal cancer involves control of a process called glycolysis.  But while that study also found reduced SIRT6 expression in PDAC tumor cells, the current investigation indicated that SIRT6 deficiency promotes PDAC through a different mechanism. Experiments in cell lines and animal models revealed that low SIRT6 levels in PDAC were correlated with increased expression of Lin28b, an oncoprotein normally expressed during fetal development.

Lin28b expression proved to be essential to the growth and survival of SIRT6-deficient PDAC cells and acted by preventing a family of tumor-suppressing mRNAs called let-7 from blocking expression of three genes previously associated with increased aggressiveness and metastasis in pancreatic cancers.  All of these hallmarks – reduced SIRT6, increased Lin28b and reduced let-7 expression – were found in tumor samples from patients who died more quickly.

“A general message from these studies is that cancer cells benefit from modulating epigenetic factors like SIRT6 by acquiring the ability to override normal cellular growth control patterns,” says Mostoslavsky, an associate professor of Medicine at Harvard Medical School and an associate member at the Broad Institute.  “Each tumor type may acquire a unique set of capabilities that may provide tumor-specific growth and survival advantages, which may need to be determined for each kind of cancer.  In terms of our findings regarding PDAC, we are intrigued by the downstream pathways controlled by Lin28b and how they increase aggressiveness and metastasis, and we are hopeful that developing in the future Lin28b inhibitors could benefit this subset of PDAC patients, who currently have very few treatment options.”


SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b

Sita Kugel, Carlos Sebastián, Julien Fitamant,…., Alon Goren, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky

Figure thumbnail fx1
  • Loss of SIRT6 cooperates with oncogenic Kras to drive pancreatic cancer
  • SIRT6 regulates the oncofetal protein Lin28b through promoter histone deacetylation
  • Lin28b drives the growth and survival of SIRT6-deficient pancreatic cancer
  • SIRT6 and Lin28b expression define prognosis in specific pancreatic cancer subsets

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at theLin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%–40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset.


The multifaceted functions of sirtuins in cancer

Angeliki Chalkiadaki & Leonard Guarente  Affiliations  Corresponding author
Nature Reviews Cancer (2015); 15:608–624

The sirtuins (SIRTs; of which there are seven in mammals) are NAD+-dependent enzymes that regulate a large number of cellular pathways and forestall the progression of ageing and age-associated diseases. In recent years, the role of sirtuins in cancer biology has become increasingly apparent, and growing evidence demonstrates that sirtuins regulate many processes that go awry in cancer cells, such as cellular metabolism, the regulation of chromatin structure and the maintenance of genomic stability. In this article, we review recent advances in our understanding of how sirtuins affect cancer metabolism, DNA repair and the tumour microenvironment and how activating or inhibiting sirtuins may be important in preventing or treating cancer.


Figure 1: Overview of the role of sirtuins in the regulation of cancer metabolism

The inhibitory effects of sirtuin 3 (SIRT3), SIRT4 and SIRT6 on metabolic pathways that drive cancer cells are depicted. In normal cells, SIRT6 functions as a co-repressor for the transcription factors hypoxia-inducible factor 1α (HIF1α…


Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion

Ping Zhanga,1, Bo Tua,1, Hua Wangb , Ziyang Caoa , Ming Tanga , … , Bin Gaob , Robert G. Roederd,2, and Wei-Guo Zhua,e,2
PNAS | July 22, 2014;111(29): 10684–10689 | 1073/pnas.1411026111/-/DCSupplemental.

In mammalian cells, tumor suppressor p53 plays critical roles in the regulation of glucose metabolism, including glycolysis and oxidative phosphorylation, but whether and how p53 also regulates gluconeogenesis is less clear. Here, we report that p53 efficiently down-regulates the expression of phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), which encode rate-limiting enzymes in gluconeogenesis. Cell-based assays demonstrate the p53-dependent nuclear exclusion of forkhead box protein O1 (FoxO1), a key transcription factor that mediates activation of PCK1 and G6PC, with consequent alleviation of FoxO1- dependent gluconeogenesis. Further mechanistic studies show that p53 directly activates expression of the NAD+-dependent histone deacetylase sirtuin 6 (SIRT6), whose interaction with FoxO1 leads to FoxO1 deacetylation and export to the cytoplasm. In support of these observations, p53-mediated FoxO1 nuclear exclusion, down-regulation of PCK1 and G6PC expression, and regulation of glucose levels were confirmed in C57BL/J6 mice and in liver-specific Sirt6 conditional knockout mice. Our results provide insights into mechanisms of metabolism-related p53 functions that may be relevant to tumor suppression.

As the “guardian of the genome,” tumor suppressor p53 has been reported to coordinate diverse cellular responses to a broad range of environment stresses (1) and to play antineoplastic roles by activating downstream target genes involved in DNA damage repair, apoptosis, and cell-cycle arrest (2). Recent studies have indicated broader roles for p53 in mediating metabolic changes in cells under various physiological and pathological conditions (3–7). For example, p53 was reported to influence the balance between glycolysis and oxidative phosphorylation by inducing the p53-induced glycolysis and apoptosis regulator (TIGAR) and by regulating the synthesis of cytochrome c oxidase 2 (SCO2) (3), respectively, thus promoting the switch from glycolysis to oxidative phosphorylation. p53 also may impede metabolism by reducing glucose import (4) or by inhibiting the pentose phosphate pathway (PPP) (5). More recently, context-dependent inhibitory (6) or stimulatory (7, 8) effects of p53 on gluconeogenesis have been reported. It thus is clear that p53 plays important roles in glucose regulation in mammalian cells. Glucose homeostasis is maintained by a delicate balance between intestinal absorption of sugar, gluconeogenesis, and the utilization of glucose by the peripheral tissues, irrespective of feeding or fasting (9). The gluconeogenesis pathway is catalyzed by several key enzymes that include the first and last rate-limiting enzymes of the process, phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), respectively. The expression of both PCK1 and G6PC is controlled mainly at the transcription level. For example, the transcription factor forkhead box protein O1 (FoxO1) activates gluconeogenesis through direct binding to the promoters of G6PC and PCK1 (10). A dominant negative FoxO1 lacking its transactivation domain significantly decreases gluconeogenesis (11) whereas FoxO1 ablation impairs fasting- and cAMP-induced PCK1 and G6PC expression (12). Therefore, factors influencing expression of FoxO1 or its binding activity to the PCK1 and G6PC promoters are potential targets for gluconeogenesis regulation. The transcription activity of FoxO family members is regulated by a sophisticated signaling network. Various environmental stimuli cause different posttranslational modifications of FoxO proteins, including phosphorylation, acetylation, ubiquitination, and methylation (13–15). The phosphorylation of FoxO proteins is known to be essential for their shuttling between the nucleus and cytoplasm. For example, kinase Akt/PKB phosphorylates FoxO1 at threonine 24 and at serines 256 and 319, which in turn leads to 14-3-3 binding and subsequent cytoplasmic sequestration. The acetylation of FoxO proteins also affects their trafficking and DNA-binding activities (15–17). Sirtuin (SIRT)1, a homolog of the yeast silent information regulator-2 (Sir2), has been identified as a deacetylase for FoxO proteins (15, 17, 18). Of the seven mammalian sirtuins, SIRT1, SIRT6, and SIRT7 are localized to the nucleus (19), and SIRT6 was recently reported to act as a central player in regulating the DNA damage response, glucose metabolism, and aging (20–26). Using a knockout mouse model, it was found that SIRT6 functions as a corepressor of the transcription factor Hif1α to suppress glucose uptake and glycolysis.

Significance: Beyond its canonical functions in processes such as cell-cycle arrest, apoptosis, and senescence, the tumor suppressor p53 has been increasingly implicated in metabolism. Here, in vitro and in vivo studies establish a role for p53 in gluconeogenesis through a previously unidentified mechanism involving (i) direct activation of the gene encoding the NAD-dependent deacetylase sirtuin 6 (SIRT6), (ii) SIRT6-dependent deacetylation and nuclear exclusion of forkhead box protein O1 (FoxO1), and (iii) downregulation of FoxO1-activated genes (G6PC and PCK1) that are rate-limiting for gluconeogenesis. These results have implications for proposed tumor-suppressor functions of p53 through regulation of metabolic pathways.

Among a variety of other functions, SIRT6 was previously connected to glucose metabolism. For example, SIRT6 acts as a corepressor of the transcription factor Hif1-α to suppress glycolysis (23). Conversely, the deletion of Sirt6 in mice results in severe hypoglycemia (33) whereas the liver-specific deletion of Sirt6 leads to increased glycolysis and triglyceride synthesis (23, 34). Our study adds further evidence that SIRT6 plays an important role in glucose metabolism by connecting p53 transcription activity and gluconeogenesis. Our data also reemphasize a previously established role for SIRT6 in regulating the acetylation state and nuclear localization of FoxO proteins, albeit in a divergent manner. Thus, the Caenorhabditis elegans SIRT6/7 homolog SIR-2.4 was implicated in DAF-16 deacetylation and consequent nuclear localization and function in stress responses (35); and the effect was reported to be indirect and to involve a stress-induced inhibition by SIR-2.4 of CBP-mediated acetylation of DAF-16 that is independent of its deacetylase activity. These results, emphasizing context-dependent SIRT6 mechanisms, contrast with the SIRT6 deacetylase activity requirement for FoxO1 nuclear exclusion in the present study and a likely direct effect of SIRT6 on FoxO1 deacetylation based on their direct interaction, the SIRT6 deacetylase activity requirement, and precedent (15, 17, 18) from direct SIRT1-mediated deacetylation of FoxO proteins.

Despite a high genetic diversity, cancer cells exhibit a common set of functional characteristics, one being the “Warburg effect”: i.e., continuous high glucose uptake and a higher rate of glycolysis than that in normal cells (36). To favor the rapid proliferation requirement for high ATP/ADP and ATP/AMP ratios, cancer cells use large amounts of glucose. p53, as one of the most important tumor suppressors, exerts its antineoplastic function through diverse pathways that include the regulation of glucose metabolism. Thus, p53 regulates glucose metabolism by activation of TIGAR (3), which lowers the intracellular concentrations of fructose-2,6-bisphosphate and decreases glycolysis. On the other hand, p53 activation causes down-regulation of several glycolysisrelated factors such as phosphoglycerate mutase (PGM) (37) and the glucose transporters (4). Expression of p53 also can limit the activity of IκBα and IκBβ, thereby restricting the activation of NFκB and dampening the expression of glycolysis-promoting genes such as GLUT3 (38). As a reverse glycolysis pathway, gluconeogenesis generates glucose from noncarbohydrate precursors and is conceivably essential for tumor cell growth. However, the current study further supports the notion (6) that p53 is also involved in a gluconeogenesis inhibition pathway, which in this case is executed by enhanced SIRT6 expression and subsequent FoxO1 nuclear exclusion. These results raise the interesting possibility that an inhibition of gluconeogenesis may contribute to the tumorsuppressive function of p53.
Keywords : Oncogenes, Tumor suppressors, Glutamine metabolism, Cancer cells … p53 is a well-known protein which is involved in many cellular functionsincluding cell … deprivation activates p53 by regulating protein phosphatase 2A ( PP2A). …. and tumor suppressors may affect glutamine metabolism in cancercells


Protein controlling glucose metabolism also a tumor suppressor

Finding supports metabolic strategies to control tumor growth    December 6, 2012

A protein known to regulate how cells process glucose also appears to be a tumor suppressor, adding to the potential that therapies directed at cellular metabolism may help suppress tumor growth.  In their report in the Dec. 7 issue of Cell, a multi-institutional research team describes finding that cells lacking the enzyme SIRT6, which controls how cells process glucose, quickly become cancerous.  They also found evidence that uncontrolled glycolysis, a stage in normal glucose metabolism, may drive tumor formation in the absence of SIRT6 and that suppressing glycolysis can halt tumor formation.

“Our study provides solid evidence that SIRT6 may function as a tumor suppressor by regulating glycolytic metabolism in cancer cells,” says Raul Mostoslavsky, MD, PhD, of the Massachusetts General Hospital (MGH) Cancer Center, senior author of the report.  “Critically, our findings indicate that, in tumors driven by low SIRT6 levels, drugs that may inhibit glycolysis – currently a hot research topic among biotechnology companies – could have therapeutic benefits.”

The hypothesis that a switch in the way cells process glucose could set off tumor formation was first proposed in the 1920s by German researcher Otto Warburg, who later received the Nobel Prize for discoveries in cellular respiration.  He observed that, while glucose metabolism is normally a two-step process involving glycolysis in the cellular cytoplasm followed by cellular respiration in the mitochondria, in cancer cells rates of glycolysis are up to 200 times higher.  Warburg’s proposition that this switch in glucose processing was a primary cause of cancer did not hold up, as subsequent research supported the role of mutations in oncogenes, which can spur tumor growth if overexpressed, and tumor suppressors, which keep cell proliferation under control.  But recent studies have suggested that alterations in cellular metabolism may be part of the process through which activated oncogenes or inactivated tumor suppressors stimulate cancer formation.

A 2010 study led by Mostoslavsky found that the absence of SIRT6 – one of a family of proteins called sirtuins that regulate many important biological pathways – appears to “flip the switch” from normal glucose processing to the excess rates of glycolysis seen in cancer cells. The current study was specifically designed to investigate whether SIRT6’s control of glucose metabolism also suppresses tumor formation.  The research team first showed that cultured skin cells from embryonic mice lacking SIRT6 proliferated rapidly and quickly formed tumors when injected into adult mice.  They also confirmed elevated glycolysis levels in both cells lacking SIRT6 and tumor cells and found that formation of tumors through SIRT6 deficiency did not appear to involve oncogene activation.

Analysis of tumor samples from patients found reduced SIRT6 expression in many – particularly in colorectal and pancreatic tumors.  Even among patients whose tumors appeared to be more aggressive, higher levels of SIRT6 expression may have delayed or, for some, prevented relapse.   In a mouse model programmed to develop numerous colon polyps, the researchers showed that lack of intestinal SIRT6 expression tripled the formation of polyps, many of which became invasive tumors.  Treating the animals with a glycolytic inhibitor significantly reduced tumor formation, even in the absence of SIRT6.

“Our results indicate that, at least in certain cancers, inhibiting glycolytic metabolism could provide a strong alternative way to halt cancer growth, possibly acting synergistically with current anti-tumor therapies,” says Mostoslavsky, an assistant professor of Medicine at Harvard Medical School.  “Cancer metabolism has only recently emerged as a hallmark of tumorigenesis, and the field is rapidly expanding.  With the current pace of research and the speed at which some basic discoveries are moving into translational studies, it is likely that drugs targeting cancer metabolism may be available to patients in the near future.”



Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivoincreases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

Cancer cells are characterized by the acquisition of several characteristics that enable them to become tumorigenic (Hanahan and Weinberg, 2000). Among them, the ability to sustain uncontrolled proliferation represents the most fundamental trait of cancer cells. This hyperproliferative state involves the deregulation of proliferative signaling pathways as well as loss of cell cycle regulation. In addition, tumor cells need to readjust their energy metabolism to fuel cell growth and division. This metabolic adaptation is directly regulated by many oncogenes and tumor suppressors, and is required to support the energetic and anabolic demands associated with cell growth and proliferation (Lunt and Vander Heiden, 2011).

Alteration in glucose metabolism is the best-known example of metabolic reprogramming in cancer cells. Under aerobic conditions, normal cells convert glucose to pyruvate through glycolysis, which enters the mitochondria to be further catabolized in the tricarboxylic acid cycle (TCA) to generate adenosine-5’-triphosphate (ATP). Under anaerobic conditions, mitochondrial respiration is abated; glucose metabolism is shifted towards glycolytic conversion of pyruvate into lactate. This metabolic reprogramming is also observed in cancer cells even in the presence of oxygen and was first described by Otto Warburg several decades ago (Warburg, 1956; Warburg et al., 1927). By switching their glucose metabolism towards “aerobic glycolysis”, cancer cells accumulate glycolytic intermediates that will be used as building blocks for macromolecular synthesis (Vander Heiden et al., 2009). Most cancer cells exhibit increased glucose uptake, which is due, in part, to the upregulation of glucose transporters, mainly GLUT1 (Yamamoto et al., 1990; Younes et al., 1996). Moreover, cancer cells display a high expression and activity of several glycolytic enzymes, including phospho-fructose kinase (PFK)-1, pyruvate kinase M2, lactate dehydrogenase (LDH)-A and pyruvate dehydrogenase kinase (PDK)-1 (Lunt and Vander Heiden, 2011), leading to the high rate of glucose catabolism and lactate production characteristic of these cells. Importantly, downregulation of either LDH-A or PDK1 decreases tumor growth (Bonnet et al., 2007; Fantin et al., 2006; Le et al., 2010) suggesting an important role for these proteins in the metabolic reprogramming of cancer cells.

Traditionally, cancer-associated alterations in metabolism have been considered a secondary response to cell proliferation signals. However, growing evidence has demonstrated that metabolic reprogramming of cancer cells is a primary function of activated oncogenes and inactivated tumor suppressors (Dang et al., 2012;DeBerardinis et al., 2008; Ward and Thompson, 2012). Despite this evidence, whether the metabolic reprogramming observed in cancer cells is a driving force for tumorigenesis remains as yet poorly understood.

Sirtuins are a family of NAD+-dependent protein deacetylases involved in stress resistance and metabolic homeostasis (Finkel et al., 2009). In mammals, there are seven members of this family (SIRT1-7). SIRT6 is a chromatin-bound factor that was first described as a suppressor of genomic instability (Mostoslavsky et al., 2006). SIRT6 also localizes to telomeres in human cells and controls cellular senescence and telomere structure by deacetylating histone H3 lysine 9 (H3K9) (Michishita et al., 2008). However, the main phenotype SIRT6 deficient mice display is an acute and severe metabolic abnormality. At 20 days of age, they develop a degenerative phenotype that includes complete loss of subcutaneous fat, lymphopenia, osteopenia, and acute onset of hypoglycemia, leading to death in less than ten days (Mostoslavsky et al., 2006). Recently, we have demonstrated that the lethal hypoglycemia exhibited by SIRT6 deficient mice is caused by an increased glucose uptake in muscle and brown adipose tissue (Zhong et al., 2010). Specifically, SIRT6 co-represses HIF-1α by deacetylating H3K9 at the promoters of several glycolytic genes and, consequently, SIRT6 deficient cells exhibit increased glucose uptake and upregulated glycolysis even under normoxic conditions (Zhong et al., 2010). Such a phenotype, reminiscent of the “Warburg Effect” in tumor cells, prompted us to investigate whether SIRT6 may protect against tumorigenesis by inhibiting glycolytic metabolism.

Here, we demonstrate that SIRT6 is a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Strikingly, SIRT6 acts as a first hit tumor suppressor and lack of this chromatin factor leads to tumor formation even in non-transformed cells. Notably, inhibition of glycolysis in SIRT6 deficient cells completely rescues their tumorigenic potential, suggesting that enhanced glycolysis is the driving force for tumorigenesis in these cells. Furthermore, we provide new data demonstrating that SIRT6 regulates cell proliferation by acting as a co-repressor of c-Myc, inhibiting the expression of ribosomal genes. Finally, SIRT6 expression is downregulated in human cancers, strongly reinforcing the idea that SIRT6 is a novel tumor suppressor.


In addition to controlling glucose metabolism in cancer cells, our current work unravels a novel function of SIRT6 as a regulator of ribosomal gene expression. One of the main features of cancer cells is their high proliferative potential. In order to proliferate, cancer cells readjust their metabolism to generate biosynthetic precursors for macromolecular synthesis (Deberardinis et al., 2008). However, protein synthesis also requires the activation of a transcriptional program leading to ribosome biogenesis and mRNA translation (van Riggelen et al., 2010). As a master regulator of cell proliferation, MYC regulates ribosome biogenesis and protein synthesis by controlling the transcription and assembly of ribosome components as well as translation initiation (Dang et al., 2012; van Riggelen et al., 2010). Our results show that SIRT6 specifically regulates the expression of ribosomal genes. In keeping with this, SIRT6-deficient tumor cells exhibit high levels of ribosomal protein gene expression. Beyond ribosome biosynthesis, MYC regulates glucose and glutamine metabolism (Dang et al., 2012). Our results show that glutamine – but not glucose – metabolism is rescued in SIRT6-deficient/MYC knockdown cells, suggesting that SIRT6 and MYC might have redundant roles in regulating glucose metabolism.

Overall, our results indicate that SIRT6 represses tumorigenesis by inhibiting a glycolytic switch required for cancer cell proliferation. Inhibition of glycolysis in SIRT6-deficient cells abrogates tumor formation, providing proof of concept that inhibition of glycolytic metabolism in tumors with low SIRT6 levels could provide putative alternative approaches to modulate cancer growth. Furthermore, we uncover a new role for SIRT6 as a regulator of ribosome biosynthesis by co-repressing MYC transcriptional activity. Our results indicate that SIRT6 sits at a critical metabolic node, modulating both glycolytic metabolism and ribosome biosynthesis (Figure 7L). SIRT6 deficiency deregulates both pathways, leading to robust metabolic reprogramming that is sufficient to promote tumorigenesis bypassing major oncogenic signaling pathway activation.


Lack of cellular enzyme triggers switch in glucose processing

Understanding mechanism underlying SIRT6 activity may help treat diabetes, cancer   January 21, 2010

A study investigating how a cellular enzyme affects blood glucose levels in mice provides clues to pathways that may be involved in processes including the regulation of longevity and the proliferation of tumor cells. In their report in the January 22 issue of Cell, a Massachusetts General Hospital (MGH)-based team of researchers describes the mechanism by which absence of the enzyme SIRT6 induces a fatal drop in blood sugar in mice by triggering a switch between two critical cellular processes.

“We found that SIRT6 functions as a master regulator of glucose levels by maintaining the normal processes by which cells convert glucose into energy,” says Raul Mostoslavsky, MD, PhD, of the MGH Cancer Center, who led the study. “Learning more about how this protein controls the way cells handle glucose could lead to new approaches to treating type 2 diabetes and even cancer.”

SIRT6 belongs to a family of proteins called sirtuins, which regulate important biological pathways in organisms from bacteria to humans. Originally discovered in yeast, sirtuins in mammals have been shown to have important roles in metabolic regulation, programmed cell death and adaptation to stress. SIRT6 is one of seven mammalian sirtuins, and Mostoslavsky’s team previously showed that mice lacking the protein die in the first month of life from acute hypoglycemia. The current study was designed to investigate exactly how lack of SIRT6 causes this radical drop in blood sugar.

Normally cells convert glucose into energy through a two-step process. The first stage called glycolysis takes place in the cytoplasm, where glucose is broken down into an acid called pyruvate and a few molecules of ATP, the enzyme that provides the energy to power most biological processes. Pyruvate is taken into cellular structures called mitochondria, where it is further processed to release much greater amounts of ATP through a process called cellular respiration.

In a series of experiments in mouse cells, the researchers showed that SIRT6-deficiency hypoglycemia is caused by increased cellular uptake of glucose and not by elevated insulin levels or defects in the absorption of glucose from food. They then found increased levels of glycolysis and reduced mitochondrial respiration in SIRT6-knockout cells, something usually seen when cells are starved for oxygen or glucose, and showed that activation of the switch from cellular respiration to glycolysis is controlled through SIRT6’s regulation of a protein called HIF1alpha. Normally, SIRT6 represses glycolytic genes through its role as a compactor of chromatin – the tightly wound combination of DNA and a protein backbone that makes up chromosomes. In the absence of SIRT6, this structure is opened, causing activation of these glycolytic genes. The investigators’ finding increased expression of glycolytic genes in living SIRT6-knockout mice – which also had elevated levels of lactic acid, characteristic of a switch to glycolytic glucose processing – supported their cellular findings.

Studies in yeast, worms and flies have suggested a role for sirtuins in aging and longevity, and while much of the enzymes’ activity in mammals is unclear, SIRT6’s control of critical glucose-metabolic pathways could signify a contribution to lifespan regulation. Elevated glycolysis also is commonly found in tumor cells, suggesting that a lack of SIRT6 could contribute to tumor growth. Conversely, since knocking out SIRT6 causes blood sugar to drop, limited SIRT6 inhibition could be a novel strategy for treating type 2 diabetes.

“There’s a lot we still don’t know about SIRT6,” adds Mostoslavsky, who is an assistant professor of Medicine at Harvard Medical School. “We need to identify the factors that interact with SIRT6 and determine how it is regulated; investigate whether it acts as a tumor suppressor and how it might help lower glucose levels in diabetes; and determine its target organs in living animals, all of which we are investigating.”


A tale of metabolites: the crosstalk between chromatin and energy metabolism

Mitochondrial metabolism influences histone and DNA modifications by retrograde signaling and activation of transcriptional programs. Considering the high number of putative sites for acetylation and methylation in chromatin, we propose in this Perspective that epigenetic modifications might impinge on cellular metabolism by affecting the pool of acetyl-CoA and SAM.

Metabolism can be defined as the sum of chemical reactions that occur within a cell to sustain life. It is also the way that a cell interacts with energy sources: in other words, it is the coordination of energy intake, its utilization and storage that ultimately allows growth and cell division. In animal cells, mitochondria have evolved to become the most efficient system to generate energy. This organelle consumes carbon sources via oxidative phosphorylation to produce ATP, the energy currency of the cell. Additionally, the mitochondria produces intermediate metabolites for the biosynthesis of DNA, proteins and lipids.

Under basic dividing conditions, uptake of nutrients is tightly regulated through growth signaling pathways, thus differentiated cells engage in oxidative metabolism, the most efficient mechanism to produce energy from nutrients. Cells metabolize glucose to pyruvate through glycolysis in the cytoplasm, and this pyruvate is then oxidized into CO2 through the mitochondrial TCA cycle. The electrochemical gradient generated across the inner mitochondrial membrane facilitates ATP production in a highly efficient manner. Studies in recent years indicate that under conditions of nutrient excess, cells increase their nutrient uptake, adopting instead what is known as aerobic glycolysis, an adaptation that convert pyruvate into lactate, enabling cells to produce intermediate metabolites to sustain growth (anabolic metabolism) (1). Interestingly, most cancer cells undergo the same metabolic switch (Warburg Effect), a unique evolutionary trait that allows them to grow unabated. Although aerobic glycolysis generates much less ATP from glucose compared to oxidative phosphorylation, it provides critical intermediate metabolites that are used for anaplerotic reactions, and therefore is an obligatory adaptation among highly proliferative cells. In response to variations in nutrient availability, cells regulate their metabolic output, coordinating biochemical reactions and mitochondrial activity by altering transcription of mitochondrial genes through both activation of transcription factors, such as PGC1α, and chromatin modulators that exert epigenetic changes on metabolic genes.

Mitochondrial dysfunction has been implicated in aging, degenerative diseases and cancer. Proper mitochondrial function can be compromised by the accumulation of mutations in mitochondrial DNA that occur during aging. In addition, reactive oxygen species (ROS) produced during oxidative phosphorylation can promote oxidative damage to DNA, protein and lipids, in turn adversely affecting global cellular functions. In recent years, several studies have illustrated a novel unexpected link between metabolism and gene activity: fluctuations in mitochondrial and cytoplasmic metabolic reactions can reprogram global metabolism by means of impacting epigenetic dynamics. These studies will be briefly summarized in the first part of this article. In the second part, we will propose a provocative novel hypothesis: the crosstalk between metabolism and epigenetics is a two-way street, and defects in chromatin modulators may affect availability of intermediate metabolites, in turn influencing energy metabolism.

Metabolism impacts epigenetics

A regulated crosstalk between metabolic pathways in the mitochondria and epigenetic mechanisms in the nucleus allows cellular adaptations to new environmental conditions. Fine-tuning of gene expression is achieved by changes in chromatin dynamics, including methylation of DNA and posttranslational modifications of histones: acetyl, methyl and phosphate groups can be added by acetyltransferases, methyltransferases and kinases, respectively, to different residues on histones. Given the number of residues that can potentially undergo modifications in histone tails and in the DNA, it is reasonable to consider that metabolic changes affecting the availability of these metabolites will impact epigenetics (as discussed below).

Recently, acetylation of proteins was revealed to be as abundant as phosphorylation (2). This posttranslational modification involves the covalent binding of an acetyl group obtained from acetyl-CoA to a lysine. In histones, acetylation can modify higher order chromatin structure and serve as a docking site for histone code readers. Recent mass spectrometry studies have uncovered the complete acetylome in human cells and revealed that protein acetylation occurs broadly in the nucleus, cytoplasm and mitochondria, affecting more than 1700 proteins (2). Acetylation of proteins depends on the availability of acetyl-CoA in each cellular compartment, but this metabolite is produced in the mitochondria and cannot cross the mitochondrial membrane. In single cell eukaryotes, the pool of acetyl groups required for histone acetylation comes from the production of acetyl-CoA by the enzyme acetyl-CoA synthetase (Acs2p), which is responsible of converting acetate into acetyl-CoA. In mammalian cells, although they have a homolog enzyme to Acs2p, AceCS1, the majority of acetyl-CoA is produced from mitochondrion-derived citrate by the enzyme adenosine triphosphate (ATP)-citrate lyase (ACL) (3). ACL is present in the cytoplasm and in the nucleus, and is responsible for the production of acetyl-CoA from citrate in both compartments. Citrate is generated in the metabolism of glucose and glutamine in the TCA cycle. In contrast to acetyl-CoA, citrate can cross the mitochondrial membrane and diffuse through the nuclear pores into the nucleus, where it can be converted into acetyl-CoA by ACL. Wellen and colleagues found that ACL is required for acetylation of histones under normal growth conditions; knockdown of ACL decreases the pool of acetyl-CoA in the nucleus and reduces the level of histone acetylation (3). Strikingly, reduction in histone acetylation occurs preferentially around glycolytic genes, leading to downregulation of their transcription and therefore inhibition of glycolysis. These observations reveal a process where glucose metabolism dictates histone acetylation that in a feedback mechanism controls the rate of glycolysis.

Notably, deacetylation of histones also exhibits a metabolic influence. Deacetylation of histones is achieved by class I and class II histone deacetylases (HDACs) and by a separate class (class III), also known as sirtuins. Sirtuins use NAD+ as a cofactor for deacetylation, and the ratio of NAD+/NADH regulates their activity. In diets rich in carbohydrates, growth factors stimulate cellular glucose uptake and the production of energy is carried out through glycolysis. In this context, the NAD+/NADH ratio decreases, in turn inhibiting, in theory, sirtuins in the cytoplasm (Sirt2) and nucleus (Sirt1, Sirt6 and Sirt7). In fact, low Sirt1 and Sirt6 activity generates a global increase in protein acetylation. Interestingly, Sirt6, which is exclusively nuclear, deacetylates H3K9 Hif1α target genes, repressing their transcription. Since most of these genes are glycolytic, deacetylation of histones by Sirt6 modulates glycolysis. Indeed, SIRT6-deficient mice experience a dramatic increase in glucose uptake for glycolysis, triggering a fatal hypoglycemia in few weeks (4).

In animal cells, both histone acetylation and deacetylation are under the control of glucose metabolism through the availability of acetyl-CoA and NAD+, respectively. However, is this metabolic control restricted to acetylation, or can other reactions in the nucleus be influenced by the energy status of the cell?

Histone methyltransferases (HMTs) use S-adenosylmethionine (SAM) to transfer a methyl group onto lysine and arginine residues on histone tails. SAM is produced from methionine by the enzyme S-adenosyl methionine transferase (MAT) in a reaction that uses ATP. The recent finding of MAT in the nucleus suggests that the SAM pool could also be controlled locally in this compartment (5). The reverse reaction catalyzed by histone demethylases (HDMs) uses flavin adenine dinucleotide (FAD+) and α-ketoglutarate as coenzymes. FAD is a common redox coenzyme that exists in two different redox states. In its reduced state, FADH2 is a carrier of energy and when oxidized, FAD+ is consumed in the oxidation of succinate to fumarate by the enzyme succinate dehydrogenase (complex II) in one of the last steps of the TCA cycle. On the other hand, α-ketoglutarate is an intermediate in the TCA cycle. It is generated from isocitrate by the enzymes isocitrate dehydrogenase 1 and 2 (IDH1-cytosolic and IDH2-mitochondrial) (Figure 1A–B). Based on these findings, it is easy to infer that the amount of coenzymes used for histone methylation and demethylation could also be controlled by metabolic reactions. Moreover, the different cellular compartments compete for the same metabolites. Indeed, changes in diet that affect the biosynthesis of SAM, FAD and α-ketoglutarate in the mitochondria and cytoplasm have been shown to impact histone methylation (6).

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Figure 1   A) Diagram depicting two-way crosstalk between metabolites in cytoplasm/mitochondria and chromatin.

More recently, some of the metabolic enzymes responsible for producing cofactors for nuclear biochemical reactions have been found mutated in cancer. For instance, IDH1 and IDH2 somatic mutations are recurrent in gliomas and acute myeloid leukemias (AML). These mutations lead not only to a decreased production of α-ketoglutarate but also to a new activity: α-ketoglutarate is in fact converted into 2-hydroxyglutarate (2-HG), a metabolite rarely found in normal cells. The new metabolite is a competitive inhibitor of α-ketoglutarate-dependent dioxygenase enzymes, including the Jumonji C (JmjC) domain containing histone demethylases and the recently discovered TET family of 5-methylcytosine (5mC) hydroxylases involved in DNA demethylation (7). By inhibiting JmjC and TET enzymes, the aberrant production of 2-HG generates a genome-wide histone and DNA hypermethylation phenotype. This is considered to be, at least in part, at the origin of tumorigenesis in IDH1 and IDH2 mutated cells and for this reason, 2-HG may earn its place as an oncometabolite. The discovery that mutations in metabolic enzymes may influence tumorigenesis by means of controlling genome-wide epigenetic changes caused a paradigm shift, indicating that such metabolic abnormalities may affect cancer beyond the Warburg Effect.  ….

Chromatin modifications and cellular metabolism are tightly connected. Thus far the only aspects that have been considered are the retrograde signaling, with mitochondrial metabolites affecting histone modifications, and the anterograde transcriptional regulation of metabolism. A third aspect of the link between nucleus and metabolism has been, in our opinion, omitted so far: a direct influence of chromatin on acetyl-CoA and SAM availability, which may have an essential role also in cancer establishment and development (Figure 1A–B). Notably, a shift towards glycolytic metabolism is now considered a hallmark of cancer cells. It is also true that multiple tumors carry mutations in chromatin modifiers. However, new studies suggest that those two processes may be much more intertwined that previously appreciated, further blurring the limits on their respective roles in tumorigenesis. There is no doubt that changes in metabolite availability can drastically impact chromatin modifications. We believe that the opposite may be true as well. At least in mouse models, deficiency in two chromatin modifiers, SIRT6 and Jhdm2, causes drastic metabolic abnormalities. Even though some of those phenotypes depend on changes in gene-expression, we would like to propose that severe attrition of metabolite pools might as well play a role, a possibility that awaits experimental proof.



Investigators at UC San Diego say that when they blocked a well known signaling molecule that plays a major role in driving colorectal cancer, an escape pathway emerged that allowed tumors to continue to grow.

The pathway they explored, ERK1/2, is a problem for about a third of all colorectal cancer patients, says Petrus R. de Jong, MD, PhD, a co-first author on the paper.

“Since we were genetically deleting the ERK1/2 pathway, we expected to see less cell proliferation,” said de Jong. “Instead, the opposite occurred. There was more cell growth and loss of organization within the cells.”

The problem was ERK5, the investigators add. And when that was blocked as well in animal models and cell lines for the disease, the combination approach proved more effective in halting cancer growth.

“If you block one pathway, cancer cells usually mutate and find another pathway that ultimately allows for a recurrence of cancer growth,” said co-first author Koji Taniguchi. “Usually, mutations occur over weeks or months. But other times, as in this case, the tumor does not need to develop mutations to find an escape route from targeted therapy. When you find the compensatory pathway and block both, there is no more escape.”


GEN News Highlights    May 18, 2016
Blocking Cancer Signaling Leads to Discovery of New Tumor-Promoting Pathway

 Immunofluorescent staining of intestinal epithelium tissue shows cell growth (green). In a normal mouse model (left), cell growth is controlled, but in a mouse model with the ERK1/2 pathway blocked (right) increased cell proliferation and loss of organization occurred. [UC San Diego Health]

An international research team lead by scientists at the University of California San Diego School of Medicine uncovered some surprising results while investigating a potential therapeutic target for the ERK1 and two pathways. These signaling pathways are widely expressed and known to drive cancer growth in one-third of patients with colorectal cancer (CRC). The UCSD team found that an alternative pathway immediately emerges when ERK1/2 is halted, thus allowing tumor cell proliferation to continue.

“Since we were genetically deleting the ERK1/2 pathway, we expected to see less cell proliferation,” explained co-lead study author Petrus R. de Jong, M.D., Ph.D., translational scientist at Sanford Burnham Prebys Medical Discovery Institute. “Instead, the opposite occurred. There was more cell growth and loss of organization within the cells.”

The exciting part of this new study is investigators found that treating both ERK1/2 and the compensatory pathway ERK5 concomitantly with a combination of drug inhibitors halted CRC growth more effectively in both mouse models and human CRC cell lines.

“We show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration, and secretory cell differentiation,” the authors wrote. “However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion ofErk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines.”

The findings from this study were published recently in Nature Communications in an article entitled “ERK5 Signalling Rescues Intestinal Epithelial Turnover and Tumour Cell Proliferation upon ERK1/2 Abrogation.”

The ERK pathway plays a critical role in embryonic development and tissue repair because it instructs cells to multiply and start dividing, but when overactivated cancer growth often occurs.

“Therapies aimed at targeting ERK1/2 likely fail because this mechanism is allowing proliferation through a different pathway,” noted senior study author Eyal Raz, M.D., professor of medicine at UC San Diego School of Medicine. “Previously, ERK5 didn’t seem important in colorectal cancer. This is an underappreciated escape pathway for tumor cells. Hence, the combination of ERK1/2 and ERK5 inhibitors may lead to more effective treatments for colorectal cancer patients.”

Currently, there are 1.2 million people living with CRC in the United States, making it the third most common cancer among men and women. In 2016 alone, an estimated 134,490 new cases are expected to be diagnosed, so understanding the molecular mechanisms that drive tumor promotion are paramount to treating this disease effectively.

“If you block one pathway, cancer cells usually mutate and find another pathway that ultimately allows for a recurrence of cancer growth,” remarked co-lead study author Koji Taniguchi, M.D., Ph.D., senior researcher at the Keio University School of Medicine in Tokyo. “Usually, mutations occur over weeks or months. But other times, as in this case, the tumor does not need to develop mutations to find an escape route from targeted therapy. When you find the compensatory pathway and block both, there is no more escape.”

The researchers were excited by their findings but urged caution at over interpretation of their initial findings and suggested that other classes of inhibitors be tested in combination with ERK5 inhibitors in human CRC cells in preclinical mouse models before any patient trial can begin.


ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation

Petrus R. de JongKoji TaniguchiAlexandra R. HarrisSamuel BertinNaoki TakahashiJen DuongAlejandro D. CamposGarth PowisMaripat CorrMichael Karin & Eyal Raz
Nature Communications 7, Article number:11551  doi:10.1038/ncomms11551

The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are part of the classical family of mammalian mitogen-activated protein kinases (MAPKs), which also include three c-Jun amino-terminal kinases (JNK1/2/3), four p38 isoforms and its lesser-known counterpart, ERK5. The serine/threonine kinases ERK1 (MAPK3, also known as p44 MAPK) and ERK2 (MAPK1, also known as p42 MAPK) show 83% amino acid identity, are ubiquitously expressed and typically activated by growth factors and phorbol esters, whereas the p38 and JNK pathways are mainly activated by inflammatory cytokines and stress1. MAPKs are involved in regulation of mitosis, gene expression, cell metabolism, cell motility and apoptosis. ERK1/2 are activated by MEK1 and MEK2, which themselves are activated by Raf-1, A-Raf or B-Raf1, 2. Ras proteins (K-Ras, H-Ras or N-Ras) are small GTPases that can be activated by receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCRs), which recruit Raf proteins to the plasma membrane where they are activated. Together, these modules constitute the Ras–Raf–MEK–ERK pathway3.

The activation of ERK1/2 results in their nuclear translocation where they can phosphorylate a variety of nuclear targets such as Elk-1, c-Fos and c-Myc1, in addition to p90 ribosomal S6 kinases (p90RSKs) and mitogen- and stress-activated protein kinases, MSK1/2. The full repertoire of substrates for ERK1/2 consists of at least 160 cellular proteins4. These proteins are typically involved in the regulation of cell proliferation—more specifically, G1/S-phase cell cycle progression—and differentiation. However, their cellular effects are context-dependent and determined by the spatial and temporal dynamics of ERK1/2 activity5, which are highly regulated by scaffolding proteins and phosphatases3, 6, 7.

Despite vast literature on the role of ERK1/2 in cell proliferation, the absolute requirement of this signalling module in rapidly dividing tissues relative to other signalling pathways is unknown. The small intestinal epithelium is particularly suitable to address this question given the short (4–8 days) and dynamic life cycle of intestinal epithelial cells (IECs). Lgr5+ intestinal stem cells at the intestinal crypt base produce transit-amplifying cells, which then undergo a number of proliferative cycles before terminal differentiation into absorptive enterocytes at the crypt–villus border. Enterocytes then migrate to the villus tip where they undergo anoikis and are shed into the gut lumen8. All of these cellular events are tightly coordinated by the Wnt, Notch, bone morphogenetic protein (BMP) and Hedgehog pathways9, whereas the roles of ERK1/2 remain to be charted. In the intestines, the ERK1/2 pathway is likely activated by autocrine and paracrine factors downstream of RTKs, such as epidermal growth factor receptor (EGFR)10, and by exogenous microbial-derived substrates that signal through the Toll-like receptor (TLR)/MyD88 pathway11.

To study the effects of ERK1/2 in the adult intestinal epithelium, we generated mice with a conditional (IEC-specific) and tamoxifen-inducible deletion of Erk2 on the Erk1−/− background, which completely abrogates this pathway. We show that the ERK1/2 signalling module, surprisingly, is dispensable for IEC proliferation. Genetic deletion of Erk1/2 in primary IEC or treatment of colorectal cancer (CRC) cell lines with MEK1/2 inhibitors results in compensatory activation of the ERK5 pathway. Moreover, the treatment of human CRC lines with a combination of MEK1/2 and ERK5 inhibitors is more efficacious in the inhibition of cancer cell growth. Thus, compensatory signalling by ERK5 suggests a potential rescue pathway that has clinical implications for targeted therapy in colorectal cancer.


Figure 1: Wasting disease associated with malabsorption in Erk1/2ΔIEC mice.

ERK1/2ΔIEC causes wasting and enterocyte dysfunction

Here we show that ERK1/2 signalling in mouse intestinal epithelium is dispensable for cell proliferation, while it resulted in abnormal differentiation of enterocytes, wasting disease and ultimately lethality (Fig. 1). Consistent with these findings, ERK1/2 MAPKs were shown to be associated with the enterocyte brush border and activated upon RTK stimulation or feeding27 or electrical field stimulation in polarized epithelium28. This seems at odds with literature that suggest that maintained ERK1/2 signalling precludes enterocyte differentiation29, 30. A possible explanation for this discrepancy could be that cycling IEC in the transit amplifying zone of the crypt require relatively high levels of active ERK1/2, which is readily blocked by pharmacological intervention, whereas a transition to low level ERK1/2 activity in IEC migrating into the villus compartment promotes the absorptive enterocyte differentiation program that is only perturbed upon complete genetic deletion of Erk1/2. Little is known about the role of ERK1/2 signalling in the life cycle of secretory cells in the gut. A recent report by Heuberger et al.15 described that IEC-specific deletion of non-receptor tyrosine phosphatase, Shp2, resulted in the loss of p-ERK1/2 levels in the small intestine. This coincided with an increased number of Paneth cells at the expense of goblet cells in the small intestine, as well as shortening of villi. They also observed the strongest staining for epithelial p-ERK1/2 in the TA zone. This p-ERK1/2+ staining pattern and the architectural organization of the TA zone were lost in Shp2 knockout mice. Interestingly, the deleterious effects of Shp2 deficiency were rescued by expression of constitutively active MEK1. A model was proposed in which the balance between Wnt/β-catenin and MAPK signalling determines Paneth cell versus goblet cell differentiation, respectively15. This proposed crucial role for ERK1/2 MAPK signalling in intestinal secretory cell differentiation is consistent with our observations inERK1/2ΔIECmice.

Migration and differentiation are functionally intertwined in the intestines, as demonstrated by the immature phenotype of mislocalized Paneth cells observed in ΔIEC mice (Fig. 2). Critical to epithelial cell migration is proper cytoskeleton reorganization mediated by the small GTPases of the Rho family, cell polarization regulated by Cdc42 and dynamic adhesion through cell–matrix and cell–cell interaction via integrin/FAK/Src signalling31. The ERK1/2 module is used as a downstream effector of many of these pathways in the intestine, including Rho GTPases32, FAK33and Src34, and has been suggested to promote cell motility33, 35. RTK signalling also contributes to cell migration, for example, Eph–Ephrin receptor interactions are crucial for correct positioning of Paneth cells36. Ephrin receptor-induced epithelial cell migration has been shown to be mediated by Src and ERK1/2 activation37, 38, which may explain the Paneth cell mislocalization observed in ΔIEC mice. In summary, the ERK1/2 module is indispensable for full maturation of both absorptive enterocytes and the secretory lineage (Fig. 7a), confirming its crucial role in the integration of cellular cues required for determination of epithelial cell fate.

Figure 7: Roles of ERK1/2 and ERK5 in intestinal homeostasis and tumorigenesis.

Roles of ERK1/2 and ERK5 in intestinal homeostasis and tumorigenesis.

(a) When the ERK1/2 pathway is intact, extracellular cues that are transduced via RTKs or GPCRs activate Ras under physiological conditions, or alternatively, Ras is constitutively active in colorectal cancer (RasΔ*), which preferentially activates the Raf–MEK1/2–ERK1/2 module. The nuclear and transcriptional targets of ERK1/2 are crucial for enterocyte and secretory cell differentiation, IEC migration, as well as cell proliferation under homeostatic and oncogenic conditions. Importantly, ERK1/2 activation also results in the activation of negative feedback mechanisms that suppress its upstream kinases (for example, RTKs, son of sevenless, Raf) and activate dual specificity phosphatases (DUSPs), resulting in the silencing of the ERK5 module. (b) Upon abrogation of MEK1/2 or genetic knockout ofErk1/2, the lack of negative feedback mechanisms (that is, feedback activation) results in upregulation of the Ras–Raf–MEK5–ERK5 module, which maintains cell proliferation under physiological conditions, or results in continued tumour cell proliferation in colorectal cancer, respectively. However, the lack of activation of ERK1/2-specific targets results in differentiation and migration defects of intestinal epithelial cells culminating in malabsorption, wasting disease and mortality. Compensatory upregulation of the ERK5 pathway in CRC can be reversed by targeted treatment with its specific inhibitor, XMD8-92.

An unexpected finding was the redundancy of ERK1/2 in the gut with regard to cell proliferation.Erk1/2 deletion was compensated by upregulated ERK5 signalling. Genetic targeting of ERK1/2 in vitro previously showed that Erk2 knockdown is more effective than Erk1 knockdown in suppressing cell proliferation, although this may be related to higher expression levels of the former39. The effect of gene dosage was demonstrated in vivo by the observations that whileErk1−/− mice are viable12 and Erk2−/− mice die in utero13, Erk2+/− mice are only viable when at least one copy of Erk1 is present. However, mice heterozygous (+/−) for both Erk1 and Erk2 alleles were born at lower than Mendelian ratio39. More recently, it was reported that transgenic expression of ERK1 can compensate for Erk2 deletion40, demonstrating functional redundancy between both family members. Deletion of Erk1/2 in adult skin tissue resulted in hypoplasia, which was associated with G2/M cell cycle arrest, without notable differentiation defects of keratinocytes41. These data differ from our observations in the intestines, which might be explained by incomplete and transient siRNA-mediated knockdown of ERK1/2 in primary keratinocyte cultures41, compared with more efficient genomic deletion of Erk1 and Erk2 that is typically achieved by the Villin-Cre-ERT2 system14, possibly resulting in different outcomes.

Both ERK1/2 and ERK5 have been described to promote cell cycle progression, although they have different upstream signalling partners, MEK1/2 and MEK5, respectively1. Furthermore, ERK2 and ERK5 proteins share only about 66% sequence identity, and MEK5 is phosphorylated by MEKK2/3, which can also activate the p38 and JNK pathways42. The ERK5 pathway is classically activated by stress stimuli, in addition to mitogens; thus, it shares features of both the ERK1/2, and p38 and JNK pathways, respectively43. ERK5 induces expression of cyclin D1 (refs 44, 45), and suppresses expression of cyclin dependent kinase inhibitors46, thereby promoting G1/S-phase cell cycle progression. Importantly, the role of ERK5 in IEC differentiation and intestinal homeostasis is currently unknown. Knockout of Erk1/2 in IEC induced activity of ERK5, which was not detectable in naive mice (Fig. 3). These data suggest that the ERK1/2 and ERK5 modules may share proximal signalling components. Although EGFR is a likely candidate in this context19, 20, we found that abrogation of EGFR signalling did not prevent enhanced ERK5 activity upon MEK1/2 inhibition. Although it was originally suggested that ERK5 signalling is independent of Ras20, other groups established that Ras, either through physiological signalling47, or by its oncogenic activity48,49, activates the MEK5–ERK5 signalling axis. Thus, rewiring of signalling networks downstream of Ras could explain the supraphysiological activity of ERK5 upon conditional deletion of Erk1/2 in the intestines. In fact, it has been shown that ERK1/2 signalling mediates negative feedback on ERK5 activity50, possibly through transcriptional activation of dual specificity phosphatases (DUSPs)51. Alternatively, ERK1/2-induced FOS-like antigen 1 (Fra-1) may negatively regulate MEK5 (ref. 52). These data suggest that ERK5 is a default bypass route downstream of RTK-Ras and activated upon loss of ERK1/2-mediated repression, thereby ensuring the transduction of mitogenic signals to the nucleus (Fig. 7b). Consistent with this concept, we found that ERK5 inhibition induces atrophy of ΔIEC intestinal organoids (Fig. 4). In addition, important downstream transcriptional targets of ERK5 and ERK1/2 overlap, such as immediate-early gene Fra1 and oncogene c-Myc, whereas c-Fos and Egr1 were specifically induced by ERK1/2 (Fig. 6 and Supplementary Fig. 7). Specificity of ERK1/2 over ERK5 and other MAPK family members for the activation of c-Fos has been previously described53, demonstrating their differential biological output despite the shared ability to transduce potent mitogenic signals.

Our findings may be relevant for the use of MAPK inhibitors in the treatment of colorectal cancer. Although there was only a mild phenotype in the colons of ΔIEC mice under homeostatic conditions, the Ras–RAF–MEK–ERK pathway is generally upregulated in malignant cells including CRC54. Targeted therapy typically results in feedback activation of upstream players of the targeted kinase, which are then able to reactivate the same pathway or utilize bypass signalling routes55. For example, on activation, ERK1/2 phosphorylates EGFR, son of sevenless56, and Raf57, thereby terminating upstream signalling activity. Knockout of Erk1/2 eliminates this negative feedback. Our data suggest that ERK5 is a putative resistance pathway in the context of targeted treatment with MEK1/2 or ERK1/2 inhibitors (Fig. 7b). Different classes of MEK1/2 inhibitors display various modes of resistance to therapy (innate, adaptive and acquired)58. Since we have only used one MEK1/2 inhibitor (PD0325901) in our studies, it will be necessary to evaluate other classes of inhibitors in combination with ERK5 inhibitors. Importantly, while treatment with either the MEK1/2 or ERK5 inhibitor suppressed tumour growth in murine Apc−/− organoids, only the latter was able to inhibit the proliferation of Apc−/−;KRASG12V organoids (Fig. 6), which are more representative of human CRC. In line with this, suppression of ERK5 expression by forced expression of miR-143/145 inhibited intestinal adenoma formation in the ApcMin/+ model59, and activated MEK5 correlated with more invasive CRC in human60. ERK5 has been previously reported to mediate resistance to cytotoxic chemotherapy-induced apoptosis61. The highly specific and bioavailable ERK5 inhibitor, XMD8-92, has shown antitumour effects in multiple preclinical cancer models by inhibiting tumour angiogenesis, metastasis and chemo-resistance62. Furthermore, ERK5 inhibition does not induce feedback activation of upstream or parallel signalling pathways62. In conclusion, the ERK1/2 and ERK5 MAPK modules display a high degree of signalling plasticity in the intestinal epithelium, which has implications for targeted treatment of colorectal cancer.


Researchers Reveal Role of Transcription Factor Isoforms in Colon Diseases









Balance between the two isoforms, P1 and P2, of nuclear receptor HNF4a in the colonic crypt influences susceptibility to colitis and colon cancer. P1 is seen here in green. P2 is seen in red. [Poonamjot Deol, Sladek lab, UC Riverside]

Scientists at the University of California, Riverside have determined the distribution of the P1 and P2 isoforms of hepatocyte nuclear factor 4α (HNF4α) in the colons of mice. They report (“Opposing Roles of Nuclear Receptor HNF4α Isoforms in Colitis and Colitis-Associated Colon Cancer”) in eLife that maintaining a balance of P1 and P2 is crucial for reducing risk of contracting colon cancer and colitis.

What is already known in the field of cell biology is that the HNF4α transcription factor plays a key role in both diseases. HNF4α comes in two major isoforms, P1-HNF4α and P2-HNF4α (P1 and P2), but just how each isoform is involved in colitis and colon cancer is not understood.

“P1 and P2 have been conserved between mice and humans for 70 million years,” said Frances M. Sladek, Ph.D., professor of cell biology, who led the research project. “Both isoforms are important and we want to keep an appropriate balance between them in our gut by avoiding foods that would disrupt this balance and consuming foods that help preserve it. What these foods are is our next focus in the lab.”

The intestine is the only adult tissue in the body that expresses both P1 and P2. Dr. Sladek and her team have shown for the first time that these isoforms perform nonredundant functions in the intestine and are relevant to colitis and colitis-associated colon cancer.

“Our study also suggests that finding a drug to stabilize one isoform should be more effective than targeting both isoforms for treating colitis and colon cancer,” said Karthikeyani Chellappa, Ph.D., the first author of the research paper and a former postdoctoral researcher in Sladek’s lab.

Dr. Sladek explained that the colonic epithelial surface has finger-like invaginations (into the colonic wall) called colonic crypts that house stem cells at their base. These stem cells help regenerate new epithelial cells that continuously migrate up toward the surface, thus ensuring complete renewal of the intestinal lining every 3–5 days.

The researchers observed that the P1-positive cells were found in the surface lining and the top portion of the crypt (green in the accompanying image) whereas P2-positive cells were mostly in the proliferative compartment in the lower half of the crypt (the proliferation marker is red in the image.) Furthermore, when transgenic mice genetically engineered to have only either P1 or P2 were subjected to a carcinogen and, subsequently, to an irritant to stress the epithelial lining of the colon, the researchers found that the P1 mice showed fewer tumors than wild-type control mice. When treated with irritant alone, these mice were resistant to colitis. In sharp contrast, mice with only P2 showed more tumors and were much more susceptible to colitis.

The researchers explain these findings by invoking the “barrier function,”  a mucosal barrier generated by the colon’s epithelial cells that prevents bacteria in the gut from entering the body. In the case of P1 mice, this barrier function was enhanced. The P2 mice, on the other hand, showed a compromised barrier function, presumably allowing bacteria to pass through.

Next, the researchers examined genes expressed in the P1 and P2 mice. They found that resistin-like molecule (RELM)-beta, a cytokine (a signaling molecule of the immune system) expressed in the gastrointestinal tract and implicated in colitis, was expressed far more in the P2 mice than the P1 mice.

“This makes sense since a reduced barrier function means bacteria can go across the barrier, which activates RELM-beta,” Dr. Sladek said. “We also found that the P2 protein transcribes RELM-beta more effectively than the P1 protein.”

Next, Poonamjot Deol, Ph.D.,  an assistant project scientist in Dr. Slaked’s lab and the second author of the eLife study, will lead a project aimed at understanding how diet affects the distribution of P1 and P2 in the gut. She and others in the lab also plan to investigate how obesity and colitis may be linked. (Diet studies performed in Dr. Sladek’s lab in the past illustrated soybean oil’s adverse effect on obesity.)

“In the case of colitis, could soybean oil be playing a part in allowing bacteria to get across the barrier function?” Dr. Deol said. “We do not know. We know its detrimental effect on obesity. But more research needs to be done where colitis is concerned.”

Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer

 Karthikeyani Chellappa, 

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb-/- mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.



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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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Innovation in Cancer Biopharmaceutical Intelligence [11.5]

Writer and Curator: Larry H. Bernstein, MD, FCAP

The content of this article, with several interesting features is as follows:

11.5.1 Carmen Drahl..A Great Organic Chemist and Science Writer

11.5.2 Anthony Melvin Crasto

11.5.3 Amgen files ‘breakthrough’ leukemia drug in the US

11.5.4 Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds

11.5.5 The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy

11.5.6 A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis

11.5.7 Pauline Lau. Biochemist, Instrumental Analysis, Molecular and Clinical Diagnostics, and Pharmaceuticals

11.5.8  Kinetic and perfusion modeling of hyperpolarized 13C pyruvate and urea in cancer with arbitrary RF flip angles

11.5.9 ZSTK 474

11.5.10 Marrow-Infiltrating Lymphocytes Safely Shrink Multiple Myelomas


The following content is a series of discussions that identify innovation in therapeutics and individuals who are leaders in pharmaceutical innovation.

11.5.1 Carmen Drahl. A Great Organic Chemist and Science Writer

Her eyes fit a stellar career path. She is a talent in organic and medicinal chemistry, and an informed reporter.

Extract from Dr. Anthony Melvin Castro,  Organic Chemistry

Carmen Drahl

Carmen Drahl


Award-winning science communicator and social media power user based in Washington, DC.

Carmen Drahl is a multimedia science journalist and chemistry communicator based in Washington, DC.



A social media evangelist, Carmen started her first chemistry blog in 2006. Today, she regularly leverages Twitter, Facebook, and Google Plus Hangouts in her reporting.

Carmen has written about how life may have originated on Earth, explained how new medications get their names, and covered the ongoing issues plaguing the forensic science community. Her video on the food science behind 3D printed cocktail garnishes won the 2014 Folio Eddie Award for Best Association Video.

Until December 2014, Carmen worked at Chemical & Engineering News magazine. Her work has also been featured at Scientific American’s blog network, SiriusXM’s Doctor Radio, and elsewhere.

Carmen holds a Ph.D. in chemistry from Princeton University.

Ph.D. with Erik J. Sorensen.  She was on a team that completed the first total synthesis of abyssomicin C, a molecule found in small quantities in nature that showed hints of promise as a potential antibiotic. I constructed molecular probes from abyssomicin for proteomics studies of its biological activity.

M.A. with George L. McLendon worked toward developing a drug conjugate as a potential treatment for cancer. I synthesized a photosensitizer dye-peptide conjugate for targeting the cell death pathway called apoptosis.

Jacobus Fellowship Recipients - Carmen Drahl - Princeton

Jacobus Fellowship Recipients – Carmen Drahl – Princeton

Jacobus Fellowship Recipients – Carmen Drahl – Princeton

At a reception before the Alumni Day luncheon, President Tilghman (third from left) congratulated the winners of the University’s highest awards for students: (from left) Pyne Prize winners Lester Mackey and Alisha Holland; and Jacobus Fellowship recipients Sarah Pourciau, Egemen Kolemen and Carmen Drahl.


interviewing, science writing, social media, Twitter, Storify, YouTube, public speaking, hosting, video production, iPhone videography, non-linear video editing, blogging (WordPress and Blogger), HTML website coding

Carmen Drahl

By the time I discovered science blogs I knew my career goals were changing. I’d already been lucky enough to audit a science writing course at Princeton taught by Mike Lemonick from TIME, and thought that maybe science writing was a good choice for me. After reading chemistry blogs for a while I realized “Hey, I can do this!” and started my own blog, She Blinded Me with Science, in July 2006. It was the typical grad student blog, a mix of posts about papers I liked and life in the lab.

Carmen Drahl pic1

Carmen Drahl

At C&E News I’ve contributed to its C&ENtral Science blog, which premiered in spring 2008. I’ve experimented with a few different kinds of posts- observations and on-the-street interviews when

I run into something chemistry-related in DC, in-depth posts from meetings, and video demos of iPod apps. One of my favorite things to do is toy with new audio/video/etc technology for the blog.

Meant to treat: tumors with loss-of-function in the tumor suppressor protein PTEN (phosphatase and tensin homolog)- 2nd most inactivated tumor suppressor after p53- cancers where this is often the case include prostate and endometrial

Mode of action: inhibitor of phosphoinositide 3-kinase-beta (PI3K-beta). Several lines of evidence suggest that proliferation in certain PTEN-deficient tumor cell lines is driven primarily by PI3K-beta.

Medicinal chemistry tidbits: The GSK team seemed boxed in because in 3 out of 4 animals used in preclinical testing, promising drug candidates had high clearance. It turned out that a carbonyl group that they thought was critical for interacting with the back pocket of the PI3K-beta enzyme wasn’t so critical after all. When they realized they could replace the carbonyl with a variety of functional groups, GSK2636771 eventually emerged. GSK2636771B (shown)



11.5.2 Anthony Melvin Crasto

Principal Scientist, Process research

Glenmark Generics Ltd.

Anthony Melvin Crasto Ph.D

Worlddrugtracker, Principal scientist, Process research, Glenmark-Generics Ltd & Founder of Several Linkedin Gps

Glenmark Generics Ltd., Glenmark Pharmaceuticals

Glenmark Pharmaceuticals, Innovassynth, RPG Life Sciences

Institute of Chemical Technology (UDCT)

December 2005 – Present (9 years 6 months) Mahape, Navimumbai, India,

Currently working with GLENMARK GENERICS LTD research centre as Principal Scientist, process research (bulk actives) at Mahape ,Navi Mumbai,and leading a team of scientists in developing APIs for regulated markets, this involves visualization and execution of novel routes, polymorphs, and developing intellectual property to protect the invention. This involves all aspects of synthesis in lab and commercialization on plant , support for DMF filing.

Currently involved in development of several targets for regulated markets. Provide support to US/European marketing team for developing and execution of new projects

Process Development :-

  • Providing guidance and support for process development for challenging of patents in regulated market.
  • Design patent non-infringing scalable synthetic routes/process and scale-up of API’s
  • Bench and Pilot scale synthesis transformations in hands on
  • Optimization of the process, ie,developing industrially feasible process.
  • Preparation of PDR, filing of patent and DMF
  • Lead a group of Scientists and Group Leaders(for docs).

Skill sets:- Technical skills:


  • Development of novel synthetic routes/process for pharmaceuticals and successful implementation of the technology in pilot plant
  • Conducted various reactions at laboratory and production scales.
  • Synthesized various classes of compounds.
  • Experienced to work under cGMP condition

EX Hoechst Marion Roussel(SANOFI AVENTIS), RPG Life Sciences,Innovassynth, SEARLE,AGREVO,IOC

Glenmark Generics Ltd.

Research Activities Covered in Entire Career

1) Extensive range of chemistry and scale of manufacture from laboratory, scale up laboratory, pilot plant, plant scale including third party activity.

Applied intellectual and synthetic skills to the process development of pharmaceutical drugs/their intermediates, and natural products, neutraceuticals, mettalocenes, speciality chemicals, flavours and fragrances in the laboratory and monitor them during plant trials.

Act as a technology transfer man and provide all data required for transfer from lab to commercialization.

Use of Internet and manual literature search methods to decide on non-infringing route

Write DHR for API before implementation of novel route in the plant and assist for all batches for the DMF purposes, very well versed with IPR issues

Ability to develop novel routes for API,s and draft patents,well versed with polymorphism issues.

Several patents filed in US/EU

Total experience 23+ in industry.

Currently working as principal scientist and leading a team of scientists in developing APIs for regulated markets, this involves novel routes, polymorphs, and developing intellectual property to protect the invention. This involves all aspects of synthesis and commercialization and assist in providing support for DMF filing.

11.5.3 Amgen files ‘breakthrough’ leukemia drug in the US

Daily News | Sept 22, 2014

Selina Mckee

Biotechnology giant Amgen has filed its investigational cancer immunotherapy blinatumomab in the US for the treatment of certain forms of acute lymphoblastic leukaemia (ALL).

Specifically, the Biologic License Application seeks approval to market the drug for patients with Philadelphia-negative (Ph-) relapsed/refractory B-precursor forms of the aggressive blood/bone marrow cancer.

Blinatumomab is the first of Amgen’s BiTE antibody constructs, a novel immunotherapy approach under which antibodies are modified to engage two different targets simultaneously. The drug has already been awarded both ‘Orphan’ and ‘Breakthrough’ status by the Food and Drug Administration, indicating that it could offer a significant advance over available therapies on at least one clinically significant endpoint.

The submission includes data from a Phase II which successfully met its primary endpoint, showing a complete response (no leukaemia cells detectable with microscopy) rate of 43% in patients with relapsed/refractory ALL, including those with resistance to previous treatment approaches.

“Currently, there is no broadly accepted standard treatment regimen for adult patients with relapsed or refractory ALL,” noted Anthony Stein, clinical professor, Haematology/Oncology at City of Hope, adding that “blinatumomab has the potential to significantly advance treatment options for patients living with this difficult-to-treat disease”.

In the US, it is estimated that more than 6,000 cases of ALL will be diagnosed in 2014. In adult patients with relapsed or refractory ALL, median overall survival is just three to five months, further highlighting the urgent need for new treatment options.

Read more at:

Follow us: @PharmaTimes on Twitter

11.5.4 Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds

By Ralph Turchiano on Aug 5, 2014 •

High doses of the herb American ginseng (Panax quinquefolius) over two months reduced cancer-related fatigue in patients more effectively than a placebo, a Mayo Clinic-led study found. Sixty percent of patients studied had breast cancer. The findings are being presented at the American Society of Clinical Oncology’s annual meeting.

Researchers studied 340 patients who had completed cancer treatment or were being treated for cancer at one of 40 community medical centers. Each day, participants received a placebo or 2,000 milligrams of ginseng administered in capsules containing pure, ground American ginseng root.

“Off-the-shelf ginseng is sometimes processed using ethanol, which can give it estrogen-like properties that may be harmful to breast cancer patients,” says researcher Debra Barton, Ph.D., of the Mayo Clinic Cancer Center.

At four weeks, the pure ginseng provided only a slight improvement in fatigue symptoms. However, at eight weeks, ginseng offered cancer patients significant improvement in general exhaustion — feelings of being “pooped,” “worn out,” “fatigued,” “sluggish,” “run-down,” or “tired” — compared to the placebo group.

11.5.5 The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy

Royal Jelly - queen larvae

Royal Jelly – queen larvae

Royal Jelly – queen larvae

Royal jelly is a honey bee secretion that is used in the nutrition of larvae, as well as adult queens.[1] It is secreted from the glands in the hypopharynx of worker bees, and fed to all larvae in the colony, regardless of sex or caste.[2]

When worker bees decide to make a new queen, because the old one is either weakening or dead, they choose several small larvae and feed them with copious amounts of royal jelly in specially constructed queen cells. This type of feeding triggers the development of queen morphology, including the fully developed ovaries needed to lay eggs.[3]

Other Common Names:  Apilak, Gelée Royale, Queen Bee Jelly

Royal Jelly has been called the “Crown Jewel” of the beehive that has become extremely popular since the 1950s as a wonderful source of energy and natural way to increase stamina; perhaps that is the reason why the Queen Bee is so strong and enduring.  It is also thought to be a great nutritional source of enzymes, proteins, sugars and amino acids, but there is no scientific proof to verify the supplement’s efficacy for its use as an overall health tonic.

Royal Jelly is a thick, milky material that is secreted from the hypopharyngea- salivary glands in the heads of the young nurse bees between the sixth and twelfth days of life, and when honey and pollen are combined and refined within the nurse bee, Royal Jelly is naturally created.  While all larvæ in a colony are fed Royal Jelly, it is the only food that is fed to the Queen Bee throughout her life; other adult bees do not consume it at all.  All female eggs may produce a Queen Bee, but this occurs only when – during the whole development of the larvæ – she is cared for and fed by this material – in large quantities.

As a result of this special nutrition, the Queen develops reproductive organs (while the worker bee develops traits that relate only to work, i.e., stronger mandibles, brood food, wax glands and pollen baskets).  The Queen develops in about fifteen days, while the workers require twenty-one; and finally, the Queen endures for several years, while workers survive only a few months. “10-2 HDA,” thought to be the principle active substance in Royal Jelly, makes the Queen Bee fifty percent larger than the other female worker bees and gives her incredible stamina, ovulation ability and longevity, living four to five years longer than worker bees who only live forty or more days.  Perhaps this is the reason why so many positive qualities have been attributed to Royal Jelly as a truly rare gift of nature, but it should be noted that there is no clinical evidence to support the claims.

There is even great controversy as to the constituents included in the supplement.  Most researchers claim that it includes all the B-vitamins and vitamins A, C, D and E; some disagree.  It does contain proteins, sugars, lipids (essential fatty acids), many essential amino acids, collagen, lecithin, enzymes and minerals, in addition to the very valuable 10-2-HDA (10-Hydroxy-2-Decenoic Acid).  It is said that Royal Jelly may be most effective when combined with honey.

The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy, etc., and recent studies indicated immuno-regulation and anti-malignancy activities.  It can promote the growth of T-lymphocyte subsets, Interleukin-2 and the generation of tumor necrosis factor.  Much research is being conducted on this valuable active constituent, which has exhibited positive physiological and pharmacological effects including vasodilative and hypotensive activities, antihypercholesterolemic activity and anti-inflammatory functions.

10-2-HDA (10-Hydroxy-2-Decenoic Acid)

10-2-HDA (10-Hydroxy-2-Decenoic Acid)

11.5.6  A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis
OCT 28, 2014

A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis,
C.H. Hornung, M.R. Mackley, I.R. Baxendale and S.V. Ley and, Org. Proc. Res. Dev., 2007, 11, 399-405.

This paper reports proof of concept, development, and trials for a novel plastic microcapillary flow disc (MFD) reactor. The MFD was constructed from a flexible, plastic microcapillary film (MCF), comprising parallel capillary channels with diameters in the range of 80−250 μm. MCFs were wound into spirals and heat treated to form solid discs, which were then capable of carrying out continuous flow reactions at elevated temperatures and pressures and with a controlled residence time. Three reaction schemes were conducted in the system, namely the synthesis of oxazoles, the formation of an allyl-ether, and a Diels−Alder reaction. Reaction scales of up to four kilograms per day could be achieved. The potential benefits of the MFD technology are compared against those of other reactor geometries including both conventional lab-scale and other microscale devices.

11.5.7 Pauline Lau. Biochemist, Instrumental Analysis, Molecular and Clinical Diagnostics, and Pharmaceuticals.

She was born on the China-Russian border, near the end of the rail line.  When they came to US her mother saw bagels and said, look – they have round bread.

At the meetings she always took us to the best Chinese restaurant, and said not to ask what’s in the food.  They always brought out a fish fresh from the tank and showed it to us.  When she went to Roche, where she became a legend. she got a house on the lake. They had to remove the roof to put a round banquet table in her house. At a meeting in Mexico, we saw the amazing too good to be true Monarch butterflies filling the trees.  Her photographic skills are suberb.  She’ll live to 100.

Carl Garber just retired and gave me the address.  I just found your photo calender!

Yes, I have been hiding in Taiwan for the past almost 10 years.  I moved from diagnostic to pharma and selling mostly biosimilar products to pharmaceutical emerging countries which has strong market growth comparing to US/EU.

Pauline Lau Group

Pauline Lau Group

Pauline Lau Group

Pauline Lau Group

I do not go back to US often now.  We have an office in Taipei.  Here is a recent magazine article about our company.  You will see few of my employees and I in front of our 28th floor office window.

I am rushing out for Singapore and will be meeting there for a few days.

11.5.8  Kinetic and perfusion modeling of hyperpolarized 13C pyruvate and urea in cancer with arbitrary RF flip angles

Naeim Bahrami, Christine Leon Swisher, Cornelius Von Morze, Daniel B. Vigneron, Peder E. Z. Larson
Department of Radiology and Biomedical Imaging, University of California – San Francisco, San Francisco, CA, USA
Quant Imaging MedSurg 2014; 4(1):24-32

Abstract: The accurate detection and characterization of cancerous tissue is still a major problem for the clinical management of individual cancer patients and for monitoring their response to therapy. MRI with hyperpolarized agents is a promising technique for cancer characterization because it can non-invasively provide a local assessment of the tissue metabolic profile. In this work, we measured the kinetics of hyperpolarized [1-13C] pyruvate and 13C-urea in prostate and liver tumor models using a compressed sensing dynamic MRSI method. A kinetic model fitting method was developed that incorporated arbitrary RF flip angle excitation and measured a pyruvate to lactate conversion rate, Kpl, of 0.050 and 0.052 (1/s) in prostate and liver tumors, respectively, which was significantly higher than Kpl in healthy tissues [Kpl =0.028 (1/s), P<0.001]. Kpl was highly correlated to the total lactate to total pyruvate signal ratio (correlation coefficient =0.95). We additionally characterized the total pyruvate and urea perfusion, as in cancerous tissue there is both existing vasculature and neovascularization as different kinds of lesions surpass the normal blood supply, including small circulation disturbance in some of the abnormal vessels. A significantly higher perfusion of pyruvate (accounting for conversion to lactate and alanine) relative to urea perfusion was seen in cancerous tissues (liver cancer and prostate cancer) compared to healthy tissues (P<0.001), presumably due to high pyruvate uptake in tumors. Keywords: Hyperpolarized carbon-13; metabolic imaging; cancer; perfusion; kinetic modeling; dynamic MRSI

Hyperpolarization is the nuclear spin polarization of a material far beyond thermal equilibrium conditions. The accurate and correct diagnosis and characterization of cancer is still a major problem for the clinical management of every kind of cancer patients, including individual prostate or liver cancer patients, and also in order to monitor their response to therapy (1-3). Magnetic resonance spectroscopic imaging (MRSI) with hyperpolarized 13C labeled substrates is a new method to study any cancers that may be able to simultaneously and noninvasively assess changes in metabolic intermediates from multiple biochemical pathways of interest. Recent studies have shown a large amount of potential applications of hyperpolarized (HP) 13C MRSI for the in vivo monitoring of cellular metabolism and the characterization of disease. The low natural abundance and sensitivity of 13C compared to protons poses a technical challenge using conventional approaches (4,5). Dynamic nuclear polarization (DNP) of 13C labeled pyruvate and subsequent rapid dissolution generates a contrast agent with a four order-of-magnitude sensitivity enhancement that is injected and gives the ability to monitor the spatial distribution of pyruvate and its conversion to lactate, alanine, and bicarbonate. The conversion of pyruvate to lactate catalyzed by the enzyme lactate dehydrogenase is of particular interest, as the kinetics of this process have been shown to be sensitive to the presence and severity of disease in preclinical models (6,7). HP MRSI can also be used to measure perfusion that in cancer can reflect spatially heterogeneous changes to existing vasculature and neovascularization as tumors surpass the normal blood supply, including microcirculatory disturbance in abnormal vessels. Tumor perfusion data in addition to the metabolic data available from spectroscopic imaging of 13C pyruvate would be of important value in exploring the complex relationship between perfusion and metabolism in cancer at both preclinical and clinical research levels (8-11). The primary purpose of this research was to study the dynamics of simultaneously injected HP [1-13C]-pyruvate and 13C-urea to provide improved characterization of cancerous tissues. To achieve rapid, 2 s temporal resolution, whole mouse MRSI we used a 18-fold accelerated compressed sensing acquisition and reconstruction with smaller flip angles for pyruvate and urea compared to lactate and alanine for efficient usage of the hyperpolarized magnetization by preserving the substrate. This flip angle scheme required using a modified kinetic model that accounts for arbitrary RF flip angles (12-15). Data was acquired in mice with prostate and liver cancer and comparisons were made to normal tissues such as kidney and healthy liver of the metabolite concentrations, including Urea, Pyruvate, and Lactate, the conversion constant (Kpl) between pyruvate to lactate, and the conversion constant (Kpa) between pyruvate to alanine. We also created novel parameterizations of the total pyruvate and urea perfusions in order to assess vascular delivery and tissue uptake. A key new feature of our modeling is the ability to detect metabolic conversion, magnetization exchange between compounds, and perfusion when using arbitrary RF flip angles for different compounds.

We observed a strong correlation between Kpl and the total lactate to total pyruvate ratio, as others have also shown. The ratio is a simpler calculation and easier to implement than the kinetic modeling. However, we have determined through simulation that the total lactate to total pyruvate ratio is highly influenced by the delivery time of pyruvate, so care should be taken when using this ratio if variable vascular delivery rates are expected. Both the kinetic modeling and metabolite ratio are highly influenced by the actual RF flip angles, and precise B1 calibration is important for quantitative measurements. Measurement of urea perfusion can be a marker vascular delivery since urea primarily stays in the vasculature. Liver is a very vascular organ and the opened capillary shape of liver vasculature likely caused high urea perfusion in liver. The kidneys are highly vascularized and are also responsible for concentrating urea for removal in the urine. In tumors, the tissue request for blood is high but in a more uncontrolled way because of the abnormality of blood vasculature and circulation inside most tumors. Thus the urea perfusion in tumors is likely more sporadic and random. Urea cannot perfuse well in some parts of tumor particularly in suspected necrotic regions. On the other hand, some parts of tumor have more metabolic activity and, therefore, these parts need more blood and more vessels, and consequently should have more urea perfusion. Our total pyruvate and urea perfusion parameterizations are different from conventional perfusion modeling, and were designed as a simple representation of the total amount of these compounds that are present in the tissue. In particular, the total pyruvate perfusion also includes any pyruvate or metabolic products that remain in the tissue, in addition to those present in the vasculature. The urea perfusion should primarily represent the vasculature delivery since it primarily stays in the vessels, while the total pyruvate perfusion can also be a marker for vascular delivery but also includes tissue uptake. We hypothesize that when the pyruvate perfusion is higher relative to urea perfusion it represents a higher amount of uptake of the pyruvate that is flowing into the tissue.

Conclusions In this study we fit metabolite T1 values, conversion rates, Kpa, and Kpl, and measured novel pyruvate and urea perfusion parameterizations across cancerous and normal tissues from data acquired with a multiband RF excitation, compressed sensing dynamic MRSI pulse sequence. Our modeling allowed for use of arbitrary RF flip angles between metabolites, which in turn allows for efficient usage of the hyperpolarized magnetization. We observed a high correlation between our Kpl fits and the total lactate to pyruvate signal ratio, suggesting either could be used to characterize pyruvate-lactate metabolism. Through the novel pyruvate and urea perfusion parameterizations we were able to quantify the increased uptake of pyruvate in cancerous tissues, which correlated with increased metabolic conversion to lactate. These provided a more complete characterization of cancerous tissue metabolism and perfusion.

11.5.9  ZSTK474

(Dr. Anthony Melvin Castro)



ZSTK474 is a cell permeable and reversible P13K inhibitor with an IC₅₀ at 6nm. It was identified as part of a screening library, selected for its ability to block tumor cell growth. ZSTK474 has shown strong antitumor activities against human cancer xenographs when administered orally to mice without a significant toxic effect.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases.

However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002.

PATENT                                                                                                          SUBMITTED GRANTED

Heterocyclic compound and antitumor agent containing the same as active ingredient [US7071189]                                                                                                                                                               2004-06-17   2006-07-04

Treatment of prostate cancer, melanoma or hepatic cancer [US2007244110]                                                                                                                                                                                                   2007-10-18

Heterocyclic compound and antitumor agent containing the same as effective ingredient [US7307077]                                                                                                                                                           2006-11-02   2007-12-11

Immunosuppressive agent and anti-tumor agent comprising heterocyclic compound as active ingredient [us7750001]                                                                                                                                   2008-05-15   2010-07-06

Pyrimidinyl and 1,3,5-triazinyl benzimidazoles and their use in cancer therapy [us2011009405]                                                                                                                                                                       2011-01-13

Substituted pyrimidines and triazines and their use in cancer therapy [us2011053907]                                                                                                                                                                                     2011-03-03

Immunosuppressive agent and anti-tumor agent comprising heterocyclic compound as active ingredient [us2010267700]                                                                                                                             2010-10-21

Amorphous body composed of heterocyclic compound, solid dispersion and pharmaceutical preparation each comprising the same, and process for production of the same [us8227463]                                                                                                                                                                                                                                                                                                                                                                                                                           2010-09-30    2012-07-24

Pyrazolo[1,5-a]pyridines and their use in cancer therapy
[us2010226881]                                                                                                                                                                                                                                                                                                 2010-09-09

Pyrimidinyl and 1,3,5-triazinyl benzimidazole sulfonamides and their use in cancer therapy [us2010249099]                                                                                                                                                   2010-09-30

11.5.10 Marrow-Infiltrating Lymphocytes Safely Shrink Multiple Myelomas

 Medical researchers at the Johns Hopkins Kimmel Cancer Center have published a report that appeared in the journal Science Translational Medicine in which they describe, for the first time, the safe use of a patient’s own immune cells to treat the white blood cell cancer multiple myeloma. There are more than 20,000 new cases of multiple myeloma and more than 10,000 deaths each year in United States. It is the second most common cancer originating in the blood.

The procedure under investigation in this study is called utilizes a specific type of tumor-targeting T cells, known as marrow-infiltrating lymphocytes (MILs). “What we learned in this small trial is that large numbers of activated MILs can selectively target and kill myeloma cells,” says Johns Hopkins immunologist Ivan Borrello, M.D., who led the clinical trial.

According to Borrello, MILs are the foot soldiers of the immune system that attack invading bacteria or viruses. Unfortunately, they are typically inactive and too few in number to have a measurable effect on cancers.

Experiments conducted is Borrello’s laboratory and in the laboratory of competing and collaborating scientists have shown that when myeloma cells are exposed to activated MILs in culture, these cells could not only selectively target the tumor cells, but they could also effectively destroy them.

To move this procedure from the laboratory into the clinic, Borrello and his collaborators enrolled 25 patients with newly diagnosed or relapsed multiple myeloma. Only 22 were able to receive this new treatment, however.

The Hopkins team extracted and purified MILs from the bone marrow of each patient and grew them in the laboratory to increase their numbers. Then they activated the MILs by exposing them to microscopic beads coated with immune activating antibodies. These antibodies bind to specific cell surface proteins on the MILs that induce profound changes in the cells. This induction step wakes the MILs up and readies them to sniff out tumor cells. These laboratory-manipulated MILs were then intravenously injected back into each patient (each of the 22 patients with their own cells). Three days before these injections of expanded MILs, all patients received high doses of chemotherapy and a stem cell transplant, which are standard treatments for multiple myeloma.

One year after receiving the MILs therapy, 13 of the 22 patients had at least a partial response to the therapy (their cancers had shrunk by at least 50 percent) Seven patients experienced at least a 90 percent reduction in tumor cell volume and lived and average of 25.1 months without cancer progression. The remaining 15 patients had an average of 11.8 progression-free months following their MIL therapy. None of the participants experienced serious side effects from the MIL therapy.

According to Borrello, several U.S. cancer centers have conducted similar experimental treatments (adoptive T cell therapy). However, only this Johns Hopkins team has used MILs. Other types of tumor-infiltrating cells can be used for such treatments, but Borrello noted that these cells are usually less plentiful in patients’ tumors and may not grow as well outside the body.

In nonblood-based tumors, such as melanoma, only about half of those patients have T cells in their tumors that can be harvested, and only about one-half of those harvested cells can be grown. “Typically, immune cells from solid tumors, called tumor-infiltrating lymphocytes, can be harvested and grown in only about 25 percent of patients who could potentially be eligible for the therapy. But in our clinical trial, we were able to harvest and grow MILs from all 22 patients,” says Kimberly Noonan, Ph.D., a research associate at the Johns Hopkins Universit School of Medicine.

This small trial helped Noonan and her colleagues learn more about which patients may benefit from MILs therapy. As an example, they were able to determine how many of the MILs grown in the lab were specifically targeted to the patient’s tumor and whether they continued to target the tumor after being infused. They also found that patients whose bone marrow before treatment contained a high number of certain immune cells, known as central memory cells, also had better response to MILs therapy. Patients who began treatment with signs of an overactive immune response did not respond as well.

Noonan says the research team has used these data to guide two other ongoing MILs clinical trials. Those studies, she says, are trying to extend anti-tumor response and tumor specificity by combining the MILs transplant with a Johns Hopkins-developed cancer vaccine called GVAX and the myeloma druglenalidomide, which stimulates T cell responses.

These trials also have elucidated new ways to grow the MILs. “In most of these trials, you see that the more cells you get, the better response you get in patients. Learning how to improve cell growth may therefore improve the therapy,” says Noonan.

Kimmel Cancer Center scientists are also developing MILs treatments to address solid tumors such as lung, esophageal and gastric cancers, as well as the pediatric cancers neuroblastoma and Ewing’s sarcoma.

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Warburg Effect and Mitochondrial Regulation -2.1.3

Writer and Curator: Larry H Bernstein, MD, FCAP 

2.1.3 Warburg Effect and Mitochondrial Regulation Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier

NM Vacanti, AS Divakaruni, CR Green, SJ Parker, RR Henry, TP Ciaraldi, et a..
Molec Cell 6 Nov 2014; 56(3):425–435


  • Oxidation of fatty acids and amino acids is increased upon MPC inhibition
    •Respiration, proliferation, and biosynthesis are maintained when MPC is inhibited
    •Glutaminolytic flux supports lipogenesis in the absence of MPC
    •MPC inhibition is distinct from hypoxia or complex I inhibition


Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied 13C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.



Graphical Abstract – Oxidation of fatty acids and amino acids is increased upon MPC inhibition

Figure 2. MPC Regulates Mitochondrial Substrate Utilization (A) Citrate mass isotopomer distribution (MID) resulting from culture with [U-13C6]glucose (UGlc). (B) Percentage of 13C-labeled metabolites from UGlc. (C) Percentage of fully labeled lactate, pyruvate, and alanine from UGlc. (D) Serine MID resulting from culture with UGlc. (E) Percentage of fully labeled metabolites derived from [U-13C5]glutamine (UGln). (F) Schematic of UGln labeling of carbon atoms in TCA cycle intermediates arising via glutaminoloysis and reductive carboxylation. Mitochondrion schematic inspired by Lewis et al. (2014). (G and H) Citrate (G) and alanine (H) MIDs resulting from culture with UGln. (I) Maximal oxygen consumption rates with or without 3 mM BPTES in medium supplemented with 1 mM pyruvate. (J) Percentage of newly synthesized palmitate as determined by ISA. (K) Contribution of UGln and UGlc to lipogenic AcCoA as determined by ISA. (L) Contribution of glutamine to lipogenic AcCoA via glutaminolysis (ISA using a [3-13C] glutamine [3Gln]) and reductive carboxylation (ISA using a [5-13C]glutamine [5Gln]) under normoxia and hypoxia. (M) Citrate MID resulting from culture with 3Gln. (N) Contribution of UGln and exogenous [3-13C] pyruvate (3Pyr) to lipogenic AcCoA. 2KD+Pyr refers to Mpc2KD cells cultured with 10 mM extracellular pyruvate. Error bars represent SD (A–E, G, H, and M), SEM(I), or 95% confidence intervals(J–L, and N).*p<0.05,**p<0.01,and ***p<0.001 by ANOVA with Dunnett’s post hoc test (A–E and G–I) or * indicates significance by non-overlapping 95% confidence intervals (J–L and N).

Figure 3. Mpc Knockdown Increases Fatty Acid Oxidation. (A) Schematic of changes in flux through metabolic pathways in Mpc2KD relative to control cells. (B) Citrate MID resulting from culture with [U-13C16] palmitate conjugated to BSA (UPalm). (C) Percentage of 13C enrichment resulting from culture with UPalm. (D) ATP-linked and maximal oxygen consumption rate, with or without 20m Metomoxir, with or without 3 mM BPTES. Culture medium supplemented with 0.5 mM carnitine. Error bars represent SD (B and C) or SEM (D). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed, equal variance, Student’s t test(B–D), or by ANOVA with Dunnett’s post hoc test (D).

Figure 4. Metabolic Reprogramming Resulting from Pharmacological Mpc Inhibition Is Distinct from Hypoxia or Complex I Inhibition Oxidation of Alpha-Ketoglutarate Is Required for Reductive Carboxylation in Cancer Cells with Mitochondrial Defects

AR Mullen, Z Hu, X Shi, L Jiang, …, WM Linehan, NS Chandel, RJ DeBerardinis
Cell Reports 12 Jun 2014; 7(5):1679–1690


  • Cells with mitochondrial defects use bidirectional metabolism of the TCA cycle
    •Glutamine supplies the succinate pool through oxidative and reductive metabolism
    •Oxidative TCA cycle metabolism is required for reductive citrate formation
    •Oxidative metabolism produces reducing equivalents for reductive carboxylation


Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse.

Proliferating cells support their growth by converting abundant extracellular nutrients like glucose and glutamine into precursors for macromolecular biosynthesis. A continuous supply of metabolic intermediates from the tricarboxylic acid (TCA) cycle is essential for cell growth, because many of these intermediates feed biosynthetic pathways to produce lipids, proteins and nucleic acids (Deberardinis et al., 2008). This underscores the dual roles of the TCA cycle for cell growth: it generates reducing equivalents for oxidative phosphorylation by the electron transport chain (ETC), while also serving as a hub for precursor production. During rapid growth, the TCA cycle is characterized by large influxes of carbon at positions other than acetyl-CoA, enabling the cycle to remain full even as intermediates are withdrawn for biosynthesis. Cultured cancer cells usually display persistence of TCA cycle activity despite robust aerobic glycolysis, and often require mitochondrial catabolism of glutamine to the TCA cycle intermediate AKG to maintain rapid rates of proliferation (Icard et al., 2012Hiller and Metallo, 2013).

Some cancer cells contain severe, fixed defects in oxidative metabolism caused by mutations in the TCA cycle or the ETC. These include mutations in fumarate hydratase (FH) in renal cell carcinoma and components of the succinate dehydrogenase (SDH) complex in pheochromocytoma, paraganglioma, and gastrointestinal stromal tumors (Tomlinson et al., 2002Astuti et al., 2001Baysal et al., 2000Killian et al., 2013Niemann and Muller, 2000). All of these mutations alter oxidative metabolism of glutamine in the TCA cycle. Recently, analysis of cells containing mutations in FH, ETC Complexes I or III, or exposed to the ETC inhibitors metformin and rotenone or the ATP synthase inhibitor oligomycin revealed that turnover of TCA cycle intermediates was maintained in all cases (Mullen et al., 2012). However, the cycle operated in an unusual fashion characterized by conversion of glutamine-derived AKG to isocitrate through a reductive carboxylation reaction catalyzed by NADP+/NADPH-dependent isoforms of isocitrate dehydrogenase (IDH). As a result, a large fraction of the citrate pool carried five glutamine-derived carbons. Citrate could be cleaved to produce acetyl-CoA to supply fatty acid biosynthesis, and oxaloacetate (OAA) to supply pools of other TCA cycle intermediates. Thus, reductive carboxylation enables biosynthesis by enabling cells with impaired mitochondrial metabolism to maintain pools of biosynthetic precursors that would normally be supplied by oxidative metabolism. Reductive carboxylation is also induced by hypoxia and by pseudo-hypoxic states caused by mutations in the von Hippel-Lindau (VHL) tumor suppressor gene (Metallo et al., 2012Wise et al., 2011).

Interest in reductive carboxylation stems in part from the possibility that inhibiting the pathway might induce selective growth suppression in tumor cells subjected to hypoxia or containing mutations that prevent them from engaging in maximal oxidative metabolism. Hence, several recent studies have sought to understand the mechanisms by which this pathway operates. In vitro studies of IDH1 indicate that a high ratio of NADPH/NADP+ and low citrate concentration activate the reductive carboxylation reaction (Leonardi et al., 2012). This is supported by data demonstrating that reductive carboxylation in VHL-deficient renal carcinoma cells is associated with a low concentration of citrate and a reduced ratio of citrate:AKG, suggesting that mass action can be a driving force to determine IDH directionality (Gameiro et al., 2013b). Moreover, interrupting the supply of mitochondrial NADPH by silencing the nicotinamide nucleotide transhydrogenase (NNT) suppresses reductive carboxylation (Gameiro et al., 2013a). This mitochondrial transmembrane protein catalyzes the transfer of a hydride ion from NADH to NADP+ to generate NAD+ and NADPH. Together, these observations suggest that reductive carboxylation is modulated in part through the mitochondrial redox state and the balance of substrate/products.

Here we used metabolomics and stable isotope tracing to better understand overall metabolic states associated with reductive carboxylation in cells with defective mitochondrial metabolism, and to identify sources of mitochondrial reducing equivalents necessary to induce the reaction. We identified high levels of succinate in some cells using reductive carboxylation, and determined that most of this succinate was formed through persistent oxidative metabolism of AKG. Silencing this oxidative flux by depleting the mitochondrial enzyme AKG dehydrogenase substantially altered the cellular redox state and suppressed reductive carboxylation. The data demonstrate that bidirectional/branched AKG metabolism occurs during reductive carboxylation in cells with mitochondrial defects, with oxidative metabolism producing reducing equivalents to supply reductive metabolism.

Shared metabolomic features among cell lines with cytb or FH mutations

To identify conserved metabolic features associated with reductive carboxylation in cells harboring defective mitochondrial metabolism, we analyzed metabolite abundance in isogenic pairs of cell lines in which one member displayed substantial reductive carboxylation and the other did not. We used a pair of previously described cybrids derived from 143B osteosarcoma cells, in which one cell line contained wild-type mitochondrial DNA (143Bwt) and the other contained a mutation in the cytb gene (143Bcytb), severely reducing complex III function (Rana et al., 2000Weinberg et al., 2010). The 143Bwt cells primarily use oxidative metabolism to supply the citrate pool while the 143Bcytb cells use reductive carboxylation (Mullen et al., 2012). The other pair, derived from FH-deficient UOK262 renal carcinoma cells, contained either an empty vector control (UOK262EV) or a stably re-expressed wild-type FH allele (UOK262FH). Metabolites were extracted from all four cell lines and analyzed by triple-quadrupole mass spectrometry. We first performed a quantitative analysis to determine the abundance of AKG and citrate in the four cell lines. Both 143Bcytb and UOK262EV cells had less citrate, more AKG, and lower citrate:AKG ratios than their oxidative partners (Fig. S1A-C), consistent with findings from VHL-deficient renal carcinoma cells (Gameiro et al., 2013b).

Next, to identify other perturbations, we profiled the relative abundance of more than 90 metabolites from glycolysis, the pentose phosphate pathway, one-carbon/nucleotide metabolism, the TCA cycle, amino acid degradation, and other pathways (Tables S1 and S2). Each metabolite was normalized to protein content, and relative abundance was determined between cell lines from each pair. Hierarchical clustering (Fig 1A) and principal component analysis (Fig 1B) revealed far greater metabolomic similarities between the members of each pair than between the two cell lines using reductive carboxylation. Only three metabolites displayed highly significant (p<0.005) differences in abundance between the two members of both pairs, and in all three cases the direction of the difference (i.e. higher or lower) was shared in the two cell lines using reductive carboxylation. Proline, a nonessential amino acid derived from glutamine in an NADPH-dependent biosynthetic pathway, was depleted in 143Bcytb and UOK262EV cells (Fig. 1C). 2-hydroxyglutarate (2HG), the reduced form of AKG, was elevated in 143Bcytb and UOK262EV cells (Fig. 1D), and further analysis revealed that while both the L- and D-enantiomers of this metabolite were increased, L-2HG was quantitatively the predominant enantiomer (Fig. S1D). It is likely that 2HG accumulation was related to the reduced redox ratio associated with cytb and FH mutations. Although the sources of 2HG are still under investigation, promiscuous activity of the TCA cycle enzyme malate dehydrogenase produces L-2HG in an NADH-dependent manner (Rzem et al., 2007). Both enantiomers are oxidized to AKG by dehydrogenases (L-2HG dehydrogenase and D-2HG dehydrogenase). It is therefore likely that elevated 2-HG is a consequence of a reduced NAD+/NADH ratio. Consistent with this model, inborn errors of the ETC result in 2-HG accumulation (Reinecke et al., 2011). Exposure to hypoxia (<1% O2) has also been demonstrated to reduce the cellular NAD+/NADH ratio (Santidrian et al., 2013) and to favor modest 2HG accumulation in cultured cells (Wise et al., 2011), although these levels were below those noted in gliomas expressing 2HG-producing mutant alleles of isocitrate dehydrogenase-1 or -2 (Dang et al., 2009).

Figure 1 Metabolomic features of cells using reductive carboxylation


Finally, the TCA cycle intermediate succinate was markedly elevated in both cell lines (Fig. 1E). We tested additional factors previously reported to stimulate reductive AKG metabolism, including a genetic defect in ETC Complex I, exposure to hypoxia, and chemical inhibitors of the ETC (Mullen et al., 2012Wise et al., 2011Metallo et al., 2012). These factors had a variable effect on succinate, with impairments of Complex III or IV strongly inducing succinate accumulation, while impairments of Complex I either had little effect or suppressed succinate (Fig. 1F).

Oxidative glutamine metabolism is the primary route of succinate formation

UOK262EV cells lack FH activity and accumulate large amounts of fumarate (Frezza et al., 2011); elevated succinate was therefore not surprising in these cells, because succinate precedes fumarate by one reaction in the TCA cycle. On the other hand, TCA cycle perturbation in 143Bcytb cells results from primary ETC dysfunction, and reductive carboxylation is postulated to be a consequence of accumulated AKG (Anastasiou and Cantley, 2012Fendt et al., 2013). Accumulation of AKG is not predicted to result in elevated succinate. We previously reported that 143Bcytb cells produce succinate through simultaneous oxidative and reductive glutamine metabolism (Mullen et al., 2012). To determine the relative contributions of these two pathways, we cultured 143Bwt and 143Bcytb with [U-13C]glutamine and monitored time-dependent 13C incorporation in succinate and other TCA cycle intermediates. Oxidative metabolism of glutamine generates succinate, fumarate and malate containing four glutamine-derived 13C nuclei on the first turn of the cycle (m+4), while reductive metabolism results in the incorporation of three 13C nuclei in these intermediates (Fig. S2). As expected, oxidative glutamine metabolism was the predominant source of succinate, fumarate and malate in 143Bwt cells (Fig. 2A-C). In 143Bcytb, fumarate and malate were produced primarily through reductive metabolism (Fig. 2E-F). Conversely, succinate was formed primarily through oxidative glutamine metabolism, with a minor contribution from the reductive carboxylation pathway (Fig. 2D). Notably, this oxidatively-derived succinate was detected prior to that formed through reductive carboxylation. This indicated that 143Bcytb cells retain the ability to oxidize AKG despite the observation that most of the citrate pool bears the labeling pattern of reductive carboxylation. Together, the labeling data in 143Bcytb cells revealed bidirectional metabolism of carbon from glutamine to produce various TCA cycle intermediates.

Figure 2  Oxidative glutamine metabolism is the primary route of succinate formation in cells using reductive carboxylation to generate citrate

Pyruvate carboxylation contributes to the TCA cycle in cells using reductive carboxylation

Because of the persistence of oxidative metabolism, we determined the extent to which other routes of metabolism besides reductive carboxylation contributed to the TCA cycle. We previously reported that silencing the glutamine-catabolizing enzyme glutaminase (GLS) depletes pools of fumarate, malate and OAA, eliciting a compensatory increase in pyruvate carboxylase (PC) to supply the TCA cycle (Cheng et al., 2011). In cells with defective oxidative phophorylation, production of OAA by PC may be preferable to glutamine oxidation because it diminishes the need to recycle reduced electron carriers generated by the TCA cycle. Citrate synthase (CS) can then condense PC-derived OAA with acetyl-CoA to form citrate. To examine the contribution of PC to the TCA cycle, cells were cultured with [3,4-13C]glucose. In this labeling scheme, glucose-derived pyruvate is labeled in carbon 1 (Fig. S3). This label is retained in OAA if pyruvate is carboxylated, but removed as CO2 during conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH).

Figure 3 Pyruvate carboxylase contributes to citrate formation in cells using reductive carboxylation

Oxidative metabolism of AKG is required for reductive carboxylation

Oxidative synthesis of succinate from AKG requires two reactions: the oxidative decarboxylation of AKG to succinyl-CoA by AKG dehydrogenase, and the conversion of succinyl-CoA to succinate by succinyl-CoA synthetase. In tumors with mutations in the succinate dehydrogenase (SDH) complex, large accumulations of succinate are associated with epigenetic modifications of DNA and histones to promote malignancy (Kaelin and McKnight, 2013Killian et al., 2013). We therefore tested whether succinate accumulation per se was required to induce reductive carboxylation in 143Bcytb cells. We used RNA interference directed against the gene encoding the alpha subunit (SUCLG1) of succinyl-CoA synthetase, the last step in the pathway of oxidative succinate formation from glutamine (Fig. 4A). Silencing this enzyme greatly reduced succinate levels (Fig. 4B), but had no effect on the labeling pattern of citrate from [U-13C]glutamine (Fig. 4C). Thus, succinate accumulation is not required for reductive carboxylation.

Figure 5 AKG dehydrogenase is required for reductive carboxylation

Figure 6 AKG dehydrogenase and NNT contribute to NAD+/NADH ratio

Finally, we tested whether these enzymes also controlled the NADP+/NADPH ratio in 143Bcytb cells. Silencing either OGDH or NNT increased the NADP+/NADPH ratio (Fig. 6F,G), whereas silencing IDH2reduced it (Fig. 6H). Together, these data are consistent with a model in which persistent metabolism of AKG by AKG dehydrogenase produces NADH that supports reductive carboxylation by serving as substrate for NNT-dependent NADPH formation, and that IDH2 is a major consumer of NADPH during reductive carboxylation (Fig. 6I).

Reductive carboxylation of AKG initiates a non-conventional form of metabolism that produces TCA cycle intermediates when oxidative metabolism is impaired by mutations, drugs or hypoxia. Because NADPH-dependent isoforms of IDH are reversible, supplying supra-physiological pools of substrates on either side of the reaction drives function of the enzyme as a reductive carboxylase or an oxidative decarboxylase. Thus, in some circumstances reductive carboxylation may operate in response to a mass effect imposed by drastic changes in the abundance of AKG and isocitrate/citrate. However, reductive carboxylation cannot occur without a source of reducing equivalents to produce NADPH. The current work demonstrates that AKG dehydrogenase, an NADH-generating enzyme complex, is required to maintain a low NAD+/NADH ratio for reductive carboxylation of AKG. Thus, reductive carboxylation not only coexists with oxidative metabolism of AKG, but depends on it. Furthermore, silencing NNT, a consumer of NADH, also perturbs the redox ratio and suppresses reductive formation of citrate. These observations suggest that the segment of the oxidative TCA cycle culminating in succinate is necessary to transmit reducing equivalents to NNT for the reductive pathway (Fig 6I).

Succinate accumulation was observed in cells with cytb or FH mutations. However, this accumulation was dispensable for reductive carboxylation, because silencing SUCLG1 expression had no bearing on the pathway as long as AKG dehydrogenase was active. Furthermore, succinate accumulation was not a universal finding of cells using reductive carboxylation. Rather, high succinate levels were observed in cells with distal defects in the ETC (complex III: antimycin, cytb mutation; complex IV: hypoxia) but not defects in complex I (rotenone, metformin, NDUFA1 mutation). These differences reflect the known suppression of SDH activity when downstream components of the ETC are impaired, and the various mechanisms by which succinate may be formed through either oxidative or reductive metabolism. Succinate has long been known as an evolutionarily conserved anaerobic end product of amino acid metabolism during prolonged hypoxia, including in diving mammals (Hochachka and Storey, 1975, Hochachka et al., 1975). The terminal step in this pathway is the conversion of fumarate to succinate using the NADH-dependent “fumarate reductase” system, essentially a reversal of succinate dehydrogenase/ETC complex II (Weinberg et al., 2000, Tomitsuka et al., 2010). However, this process requires reducing equivalents to be passed from NADH to complex I, then to Coenzyme Q, and eventually to complex II to drive the reduction of fumarate to succinate. Hence, producing succinate through reductive glutamine metabolism would require functional complex I. Interestingly, the fumarate reductase system has generally been considered as a mechanism to maintain a proton gradient under conditions of defective ETC activity. Our data suggest that the system is part of a more extensive reorganization of the TCA cycle that also enables reductive citrate formation.

In summary, we demonstrated that branched AKG metabolism is required to sustain levels of reductive carboxylation observed in cells with mitochondrial defects. The organization of this branched pathway suggests that it serves as a relay system to maintain the redox requirements for reductive carboxylation, with the oxidative arm producing reducing equivalents at the level of AKG dehydrogenase and NNT linking this activity to the production of NADPH to be used in the reductive carboxylation reaction. Hence, impairment of the oxidative arm prevents maximal engagement of reductive carboxylation. As both NNT and AKG dehydrogenase are mitochondrial enzymes, the work emphasizes the flexibility of metabolic systems in the mitochondria to fulfill requirements for redox balance and precursor production even when the canonical oxidative function of the mitochondria is impaired. Rewiring Mitochondrial Pyruvate Metabolism. Switching Off the Light in Cancer Cells

Peter W. Szlosarek, Suk Jun Lee, Patrick J. Pollard
Molec Cell 6 Nov 2014; 56(3): 343–344

Figure 1. MPC Expression and Metabolic Targeting of Mitochondrial Pyruvate High MPC expression (green) is associated with more favorable tumor prognosis, increased pyruvate oxidation, and reduced lactate and ROS, whereas low expression or mutated MPC is linked to poor tumor prognosis and increased anaplerotic generation of OAA. Dual targeting of MPC and GDH with small molecule inhibitors may ameliorate tumorigenesis in certain cancer types.

The study by Yang et al., (2014) provides evidence for the metabolic flexibility to maintain TCA cycle function. Using isotopic labeling, the authors demonstrated that inhibition of MPCs by a specific compound (UK5099) induced glutamine-dependent acetyl-CoA formation via glutamate dehydrogenase (GDH). Consequently, and in contrast to single agent treatment, simultaneous administration of MPC and GDH inhibitors drastically abrogated the growth of cancer cells (Figure 1). These studies have also enabled a fresh perspective on metabolism in the clinic and emphasized a need for high-quality translational studies to assess the role of mitochondrial pyruvate transport in vivo. Thus, integrating the biomarker of low MPC expression with dual inhibition of

MPC and GDH as a synthetic lethal strategy (Yang et al., 2014) is testable and may offer a novel therapeutic window for patients (DeBerardinis and Thompson, 2012). Indeed, combinatorial targeting of cancer metabolism may prevent early drug resistance and lead to enhanced tumor control, as shown recently for antifolate agents combined with arginine deprivation with modulation of intracellular glutamine (Szlosarek, 2014). Moreover, it will be important to assess both intertumoral and intratumoral metabolic heterogeneity going forward, as tumor cells are highly adaptable with respect to the precursors used to fuel the TCA cycle in the presence of reduced pyruvate transport. The observation by Vacanti et al. (2014) that the flux of BCAAs increased following inhibition of MPC activity may also underlie the increase in BCAAs detected in the plasma of patients several years before a clinical diagnosis of pancreatic cancer (Mayers et al., 2014). Since measuring pyruvate transport via the MPC is technically challenging, the use of 18-FDG positron emission tomography and more recently magnetic spectroscopy with hyperpolarized 13C-labeled pyruvate will need to be incorporated into these future studies (Brindle et al., 2011).


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Yang, C., Ko, B., Hensley, C.T., Jiang, L., Wasti, A.T., et al. (2014). Mol. Cell 56, this issue, 414–424. Betaine is a positive regulator of mitochondrial respiration

Lee I
Biochem Biophys Res Commun. 2015 Jan 9; 456(2):621-5.


  • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration.
    • Betaine increases mitochondrial membrane potential and cellular energy levels.
    • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect.

Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect. Mitochondrial dysfunction in human non-small-cell lung cancer cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Y-L Shi, S Feng, W Chen, Z-C Hua, J-J Bian and W Yin
Cell Death and Disease (2014) 5, e1579

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-XL overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-XL downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-XL and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-XL and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.

Abbreviations: c-FLIP, cellular FLICE-like inhibitory protein; DHE, dihydroethidium; DISC, death-inducing signaling complex; DPI, diphenylene iodonium; DR4/DR5, death receptor 4/5; EB, ethidium bromide; FADD, Fas-associated protein with death domain; MnSOD, manganese superoxide; NAC, N-acetylcysteine; NSCLC, non-small-cell lung carcinoma; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; UPR, unfolded protein response.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.1 However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.23 Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,4 which highlights the need for more effective treatment strategies.

TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.5 Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.6 The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.7 Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.891011

Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,1213overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.2 Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.5 Many sensitizing agents have been developed in a variety of tumor cell models.2 Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.

Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.141516 Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.1718192021 However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.22

In our previous investigation, we found that rotenone was able to suppress membrane Na+,K+-ATPase activity and enhance ouabain-induced cancer cell death.23 Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.

Rotenone sensitizes NSCLC cell lines to TRAIL-induced apoptosis

Four NSCLC cell lines including A549, H522, H157 and Calu-1 were used in this study. As shown in Figure 1a, the apoptosis induced by TRAIL alone at 50 or 100 ng/ml on A549, H522, H157 and Calu-1 cells was non-prevalent, indicating that these NSCLC cell lines are relatively TRAIL resistant. Interestingly, when these cells were treated with TRAIL combined with rotenone, significant increase in cell apoptosis was observed. To examine whether rotenone was also able to sensitize normal cells to TRAIL-mediated apoptosis, peripheral blood mononuclear cell (PBMC) isolated from human blood were used. As a result, rotenone failed to sensitize human PBMC to TRAIL-induced apoptosis, indicating that the sensitizing effect of rotenone is tumor cell specific. Of note, the apoptosis-enhancing effect of rotenone occurred independent of its cytotoxicity, because the minimal dosage required for rotenone to cause toxic effect on NSCLC cell lines was 10 μM, however, rotenone augmented TRAIL-mediated apoptosis when it was used as little as 10 nM.

Figure 1.

Full figure and legend (310K)
To further confirm the effect of rotenone, cells were stained with Hoechst and observed under fluorescent microscope (Figure 1b). Consistently, the combined treatment of rotenone with TRAIL caused significant nuclear fragmentation in A549, H522, H157 and Calu-1 cells. Rotenone or TRAIL treatment alone, however, had no significant effect.

Caspases activation is a hallmark of cell apoptosis. In this study, the enzymatic activities of caspases including caspase-3, -8 and -9 were measured by flow cytometry by using FITC-conjugated caspases substrate (Figure 1c). As a result, rotenone used at 1 μM or TRAIL used at 100 ng/ml alone did not cause caspase-3, -8 and -9 activation. The combined treatment, however, significantly increased the enzymatic activities of them. Moreover, A549 or H522 cell apoptosis by TRAIL combined with rotenone was almost completely suppressed in the presence of z-VAD.fmk, a pan-caspase inhibitor (Figure 1d). All of these data indicate that both intrinsic and extrinsic pathways are involved in the sensitizing effect of rotenone on TRAIL-mediated apoptosis in NSCLC.

Upregulation of death receptors expression is required for rotenone-mediated sensitization to TRAIL-induced apoptosis

Sensitization to TRAIL-induced apoptosis has been explained in some studies by upregulation of death receptors,24 whereas other results show that sensitization can occur without increased TRAIL receptor expression.25 As such, we examined TRAIL receptors expression on NSCLC cells after treatment with rotenone. Rotenone increased DR4 and DR5 mRNA levels in A549 cells in a time or concentration-dependent manner (Figures 2a and b), also increased DR4 and DR5 protein expression levels (Supplementary Figure S1). Notably, rotenone failed to increase DR5 mRNA levels in H157 and Calu-1 cells (Supplementary Figure S2). To observe whether the increased DR4 and DR5 mRNA levels finally correlated with the functional molecules, we examined the surface expression levels of DR4 and DR5 by flow cytometry. The results, as shown in Figure 2c demonstrated that the cell surface expression levels of DR4 and DR5 were greatly upregulated by rotenone in either A549 cells or H522 cells.

Figure 2.

Full figure and legend (173K)

To analyze whether the upregulation of DR4 and DR5 is a ‘side-effect’, or contrarily, necessary for rotenone-mediated sensitization to TRAIL-induced apoptosis, we blocked upregulation of the death receptors by small interfering RNAs (siRNAs) against DR4 and DR5 (Supplementary Figure S3). The results showed that blocking DR4 and DR5 expression alone significantly reduced the rate of cell apoptosis in A549 cells (Figure 2d). However, the highest inhibition of apoptosis was observed when upregulation of both receptors was blocked in parallel, thus showing an additive effect of blocking DR4 and DR5 at the same time. Similar results were also obtained in H522 cells

To analyze whether the upregulation of DR4 and DR5 is a ‘side-effect’, or contrarily, necessary for rotenone-mediated sensitization to TRAIL-induced apoptosis, we blocked upregulation of the death receptors by small interfering RNAs (siRNAs) against DR4 and DR5 (Supplementary Figure S3). The results showed that blocking DR4 and DR5 expression alone significantly reduced the rate of cell apoptosis in A549 cells (Figure 2d). However, the highest inhibition of apoptosis was observed when upregulation of both receptors was blocked in parallel, thus showing an additive effect of blocking DR4 and DR5 at the same time. Similar results were also obtained in H522 cells.

Rotenone-induced p53 activation regulates death receptors upregulation

TRAIL receptors DR4 and DR5 are regulated at multiple levels. At transcriptional level, studies suggest that several transcriptional factors including NF-κB, p53 and AP-1 are involved in DR4 or DR5 gene transcription.2 The NF-κB or AP-1 transcriptional activity was further modulated by ERK1/2, JNK and p38 MAP kinase activity. Unexpectedly, we found here that none of these MAP kinases inhibitors were able to suppress the apoptosis mediated by TRAIL plus rotenone (Figure 3a). To find out other possible mechanisms, we observed that rotenone was able to stimulate p53 phosphorylation as well as p53 protein expression in A549 and H522 cells (Figure 3b). As a p53-inducible gene, p21 mRNA expression was also upregulated by rotenone treatment in a time-dependent manner (Figure 3c). To characterize the effect of p53, A549 cells were transfected with p53 siRNA. The results, as shown in Figure 3d-1 demonstrated that rotenone-mediated surface expression levels of DR4 and DR5 in A549 cells were largely attenuated by siRNA-mediated p53 expression silencing. Control siRNA, however, failed to reveal such effect. Similar results were also obtained in H522 cells (Figure 3d-2). Silencing of p53 expression in A549 cells also partially suppressed the apoptosis induced by TRAIL plus rotenone (Figure 3e).

Rotenone suppresses c-FLIP expression and increases the sensitivity of A549 cells to TRAIL-induced apoptosis

The c-FLIP protein has been commonly appreciated as an anti-apoptotic molecule in death receptor-mediated cell apoptosis. In this study, rotenone treatment led to dose-dependent downregulation of c-FLIP expression, including c-FLIPL and c-FLIPs in A549 cells (Figure 4a-1), H522 cells (Figure 4a-2), H441 and Calu-1 cells (Supplementary Figure S4). To test whether c-FLIP is essential for the apoptosis enhancement, A549 cells were transfected with c-FLIPL-overexpressing plasmids. As shown in Figure 4b-1, the apoptosis of A549 cells after the combined treatment was significantly reduced when c-FLIPL was overexpressed. Similar results were also obtained in H522 cells (Figure 4b-2).

Bcl-XL is involved in the apoptosis enhancement by rotenone

Notably, c-FLIP downregulation by rotenone in NSCLC cells was irrelevant to p53 signaling (data not shown). To identify other mechanism involved, we found that anti-apoptotic molecule Bcl-XL was also found to be downregulated by rotenone in a dose-dependent manner (Figure 5a). Notably, both Bcl-XL and c-FLIPL mRNA levels remained unchanged in cells after rotenone treatment (Supplementary Figure S5). Bcl-2 is homolog to Bcl-XL. But surprisingly, Bcl-2 expression was almost undetectable in A549 cells. To examine whether Bcl-XL is involved, A549 cells were transfected with Bcl-XL-overexpressing plasmid. As compared with mock transfectant, cell apoptosis induced by TRAIL plus rotenone was markedly suppressed under the condition of Bcl-XL overexpression (Figure 5b). To characterize the mechanisms, surface expression levels of DR4 and DR5 were examined. As shown in Figure 5c, the increased surface expression of DR4 and DR5 in A549 cells, or in H522 cells were greatly reduced after Bcl-XLoverexpression (Figure 5c). In addition, Bcl-XL overexpression also significantly prevented the downregulation of c-FLIPL and c-FLIPs expression in A549 cells by rotenone treatment (Figure 5d).

Rotenone suppresses the interaction between BCL-XL/p53 and increases PUMA transcription

Lines of evidence suggest that Bcl-XL has a strong binding affinity with p53, and can suppress p53-mediated tumor cell apoptosis.26 In this study, FLAG-tagged Bcl-XL and HA-tagged p53 were co-transfected into cells; immunoprecipitation experiment was performed by using FLAG antibody to immunoprecipitate HA-tagged p53. As a result, we found that at the same amount of p53 protein input, rotenone treatment caused a concentration-dependent suppression of the protein interaction between Bcl-XL and p53 (Figure 6a). Rotenone also significantly suppressed the interaction between endogenous Bcl-XL and p53 when polyclonal antibody against p53 was used to immunoprecipitate cellular Bcl-XL (Figure 6b). Recent study highlighted the importance of PUMA in BCL-XL/p53 interaction and cell apoptosis.27 We found here that rotenone significantly increased PUMA gene transcription (Figure 6c) and protein expression (Figure 6d) in NSCLC cells, but not in transformed 293T cell. Meanwhile, this effect was attenuated by silencing of p53 expression (Figure 6e).

Mitochondria-derived ROS are responsible for the apoptosis-enhancing effect of rotenone

As an inhibitor of mitochondrial respiration, rotenone was found to induce reactive oxygen species (ROS) generation in a variety of transformed or non-transformed cells.2022 Consistently, by using 2′,7′-dichlorofluorescin diacetate (DCFH) for the measurement of intracellular H2O2 and dihydroethidium (DHE) for O2.−, we found that rotenone significantly triggered the .generation of H2O2(Figure 7a) and O2.− (Figure 7b) in A549 and H522 cells. To identify the origin of ROS production, we first incubated cells with diphenylene iodonium (DPI), a potent inhibitor of plasma membrane NADP/NADPH oxidase. The results showed that DPI failed to suppress rotenone-induced ROS generation (Figure 7c). Then, we generated A549 cells deficient in mitochondria DNA by culturing cells in medium supplemented with ethidium bromide (EB). These mtDNA-deficient cells were subject to rotenone treatment, and the result showed that rotenone-induced ROS production were largely attenuated in A549 ρ° cells, but not wild-type A549 cells, suggesting ROS are mainly produced from mitochondria (Figure 7d). Notably, the sensitizing effect of rotenone on TRAIL-induced apoptosis in A549 cells was largely dependent on ROS, because the antioxidant N-acetylcysteine (NAC) treatment greatly suppressed the cell apoptosis, as shown in annexin V/PI double staining experiment (Figure 7e), cell cycle analysis (Figure 7f) and caspase-3 cleavage activity assay (Figure 7g). Finally, in A549 cells stably transfected with manganese superoxide (MnSOD) and catalase, apoptosis induced by TRAIL and rotenone was partially reversed (Figure 7h). All of these data suggest that mitochondria-derived ROS, including H2O2 and O2.−, are responsible for the apoptosis-enhancing effect of rotenone.

Rotenone promotes BCl-XL degradation and PUMA transcription in ROS-dependent manner

To understand why ROS are responsible for the apoptosis-enhancing effect of rotenone, we found that rotenone-induced suppression of BCL-XL expression can be largely reversed by NAC treatment (Figure 8a). To examine whether this effect of rotenone occurs at posttranslational level, we used cycloheximide (CHX) to halt protein synthesis, and found that the rapid degradation of Bcl-XL by rotenone was largely attenuated in A549 ρ0 cells (Figure 8b). Similarly, rotenone-induced PUMA upregulation was also significantly abrogated in A549 ρ0 cells (Figure 8c). Finally, A549 cells were inoculated into nude mice to produce xenografts tumor model. In this model, the therapeutic effect of TRAIL combined with rotenone was evaluated. Notably, in order to circumvent the potential neurotoxic adverse effect of rotenone, mice were challenged with rotenone at a low concentration of 0.5 mg/kg. The results, as shown in Figure 8d revealed that while TRAIL or rotenone alone remained unaffected on A549 tumor growth, the combined therapy significantly slowed down the tumor growth. Interestingly, the tumor-suppressive effect of TRAIL plus rotenone was significantly attenuated by NAC (P<0.01). After experiment, tumors were removed and the caspase-3 activity in tumor cells was analyzed by flow cytometry. Consistently, the caspase-3 cleavage activities were significantly activated in A549 cells from animals challenged with TRAIL plus rotenone, meanwhile, this effect was attenuated by NAC (Figure 8e). The similar effect of rotenone also occurred in NCI-H441 xenografts tumor model (Supplementary Figure S6).

Restoration of cancer cells susceptibility to TRAIL-induced apoptosis is becoming a very useful strategy for cancer therapy. In this study, we provided evidence that rotenone increased the apoptosis sensitivity of NSCLC cells toward TRAIL by mechanisms involving ROS generation, p53 upregulation, Bcl-XL and c-FLIP downregulation, and death receptors upregulation. Among them, mitochondria-derived ROS had a predominant role. Although rotenone is toxic to neuron, increasing evidence also demonstrated that it was beneficial for improving inflammation, reducing reperfusion injury, decreasing virus infection or triggering cancer cell death. We identified here another important characteristic of rotenone as a tumor sensitizer in TRAIL-based cancer therapy, which widens the application potential of rotenone in disease therapy.

As Warburg proposed the cancer ‘respiration injury’ theory, increasing evidence suggest that cancer cells may have mitochondrial dysfunction, which causes cancer cells, compared with the normal cells, are under increased generation of ROS.33 The increased ROS in cancer cells have a variety of biological effects. We found here that rotenone preferentially increased the apoptosis sensitivity of cancer cells toward TRAIL, further confirming the concept that although tumor cells have a high level of intracellular ROS, they are more sensitive than normal cells to agents that can cause further accumulation of ROS.

Cancer cells stay in a stressful tumor microenvironment including hypoxia, low nutrient availability and immune infiltrates. These conditions, however, activate a range of stress response pathways to promote tumor survival and aggressiveness. In order to circumvent TRAIL-mediated apoptotic clearance, the expression levels of DR4 and DR5 in many types of cancer cells are nullified, but interestingly, they can be reactivated when cancer cells are challenged with small chemical molecules. Furthermore, those small molecules often take advantage of the stress signaling required for cancer cells survival to increase cancer cells sensitivity toward TRAIL. For example, the unfolded protein response (UPR) has an important role in cancer cells survival, SHetA2, as a small molecule, can induce UPR in NSCLC cell lines and augment TRAIL-induced apoptosis by upregulating DR5 expression in CHOP-dependent manner. Here, we found rotenone manipulated the oxidative stress signaling of NSCLC cells to increase their susceptibility to TRAIL. These facts suggest that cellular stress signaling not only offers opportunity for cancer cells to survive, but also renders cancer cells eligible for attack by small molecules. A possible explanation is that depending on the intensity of stress, cellular stress signaling can switch its role from prosurvival to death enhancement. As described in this study, although ROS generation in cancer cells is beneficial for survival, rotenone treatment further increased ROS production to a high level that surpasses the cell ability to eliminate them; as a result, ROS convert its role from survival to death. PPARs and ERRs. molecular mediators of mitochondrial metabolism

Weiwei Fan, Ronald Evans
Current Opinion in Cell Biology Apr 2015; 33:49–54

Since the revitalization of ‘the Warburg effect’, there has been great interest in mitochondrial oxidative metabolism, not only from the cancer perspective but also from the general biomedical science field. As the center of oxidative metabolism, mitochondria and their metabolic activity are tightly controlled to meet cellular energy requirements under different physiological conditions. One such mechanism is through the inducible transcriptional co-regulators PGC1α and NCOR1, which respond to various internal or external stimuli to modulate mitochondrial function. However, the activity of such co-regulators depends on their interaction with transcriptional factors that directly bind to and control downstream target genes. The nuclear receptors PPARs and ERRs have been shown to be key transcriptional factors in regulating mitochondrial oxidative metabolism and executing the inducible effects of PGC1α and NCOR1. In this review, we summarize recent gain-of-function and loss-of-function studies of PPARs and ERRs in metabolic tissues and discuss their unique roles in regulating different aspects of mitochondrial oxidative metabolism.

Energy is vital to all living organisms. In humans and other mammals, the vast majority of energy is produced by oxidative metabolism in mitochondria [1]. As a cellular organelle, mitochondria are under tight control of the nucleus. Although the majority of mitochondrial proteins are encoded by nuclear DNA (nDNA) and their expression regulated by the nucleus, mitochondria retain their own genome, mitochondrial DNA (mtDNA), encoding 13 polypeptides of the electron transport chain (ETC) in mammals. However, all proteins required for mtDNA replication, transcription, and translation, as well as factors regulating such activities, are encoded by the nucleus [2].

The cellular demand for energy varies in different cells under different physiological conditions. Accordingly, the quantity and activity of mitochondria are differentially controlled by a transcriptional regulatory network in both the basal and induced states. A number of components of this network have been identified, including members of the nuclear receptor superfamily, the peroxisome proliferator-activated receptors (PPARs) and the estrogen-related receptors (ERRs) [34 and 5].

The Yin-Yang co-regulators

A well-known inducer of mitochondrial oxidative metabolism is the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) [6], a nuclear cofactor which is abundantly expressed in high energy demand tissues such as heart, skeletal muscle, and brown adipose tissue (BAT) [7]. Induction by cold-exposure, fasting, and exercise allows PGC1α to regulate mitochondrial oxidative metabolism by activating genes involved in the tricarboxylic acid cycle (TCA cycle), beta-oxidation, oxidative phosphorylation (OXPHOS), as well as mitochondrial biogenesis [6 and 8] (Figure 1).

Figure 1.  PPARs and ERRs are major executors of PGC1α-induced regulation of oxidative metabolism. Physiological stress such as exercise induces both the expression and activity of PGC1α, which stimulates energy production by activating downstream genes involved in fatty acid and glucose metabolism, TCA cycle, β-oxidation, OXPHOS, and mitochondrial biogenesis. The transcriptional activity of PGC1α relies on its interactions with transcriptional factors such as PPARs (for controlling fatty acid metabolism) and ERRs (for regulating mitochondrial OXPHOS).

The effect of PGC1α on mitochondrial regulation is antagonized by transcriptional corepressors such as the nuclear receptor corepressor 1 (NCOR1) [9 and 10]. In contrast to PGC1α, the expression of NCOR1 is suppressed in conditions where PGC1α is induced such as during fasting, high-fat-diet challenge, and exercise [9 and 11]. Moreover, the knockout of NCOR1 phenotypically mimics PGC1α overexpression in regulating mitochondrial oxidative metabolism [9]. Therefore, coactivators and corepressors collectively regulate mitochondrial metabolism in a Yin-Yang fashion.

However, both PGC1α and NCOR1 lack DNA binding activity and rather act via their interaction with transcription factors that direct the regulatory program. Therefore the transcriptional factors that partner with PGC1α and NCOR1 mediate the molecular signaling cascades and execute their inducible effects on mitochondrial regulation.

PPARs: master executors controlling fatty acid oxidation

Both PGC1α and NCOR1 are co-factors for the peroxisome proliferator-activated receptors (PPARα, γ, and δ) [71112 and 13]. It is now clear that all three PPARs play essential roles in lipid and fatty acid metabolism by directly binding to and modulating genes involved in fat metabolism [1314151617,18 and 19]. While PPARγ is known as a master regulator for adipocyte differentiation and does not seem to be involved with oxidative metabolism [14 and 20], both PPARα and PPARδ are essential regulators of fatty acid oxidation (FAO) [3131519 and 21] (Figure 1).

PPARα was first cloned as the molecular target of fibrates, a class of cholesterol-lowering compounds that increase hepatic FAO [22]. The importance of PPARα in regulating FAO is indicated in its expression pattern which is restricted to tissues with high capacity of FAO such as heart, liver, BAT, and oxidative muscle [23]. On the other hand, PPARδ is ubiquitously expressed with higher levels in the digestive tract, heart, and BAT [24]. In the past 15 years, extensive studies using gain-of-function and loss-of-function models have clearly demonstrated PPARα and PPARδ as the major drivers of FAO in a wide variety of tissues.

ERRS: master executors controlling mitochondrial OXPHOS

ERRs are essential regulators of mitochondrial energy metabolism [4]. ERRα is ubiquitously expressed but particularly abundant in tissues with high energy demands such as brain, heart, muscle, and BAT. ERRβ and ERRγ have similar expression patterns, both are selectively expressed in highly oxidative tissues including brain, heart, and oxidative muscle [45]. Instead of endogenous ligands, the transcriptional activity of ERRs is primarily regulated by co-factors such as PGC1α and NCOR1 [4 and 46] (Figure 1).

Of the three ERRs, ERRβ is the least studied and its role in regulating mitochondrial function is unclear [4 and 47]. In contrast, when PGC1α is induced, ERRα is the master regulator of the mitochondrial biogenic gene network. As ERRα binds to its own promoter, PGC1α can also induce an autoregulatory loop to enhance overall ERRα activity [48]. Without ERRα, the ability of PGC1α to induce the expression of mitochondrial genes is severely impaired. However, the basal-state levels of mitochondrial target genes are not affected by ERRα deletion, suggesting induced mitochondrial biogenesis is a transient process and that other transcriptional factors such as ERRγ may be important maintaining baseline mitochondrial OXPHOS [41•42 and 43]. Consistent with this idea, ERRγ (which is active even when PGC1α is not induced) shares many target genes with ERRα [49 and 50].

Conclusion and perspectives

Taken together, recent studies have clearly demonstrated the essential roles of PPARs and ERRs in regulating mitochondrial oxidative metabolism and executing the inducible effects of PGC1α (Figure 1). Both PPARα and PPARδ are key regulators for FA oxidation. While the function of PPARα seems more restricted in FA uptake, beta-oxidation, and ketogenesis, PPARδ plays a broader role in controlling oxidative metabolism and fuel preference, with its target genes involved in FA oxidation, mitochondrial OXPHOS, and glucose utilization. However, it is still not clear how much redundancy exists between PPARα and PPARδ, a question which may require the generation of a double knockout model. In addition, more effort is needed to fully understand how PPARα and PPARδ control their target genes in response to environmental changes.

Likewise, ERRα and ERRγ have been shown to be key regulators of mitochondrial OXPHOS. Knockout studies of ERRα suggest it to be the principal executor of PGC1α induced up-regulation of mitochondrial genes, though its role in exercise-dependent changes in skeletal muscle needs further investigation. Transgenic models have demonstrated ERRγ’s powerful induction of mitochondrial biogenesis and its ability to act in a PGC1α-independent manner. However, it remains to be elucidated whether ERRγ is sufficient for basal-state mitochondrial function in general, and whether ERRα can compensate for its function. Metabolic control via the mitochondrial protein import machinery

Opalińska M, Meisinger C.
Curr Opin Cell Biol. 2015 Apr; 33:42-48

Mitochondria have to import most of their proteins in order to fulfill a multitude of metabolic functions. Sophisticated import machineries mediate targeting and translocation of preproteins from the cytosol and subsequent sorting into their suborganellar destination. The mode of action of these machineries has been considered for long time as a static and constitutively active process. However, recent studies revealed that the mitochondrial protein import machinery is subject to intense regulatory mechanisms that include direct control of protein flux by metabolites and metabolic signaling cascades. The Protein Import Machinery of Mitochondria—A Regulatory Hub

AB Harbauer, RP Zahedi, A Sickmann, N Pfanner, C Meisinger
Cell Metab 4 Mar 2014; 19(3):357–372

Mitochondria are essential cell. They are best known for their role as cellular powerhouses, which convert the energy derived from food into an electrochemical proton gradient across the inner membrane. The proton gradient drives the mitochondrial ATP synthase, thus providing large amounts of ATP for the cell. In addition, mitochondria fulfill central functions in the metabolism of amino acids and lipids and the biosynthesis of iron-sulfur clusters and heme. Mitochondria form a dynamic network that is continuously remodeled by fusion and fission. They are involved in the maintenance of cellular ion homeostasis, play a crucial role in apoptosis, and have been implicated in the pathogenesis of numerous diseases, in particular neurodegenerative disorders.

Mitochondria consist of two membranes, outer membrane and inner membrane, and two aqueous compartments, intermembrane space and matrix (Figure 1). Proteomic studies revealed that mitochondria contain more than 1,000 different proteins (Prokisch et al., 2004Reinders et al., 2006Pagliarini et al., 2008 and Schmidt et al., 2010). Based on the endosymbiotic origin from a prokaryotic ancestor, mitochondria contain a complete genetic system and protein synthesis apparatus in the matrix; however, only ∼1% of mitochondrial proteins are encoded by the mitochondrial genome (13 proteins in humans and 8 proteins in yeast). Nuclear genes code for ∼99% of mitochondrial proteins. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated into mitochondria by a multicomponent import machinery. The protein import machinery is essential for the viability of eukaryotic cells. Numerous studies on the targeting signals and import components have been reported (reviewed in Dolezal et al., 2006,Neupert and Herrmann, 2007Endo and Yamano, 2010 and Schmidt et al., 2010), yet for many years little has been known on the regulation of the import machinery. This led to the general assumption that the protein import machinery is constitutively active and not subject to detailed regulation.

Figure 1. Protein Import Pathways of Mitochondria.  Most mitochondrial proteins are synthesized as precursors in the cytosol and are imported by the translocase of the outer mitochondrial membrane (TOM complex). (A) Presequence-carrying (cleavable) preproteins are transferred from TOM to the presequence translocase of the inner membrane (TIM23 complex), which is driven by the membrane potential (Δψ). The proteins either are inserted into the inner membrane (IM) or are translocated into the matrix with the help of the presequence translocase-associated motor (PAM). The presequences are typically cleaved off by the mitochondrial processing peptidase (MPP). (B) The noncleavable precursors of hydrophobic metabolite carriers are bound to molecular chaperones in the cytosol and transferred to the receptor Tom70. After translocation through the TOM channel, the precursors bind to small TIM chaperones in the intermembrane space and are membrane inserted by the Δψ-dependent carrier translocase of the inner membrane (TIM22 complex).
(C) Cysteine-rich proteins destined for the intermembrane space (IMS) are translocated through the TOM channel in a reduced conformation and imported by the mitochondrial IMS import and assembly (MIA) machinery. Mia40 functions as precursor receptor and oxidoreductase in the IMS, promoting the insertion of disulfide bonds into the imported proteins. The sulfhydryl oxidase Erv1 reoxidizes Mia40 for further rounds of oxidative protein import and folding. (D) The precursors of outer membrane β-barrel proteins are imported by the TOM complex and small TIM chaperones and are inserted into the outer membrane by the sorting and assembly machinery (SAM complex). (E) Outer membrane (OM) proteins with α-helical transmembrane segments are inserted into the membrane by import pathways that have only been partially characterized. Shown is an import pathway via the mitochondrial import (MIM) complex

Studies in recent years, however, indicated that different steps of mitochondrial protein import are regulated, suggesting a remarkable diversity of potential mechanisms. After an overview on the mitochondrial protein import machinery, we will discuss the regulatory processes at different stages of protein translocation into mitochondria. We propose that the mitochondrial protein import machinery plays a crucial role as regulatory hub under physiological and pathophysiological conditions. Whereas the basic mechanisms of mitochondrial protein import have been conserved from lower to higher eukaryotes (yeast to humans), regulatory processes may differ between different organisms and cell types. So far, many studies on the regulation of mitochondrial protein import have only been performed in a limited set of organisms. Here we discuss regulatory principles, yet it is important to emphasize that future studies will have to address which regulatory processes have been conserved in evolution and which processes are organism specific.

Protein Import Pathways into Mitochondria

The classical route of protein import into mitochondria is the presequence pathway (Neupert and Herrmann, 2007 and Chacinska et al., 2009). This pathway is used by more than half of all mitochondrial proteins (Vögtle et al., 2009). The proteins are synthesized as precursors with cleavable amino-terminal extensions, termed presequences. The presequences form positively charged amphipathic α helices and are recognized by receptors of the translocase of the outer mitochondrial membrane (TOM complex) (Figure 1A) (Mayer et al., 1995Brix et al., 1997van Wilpe et al., 1999Abe et al., 2000Meisinger et al., 2001 and Saitoh et al., 2007). Upon translocation through the TOM channel, the cleavable preproteins are transferred to the presequence translocase of the inner membrane (TIM23 complex). The membrane potential across the inner membrane (Δψ, negative on the matrix side) exerts an electrophoretic effect on the positively charged presequences (Martin et al., 1991). The presequence translocase-associated motor (PAM) with the ATP-dependent heat-shock protein 70 (mtHsp70) drives preprotein translocation into the matrix (Chacinska et al., 2005 and Mapa et al., 2010). Here the presequences are typically cleaved off by the mitochondrial processing peptidase (MPP). Some cleavable preproteins contain a hydrophobic segment behind the presequence, leading to arrest of translocation in the TIM23 complex and lateral release of the protein into the inner membrane (Glick et al., 1992Chacinska et al., 2005 and Meier et al., 2005). In an alternative sorting route, some cleavable preproteins destined for the inner membrane are fully or partially translocated into the matrix, followed by insertion into the inner membrane by the OXA export machinery, which has been conserved from bacteria to mitochondria (“conservative sorting”) (He and Fox, 1997Hell et al., 1998Meier et al., 2005 and Bohnert et al., 2010).  …

Regulatory Processes Acting at Cytosolic Precursors of Mitochondrial Proteins

Two properties of cytosolic precursor proteins are crucial for import into mitochondria. (1) The targeting signals of the precursors have to be accessible to organellar receptors. Modification of a targeting signal by posttranslational modification or masking of a signal by binding partners can promote or inhibit import into an organelle. (2) The protein import channels of mitochondria are so narrow that folded preproteins cannot be imported. Thus preproteins should be in a loosely folded state or have to be unfolded during the import process. Stable folding of preprotein domains in the cytosol impairs protein import.  …

Import Regulation by Binding of Metabolites or Partner Proteins to Preproteins

Binding of a metabolite to a precursor protein can represent a direct means of import regulation (Figure 2A, condition 1). A characteristic example is the import of 5-aminolevulinate synthase, a mitochondrial matrix protein that catalyzes the first step of heme biosynthesis (Hamza and Dailey, 2012). The precursor contains heme binding motifs in its amino-terminal region, including the presequence (Dailey et al., 2005). Binding of heme to the precursor inhibits its import into mitochondria, likely by impairing recognition of the precursor protein by TOM receptors (Lathrop and Timko, 1993González-Domínguez et al., 2001,Munakata et al., 2004 and Dailey et al., 2005). Thus the biosynthetic pathway is regulated by a feedback inhibition of mitochondrial import of a crucial enzyme, providing an efficient and precursor-specific means of import regulation dependent on the metabolic situation.

Figure 2. Regulation of Cytosolic Precursors of Mitochondrial Proteins

(A) The import of a subset of mitochondrial precursor proteins can be positively or negatively regulated by precursor-specific reactions in the cytosol. (1) Binding of ligands/metabolites can inhibit mitochondrial import. (2) Binding of precursors to partner proteins can stimulate or inhibit import into mitochondria. (3) Phosphorylation of precursors in the vicinity of targeting signals can modulate dual targeting to the endoplasmic reticulum (ER) and mitochondria. (4) Precursor folding can mask the targeting signal. (B) Cytosolic and mitochondrial fumarases are derived from the same presequence-carrying preprotein. The precursor is partially imported by the TOM and TIM23 complexes of the mitochondrial membranes and the presequence is removed by the mitochondrial processing peptidase (MPP). Folding of the preprotein promotes retrograde translocation of more than half of the molecules into the cytosol, whereas the other molecules are completely imported into mitochondria.

Regulation of Mitochondrial Protein Entry Gate by Cytosolic Kinases

Figure 3. Regulation of TOM Complex by Cytosolic Kinases

(A) All subunits of the translocase of the outer mitochondrial membrane (TOM complex) are phosphorylated by cytosolic kinases (phosphorylated amino acid residues are indicated by stars with P). Casein kinase 1 (CK1) stimulates the assembly of Tom22 into the TOM complex. Casein kinase 2 (CK2) stimulates the biogenesis of Tom22 as well as the mitochondrial import protein 1 (Mim1). Protein kinase A (PKA) inhibits the biogenesis of Tom22 and Tom40, and inhibits the activity of Tom70 (see B). Cyclin-dependent kinases (CDK) are possibly involved in regulation of TOM. (B) Metabolic shift-induced regulation of the receptor Tom70 by PKA. Carrier precursors bind to cytosolic chaperones (Hsp70 and/or Hsp90). Tom70 has two binding pockets, one for the precursor and one for the accompanying chaperone (shown on the left). When glucose is added to yeast cells (fermentable conditions), the levels of intracellular cAMP are increased and PKA is activated (shown on the right). PKA phosphorylates a serine of Tom70 in vicinity of the chaperone binding pocket, thus impairing chaperone binding to Tom70 and carrier import into mitochondria.

Casein Kinase 2 Stimulates TOM Biogenesis and Protein Import

Metabolic Switch from Respiratory to Fermentable Conditions Involves Protein Kinase A-Mediated Inhibition of TOM

Network of Stimulatory and Inhibitory Kinases Acts on TOM Receptors, Channel, and Assembly Factors

Protein Import Activity as Sensor of Mitochondrial Stress and Dysfunction

Figure 4. Mitochondrial Quality Control and Stress Response

(A) Import and quality control of cleavable preproteins. The TIM23 complex cooperates with several machineries: the TOM complex, a supercomplex consisting of the respiratory chain complexes III and IV, and the presequence translocase-associated motor (PAM) with the central chaperone mtHsp70. Several proteases/peptidases involved in processing, quality control, and/or degradation of imported proteins are shown, including mitochondrial processing peptidase (MPP), intermediate cleaving peptidase (XPNPEP3/Icp55), mitochondrial intermediate peptidase (MIP/Oct1), mitochondrial rhomboid protease (PARL/Pcp1), and LON/Pim1 protease. (B) The transcription factor ATFS-1 contains dual targeting information, a mitochondrial targeting signal at the amino terminus, and a nuclear localization signal (NLS). In normal cells, ATFS-1 is efficiently imported into mitochondria and degraded by the Lon protease in the matrix. When under stress conditions the protein import activity of mitochondria is reduced (due to lower Δψ, impaired mtHsp70 activity, or peptides exported by the peptide transporter HAF-1), some ATFS-1 molecules accumulate in the cytosol and can be imported into the nucleus, leading to induction of an unfolded protein response (UPRmt).

Regulation of PINK1/Parkin-Induced Mitophagy by the Activity of the Mitochondrial Protein Import Machinery

Figure 5.  Mitochondrial Dynamics and Disease

(A) In healthy cells, the kinase PINK1 is partially imported into mitochondria in a membrane potential (Δψ)-dependent manner and processed by the inner membrane rhomboid protease PARL, which cleaves within the transmembrane segment and generates a destabilizing N terminus, followed by retro-translocation of cleaved PINK1 into the cytosol and degradation by the ubiquitin-proteasome system (different views have been reported if PINK1 is first processed by MPP or not; Greene et al., 2012, Kato et al., 2013 and Yamano and Youle, 2013). Dissipation of Δψ in damaged mitochondria leads to an accumulation of unprocessed PINK1 at the TOM complex and the recruitment of the ubiquitin ligase Parkin to mitochondria. Mitofusin 2 is phosphorylated by PINK1 and likely functions as receptor for Parkin. Parkin mediates ubiquitination of mitochondrial outer membrane proteins (including mitofusins), leading to a degradation of damaged mitochondria by mitophagy. Mutations of PINK1 or Parkin have been observed in monogenic cases of Parkinson’s disease. (B) The inner membrane fusion protein OPA1/Mgm1 is present in long and short isoforms. A balanced formation of the isoforms is a prerequisite for the proper function of OPA1/Mgm1. The precursor of OPA1/Mgm1 is imported by the TOM and TIM23 complexes. A hydrophobic segment of the precursor arrests translocation in the inner membrane, and the amino-terminal targeting signal is cleaved by MPP, generating the long isoforms. In yeast mitochondria, the import motor PAM drives the Mgm1 precursor further toward the matrix such that a second hydrophobic segment is cleaved by the inner membrane rhomboid protease Pcp1, generating the short isoform (s-Mgm1). In mammals, the m-AAA protease is likely responsible for the balanced formation of long (L) and short (S) isoforms of OPA1. A further protease, OMA1, can convert long isoforms into short isoforms in particular under stress conditions, leading to an impairment of mitochondrial fusion and thus to fragmentation of mitochondria.


Mitochondrial research is of increasing importance for the molecular understanding of numerous diseases, in particular of neurodegenerative disorders. The well-established connection between the pathogenesis of Parkinson’s disease and mitochondrial protein import has been discussed above. Several observations point to a possible connection of mitochondrial protein import with the pathogenesis of Alzheimer’s disease, though a direct role of mitochondria has not been demonstrated so far. The amyloid-β peptide (Aβ), which is generated from the amyloid precursor protein (APP), was found to be imported into mitochondria by the TOM complex, to impair respiratory activity, and to enhance ROS generation and fragmentation of mitochondria (Hansson Petersen et al., 2008, Ittner and Götz, 2011 and Itoh et al., 2013). An accumulation of APP in the TOM and TIM23 import channels has also been reported (Devi et al., 2006). The molecular mechanisms of how mitochondrial activity and dynamics may be altered by Aβ (and possibly APP) and how mitochondrial alterations may impact on the pathogenesis of Alzheimer’s disease await further analysis.

It is tempting to speculate that regulatory changes in mitochondrial protein import may be involved in tumor development. Cancer cells can shift their metabolism from respiration toward glycolysis (Warburg effect) (Warburg, 1956, Frezza and Gottlieb, 2009, Diaz-Ruiz et al., 2011 and Nunnari and Suomalainen, 2012). A glucose-induced downregulation of import of metabolite carriers into mitochondria may represent one of the possible mechanisms during metabolic shift to glycolysis. Such a mechanism has been shown for the carrier receptor Tom70 in yeast mitochondria (Schmidt et al., 2011). A detailed analysis of regulation of mitochondrial preprotein translocases in healthy mammalian cells as well as in cancer cells will represent an important task for the future.


In summary, the concept of the “mitochondrial protein import machinery as regulatory hub” will promote a rapidly developing field of interdisciplinary research, ranging from studies on molecular mechanisms to the analysis of mitochondrial diseases. In addition to identifying distinct regulatory mechanisms, a major challenge will be to define the interactions between different machineries and regulatory processes, including signaling networks, preprotein translocases, bioenergetic complexes, and machineries regulating mitochondrial membrane dynamics and contact sites, in order to understand the integrative system controlling mitochondrial biogenesis and fitness. Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

MC Boelens, Tony J. Wu, Barzin Y. Nabet, et al.
Cell 23 Oct 2014; 159(3): 499–513


  • Exosome transfer from stromal to breast cancer cells instigates antiviral signaling
    • RNA in exosomes activates antiviral STAT1 pathway through RIG-I
    • STAT1 cooperates with NOTCH3 to expand therapy-resistant cells
    • Antiviral/NOTCH3 pathways predict NOTCH activity and resistance in primary tumors


Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.



Graphical Abstract Emerging concepts in bioenergetics and cancer research

Obre E, Rossignol R
Int J Biochem Cell Biol. 2015 Feb; 59:167-81

The field of energy metabolism dramatically progressed in the last decade, owing to a large number of cancer studies, as well as fundamental investigations on related transcriptional networks and cellular interactions with the microenvironment. The concept of metabolic flexibility was clarified in studies showing the ability of cancer cells to remodel the biochemical pathways of energy transduction and linked anabolism in response to glucose, glutamine or oxygen deprivation. A clearer understanding of the large-scale bioenergetic impact of C-MYC, MYCN, KRAS and P53 was obtained, along with its modification during the course of tumor development. The metabolic dialog between different types of cancer cells, but also with the stroma, also complexified the understanding of bioenergetics and raised the concepts of metabolic symbiosis and reverse Warburg effect. Signaling studies revealed the role of respiratory chain-derived reactive oxygen species for metabolic remodeling and metastasis development. The discovery of oxidative tumors in human and mice models related to chemoresistance also changed the prevalent view of dysfunctional mitochondria in cancer cells. Likewise, the influence of energy metabolism-derived oncometabolites emerged as a new means of tumor genetic regulation. The knowledge obtained on the multi-site regulation of energy metabolism in tumors was translated to cancer preclinical studies, supported by genetic proof of concept studies targeting LDHA, HK2, PGAM1, or ACLY. Here, we review those different facets of metabolic remodeling in cancer, from its diversity in physiology and pathology, to the search of the genetic determinants, the microenvironmental regulators and pharmacological modulators. Protecting the mitochondrial powerhouse

M Scheibye-Knudsen, EF Fang, DL Croteau, DM Wilson III, VA Bohr
Trends in Cell Biol, Mar 2015; 25(3):158–170


  • Mitochondrial maintenance is essential for cellular and organismal function.
    • Maintenance includes reactive oxygen species (ROS) regulation, DNA repair, fusion–fission, and mitophagy.
    • Loss of function of these pathways leads to disease.

Mitochondria are the oxygen-consuming power plants of cells. They provide a critical milieu for the synthesis of many essential molecules and allow for highly efficient energy production through oxidative phosphorylation. The use of oxygen is, however, a double-edged sword that on the one hand supplies ATP for cellular survival, and on the other leads to the formation of damaging reactive oxygen species (ROS). Different quality control pathways maintain mitochondria function including mitochondrial DNA (mtDNA) replication and repair, fusion–fission dynamics, free radical scavenging, and mitophagy. Further, failure of these pathways may lead to human disease. We review these pathways and propose a strategy towards a treatment for these often untreatable disorders.


Radoslav Bozov –

Larry, pyruvate is a direct substrate for synthesizing pyrimidine rings, as well as C-13 NMR study proven source of methyl groups on SAM! Think about what cancer cells care for – dis-regulated growth through ‘escaped’ mutability of proteins, ‘twisting’ pathways of ordered metabolism space-time wise! mtDNA is a back up, evolutionary primitive, however, primary system for pulling strings onto cell cycle events. Oxygen (never observed single molecule) pulls up electron negative light from emerging super rich energy carbon systems. Therefore, ATP is more acting like a neutralizer – resonator of space-energy systems interoperability! You cannot look at a compartment / space independently , as dimension always add 1 towards 3+1.

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Refined Warburg hypothesis -2.1.2

Writer and Curator: Larry H. Bernstein, MD, FCAP

Refined Warburg Hypothesis -2.1.2

The Warburg discoveries from 1922 on, and the influence on metabolic studies for the next 50 years was immense, and then the revelations of the genetic code took precedence.  Throughout this period, however, the brilliant work of Briton Chance, a giant of biochemistry at the University of Pennsylvania, opened new avenues of exploration that led to a recent resurgence in this vital need for answers in cancer research. The next two series of presentations will open up this resurgence of fundamental metabolic research in cancer and even neurodegenerative diseases. Cancer Cell Metabolism. Warburg and Beyond

Hsu PP, Sabatini DM
Cell, Sep 5, 2008; 134:703-707

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

It is hard to begin a discussion of cancer cell metabolism without first mentioning Otto Warburg. A pioneer in the study of respiration, Warburg made a striking discovery in the 1920s. He found that, even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis, a seeming paradox as glycolysis, when compared to oxidative phosphorylation, is a less efficient pathway for producing ATP (Warburg, 1956). The Warburg effect has since been demonstrated in different types of tumors and the concomitant increase in glucose uptake has been exploited clinically for the detection of tumors by fluorodeoxyglucose positron emission tomography (FDG-PET). Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer, its causal relationship with cancer progression is still unclear. In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

Figure 1. The Altered Metabolism of Cancer Cells

Drivers (A and B). The metabolic derangements in cancer cells may arise either from the selection of cells that have adapted to the tumor microenvironment or from aberrant signaling due to oncogene activation. The tumor microenvironment is spatially and temporally heterogeneous, containing regions of low oxygen and low pH (purple). Moreover, many canonical cancer-associated signaling pathways induce metabolic reprogramming. Target genes activated by hypoxia inducible factor (HIF) decrease the dependence of the cell on oxygen, whereas Ras, Myc, and Akt can also upregulate glucose consumption and glycolysis. Loss of p53 may also recapitulate the features of the Warburg effect, that is, the uncoupling of glycolysis from oxygen levels. Advantages (C–E). The altered metabolism of cancer cells is likely to imbue them with several proliferative and survival advantages, such as enabling cancer cells to execute the biosynthesis of macromolecules (C), to avoid apoptosis (D), and to engage in local metabolite-based paracrine and autocrine signaling (E). Potential Liabilities (F and G). This altered metabolism, however, may also confer several vulnerabilities on cancer cells. For example, an upregulated metabolism may result in the build up of toxic metabolites, including lactate and noncanonical nucleotides, which must be disposed of (F). Moreover, cancer cells may also exhibit a high energetic demand, for which they must either increase flux through normal ATP-generating processes, or else rely on an increased diversity of fuel sources (G).

The Tumor Microenvironment Selects for Altered Metabolism

One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment (Wiesener et al., 2001; Zhong et al., 1999). Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF.

Recent work has demonstrated that the key components of the Warburg effect—increased glucose consumption, decreased oxidative phosphorylation, and accompanying lactate production—are also distinguishing features of oncogene activation. The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Samanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes. Loss of the tumor suppressor protein p53 prevents expression of the gene encoding SCO2 (the synthesis of cytochrome c oxidase protein), which interferes with the function of the mitochondrial respiratory chain (Matoba et al., 2006). A second p53 effector, TIGAR (TP53-induced glycolysis and apoptosis regulator), inhibits glycolysis by decreasing levels of fructose-2,6-bisphosphate, a potent stimulator of glycolysis and inhibitor of gluconeogenesis (Bensaad et al., 2006). Other work also suggests that p53-mediated regulation of glucose metabolism may be dependent on the transcription factor NF-κB (Kawauchi et al., 2008).
It has been shown that inhibition of lactate dehydrogenase A (LDH-A) prevents the Warburg effect and forces cancer cells to revert to oxidative phosphorylation in order to reoxidize NADH and produce ATP (Fantin et al., 2006; Shim et al., 1997). While the cells are respiratory competent, they exhibit attenuated tumor growth, suggesting that aerobic glycolysis might be essential for cancer progression. In a primary fibroblast cell culture model of stepwise malignant transformation through overexpression of telomerase, large and small T antigen, and the H-Ras oncogene, increasing tumorigenicity correlates with sensitivity to glycolytic inhibition. This finding suggests that the Warburg effect might be inherent to the molecular events of transformation (Ramanathan et al., 2005). However, the introduction of similar defined factors into human mesenchymal stem cells (MSCs) revealed that transformation can be associated with increased dependence on oxidative phosphorylation (Funes et al., 2007). Interestingly, when introduced in vivo these transformed MSCs do upregulate glycolytic genes, an effect that is reversed when the cells are explanted and cultured under normoxic conditions. These contrasting models suggest that the Warburg effect may be context dependent, in some cases driven by genetic changes and in others by the demands of the microenvironment. Regardless of whether the tumor microenvironment or oncogene activation plays a more important role in driving the development of a distinct cancer metabolism, it is likely that the resulting alterations confer adaptive, proliferative, and survival advantages on the cancer cell.

Altered Metabolism Provides Substrates for Biosynthetic Pathways

Although studies in cancer metabolism have largely been energy-centric, rapidly dividing cells have diverse requirements. Proliferating cells require not only ATP but also nucleotides, fatty acids, membrane lipids, and proteins, and a reprogrammed metabolism may serve to support synthesis of macromolecules. Recent studies have shown that several steps in lipid synthesis are required for and may even actively promote tumorigenesis. Inhibition of ATP citrate lyase, the distal enzyme that converts mitochondrial-derived citrate into cytosolic acetyl coenzyme A, the precursor for many lipid species, prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al., 2005). Fatty acid synthase, expressed at low levels in normal tissues, is upregulated in cancer and may also be required for tumorigenesis (reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active in the conversion of PEP to pyruvate and thus less efficient at ATP production (reviewed in Mazurek et al., 2005). A major advantage to the cancer cell, however, is that the glycolytic intermediates upstream of PEP might be shunted into synthetic processes.

Biosynthesis, in addition to causing an inherent increase in ATP demand in order to execute synthetic reactions, should also cause a decrease in ATP supply as various glycolytic and Krebs cycle intermediates are diverted. Lipid synthesis, for example, requires the cooperation of glycolysis, the Krebs cycle, and the pentose phosphate shunt. As pyruvate must enter the mitochondria in this case, it avoids conversion to lactate and therefore cannot contribute to glycolysis-derived ATP. Moreover, whereas increased biosynthesis may explain the glucose hunger of cancer cells, it cannot explain the increase in lactic acid production originally described by Warburg, suggesting that lactate must also result from the metabolism of non-glucose substrates. Recently, it has been demonstrated that glutamine may be metabolized by the citric acid cycle in cancer cells and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates (DeBerardinis et al., 2007).

Metabolic Pathways Regulate Apoptosis

In addition to involvement in proliferation, altered metabolism may promote another cancer-essential function: the avoidance of apoptosis. Loss of the p53 target TIGAR sensitizes cancer cells to apoptosis, most likely by causing an increase in reactive oxygen species (Bensaad et al., 2006). On the other hand, overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents caspase-independent cell death, presumably by stimulating glycolysis, increasing cellular ATP levels, and promoting autophagy (Colell et al., 2007). Whether or not GAPDH plays a physiological role in the regulation of cell death remains to be determined. Intriguingly, Bonnet et al. (2007) have reported that treating cancer cells with dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has striking effects on their survival and on xenograft tumor growth.

DCA, a currently approved treatment for congenital lactic acidosis, activates oxidative phosphorylation and promotes apoptosis by two mechanisms. First, increased flux through the electron transport chain causes depolarization of the mitochondrial membrane potential (which the authors found to be hyperpolarized specifically in cancer cells) and release of the apoptotic effector cytochrome c. Second, an increase in reactive oxygen species generated by oxidative phosphorylation upregulates the voltage-gated K+ channel, leading to potassium ion efflux and caspase activation. Their work suggests that cancer cells may shift their metabolism to glycolysis in order to prevent cell death and that forcing cancer cells to respire aerobically can counteract this adaptation.

Cancer Cells May Signal Locally in the Tumor Microenvironment

Cancer cells may rewire metabolic pathways to exploit the tumor microenvironment and to support cancer-specific signaling. Without access to the central circulation, it is possible that metabolites can be concentrated locally and reach suprasystemic levels, allowing cancer cells to engage in metabolite-mediated autocrine and paracrine signaling that does not occur in normal tissues. So called androgen-independent prostate cancers may only be independent from exogenous, adrenal-synthesized androgens. Androgen-independent prostate cancer cells still express the androgen receptor and may be capable of autonomously synthesizing their own androgens (Stanbrough et al., 2006).

Metabolism as an Upstream Modulator of Signaling Pathways

Not only is metabolism downstream of oncogenic pathways, but an altered upstream metabolism may affect the activity of signaling pathways that normally sense the state of the cell. Individuals with inherited mutations in succinate dehydrogenase and fumarate hydratase develop highly angiogenic tumors, not unlike those exhibiting loss of the VHL tumor suppressor protein that acts upstream of HIF (reviewed in Kaelin and Ratcliffe, 2008). The mechanism of tumorigenesis in these cancer syndromes is still contentious. However, it has been proposed that loss of succinate dehydrogenase and fumarate hydratase causes an accumulation of succinate or fumarate, respectively, leading to inhibition of the prolyl hydroxylases that mark HIF for VHL-mediated degradation (Isaacs et al., 2005; Pollard et al., 2005; Selak et al., 2005). In this rare case, succinate dehydrogenase and fumarate hydratase are acting as bona fide tumor suppressors.

There are many complex questions to be answered: Is it possible that cancer cells exhibit “metabolite addiction”? Are there unique cancer-specific metabolic pathways, or combinations of pathways, utilized by the cancer cell but not by normal cells? Are different stages of metabolic adaptations required for the cancer cell to progress from the primary tumor stage to invasion to metastasis? How malleable is cancer metabolism? Cancer metabolism. The Warburg effect today

Ferreira LMR
Exp Molec Pathol 2010; 89:372-383.

One of the first studies on the energy metabolism of a tumor was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.

Research highlights

► Warburg first established a tumor metabolic pattern in the 1920s. ► Tumors’ increased glucose uptake has been studied since then. ► Cancer bioenergetics’ study provides insights in all its hallmarks. ► New cancer diagnostic and therapeutic techniques focus on cancer metabolism.

Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives New aspects of the Warburg effect in cancer cell biology

Bensinger SJ, Cristofk HR
Sem Cell Dev Biol 2012; 23:352-361

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis–also known as the Warburg effect–characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.


► This review discusses regulatory mechanisms that contribute to the Warburg effect in cancer. ► We list cancers for which FDG-PET has established applications as well as those cancers for which FDG-PET has not been established. ► PKM2 is highlighted as an important integrator of diverse cellular stimuli to modulate metabolic flux and cancer cell proliferation. ► We discuss how cancer metabolism can directly influence gene expression programs. ► Contribution of aerobic glycolysis to the cancer microenvironment and chemotherapeutic resistance/susceptibility is also discussed.

Regulation of the Warburg effect

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Fig. 1. PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Metabolism can directly influence gene expression programs

Metabolism can directly influence gene expression programs

Fig. 2. Metabolism can directly influence gene expression programs. A schematic representation of how metabolism can intrinsically influence epigenetics resulting in durable and heritable gene expression programs in progeny. Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma

Jose C, Ballance N, Rossignal R
Biochim Biophys Acta 201; 1807(6): 552-561.

A considerable amount of knowledge has been produced during the last five years on the bioenergetics of cancer cells, leading to a better understanding of the regulation of energy metabolism during oncogenesis, or in adverse conditions of energy substrate intermittent deprivation. The general enhancement of the glycolytic machinery in various cancer cell lines is well described and recent analyses give a better view of the changes in mitochondrial oxidative phosphorylation during oncogenesis. While some studies demonstrate a reduction of oxidative phosphorylation (OXPHOS) capacity in different types of cancer cells, other investigations revealed contradictory modifications with the upregulation of OXPHOS components and a larger dependency of cancer cells on oxidative energy substrates for anabolism and energy production. This apparent conflictual picture is explained by differences in tumor size, hypoxia, and the sequence of oncogenes activated. The role of p53, C-MYC, Oct and RAS on the control of mitochondrial respiration and glutamine utilization has been explained recently on artificial models of tumorigenesis. Likewise, the generation of induced pluripotent stem cells from oncogene activation also showed the role of C-MYC and Oct in the regulation of mitochondrial biogenesis and ROS generation. In this review article we put emphasis on the description of various bioenergetic types of tumors, from exclusively glycolytic to mainly OXPHOS, and the modulation of both the metabolic apparatus and the modalities of energy substrate utilization according to tumor stage, serial oncogene activation and associated or not fluctuating microenvironmental substrate conditions. We conclude on the importance of a dynamic view of tumor bioenergetics.

Research Highlights

►The bioenergetics of cancer cells differs from normals. ►Warburg hypothesis is not verified in tumors using mitochondria to synthesize ATP. ►Different oncogenes can either switch on or switch off OXPHOS. ►Bioenergetic profiling is a prerequisite to metabolic therapy. ►Aerobic glycolysis and OXPHOS cooperate during cancer progression.

  1. Cancer cell variable bioenergetics

Cancer cells exhibit profound genetic, bioenergetic and histological differences as compared to their non-transformed counterpart. All these modifications are associated with unlimited cell growth, inhibition of apoptosis and intense anabolism. Transformation from a normal cell to a malignant cancer cell is a multi-step pathogenic process which includes a permanent interaction between cancer gene activation (oncogenes and/or tumor-suppressor genes), metabolic reprogramming and tumor-induced changes in microenvironment. As for the individual genetic mapping of human tumors, their metabolic characterization (metabolic–bioenergetic profiling) has evidenced a cancer cell-type bioenergetic signature which depends on the history of the tumor, as composed by the sequence of oncogenes activated and the confrontation to intermittent changes in oxygen, glucose and amino-acid delivery.

In the last decade, bioenergetic studies have highlighted the variability among cancer types and even inside a cancer type as regards to the mechanisms and the substrates preferentially used for deriving the vital energy. The more popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and a high lactate production, along with the absence of respiration despite the presence of high oxygen concentration (Warburg effect) [1]. To explain this abnormal bioenergetic phenotype pioneering hypotheses proposed the impairment of mitochondrial function in rapidly growing cancer cells [2].

Although the increased consumption of glucose by tumor cells was confirmed in vivo by positron emission tomography (PET) using the glucose analog 2-(18F)-fluoro-2-deoxy-d-glucose (FDG), the actual utilization of glycolysis and oxidative phosphorylation (OXPHOS) cannot be evaluated with this technique. Nowadays, Warburg’s “aerobic-glycolysis” hypothesis has been challenged by a growing number of studies showing that mitochondria in tumor cells are not inactive per se but operate at low capacity [3] or, in striking contrast, supply most of the ATP to the cancer cells [4]. Intense glycolysis is effectively not observed in all tumor types. Indeed not all cancer cells grow fast and intense anabolism is not mandatory for all cancer cells. Rapidly growing tumor cells rely more on glycolysis than slowly growing tumor cells. This is why a treatment with bromopyruvate, for example is very efficient only on rapidly growing cells and barely useful to decrease the growth rate of tumor cells when their normal proliferation is slow. Already in 1979, Reitzer and colleagues published an article entitled “Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells”, which demonstrated that oxidative phosphorylation was used preferentially to produce ATP in cervical carcinoma cells [5]. Griguer et al. also identified several glioma cell lines that were highly dependent on mitochondrial OXPHOS pathway to produce ATP [6]. Furthermore, a subclass of glioma cells which utilize glycolysis preferentially (i.e., glycolytic gliomas) can also switch from aerobic glycolysis to OXPHOS under limiting glucose conditions  [7] and [8], as observed in cervical cancer cells, breast carcinoma cells, hepatoma cells and pancreatic cancer cells [9][10] and [11]. This flexibility shows the interplay between glycolysis and OXPHOS to adapt the mechanisms of energy production to microenvironmental changes as well as differences in tumor energy needs or biosynthetic activity. Herst and Berridge also demonstrated that a variety of human and mouse leukemic and tumor cell lines (HL60, HeLa, 143B, and U937) utilize mitochondrial respiration to support their growth [12]. Recently, the measurement of OXPHOS contribution to the cellular ATP supply revealed that mitochondria generate 79% of the cellular ATP in HeLa cells, and that upon hypoxia this contribution is reduced to 30% [4]. Again, metabolic flexibility is used to survive under hypoxia. All these studies demonstrate that mitochondria are efficient to synthesize ATP in a large variety of cancer cells, as reviewed by Moreno-Sanchez [13]. Despite the observed reduction of the mitochondrial content in tumors [3][14][15][16][17][18] and [19], cancer cells maintain a significant level of OXPHOS capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis. This switch is also observed at the level of glutamine oxidation which can occur through two modes, “OXPHOS-linked” or “anoxic”, allowing to derive energy from glutamine or serine regardless of hypoxia or respiratory chain reduced activity [20].
While glutamine, glycine, alanine, glutamate, and proline are typically oxidized in normal and tumor mitochondria, alternative substrate oxidations may also contribute to ATP supply by OXPHOS. Those include for instance the oxidation of fatty-acids, ketone bodies, short-chain carboxylic acids, propionate, acetate and butyrate (as recently reviewed in [21]).

  1. Varying degree of mitochondrial utilization during tumorigenesis

In vivo metabolomic analyses suggest the existence of a continuum of bioenergetic remodeling in rat tumors according to tumor size and its rate of growth [22]. Peter Vaupel’s group showed that small tumors were characterized by a low conversion of glucose to lactate whereas the conversion of glutamine to lactate was high. In medium sized tumors the flow of glucose to lactate as well as oxygen utilization was increased whereas glutamine and serine consumption were reduced. At this stage tumor cells started with glutamate and alanine production. Large tumors were characterized by a low oxygen and glucose supply but a high glucose and oxygen utilization rate. The conversion of glucose to glycine, alanine, glutamate, glutamine, and proline reached high values and the amino acids were released [22]. Certainly, in the inner layers constituting solid tumors, substrate and oxygen limitation is frequently observed. Experimental studies tried to reproduce these conditions in vitro and revealed that nutrients and oxygen limitation does not affect OXPHOS and cellular ATP levels in human cervix tumor [23]. Furthermore, the growth of HeLa cells, HepG2 cells and HTB126 (breast cancer) in aglycemia and/or hypoxia even triggered a compensatory increase in OXPHOS capacity, as discussed above. Yet, the impact of hypoxia might be variable depending on cell type and both the extent and the duration of oxygen limitation.
In two models of sequential oncogenesis, the successive activation of specific oncogenes in non-cancer cells evidenced the need for active OXPHOS to pursue tumorigenesis. Funes et al. showed that the transformation of human mesenchymal stem cells increases their dependency on OXPHOS for energy production [24], while Ferbeyre et al. showed that cells expressing oncogenic RAS display an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest [25]. Such observations suggest that waves of gene regulation could suppress and then restore OXPHOS in cancer cells during tumorigenesis [20]. Therefore, the definition of cancer by Hanahan and Weinberg [26] restricted to six hallmarks (1—self-sufficiency in growth signals, 2—insensitivity to growth-inhibitory (antigrowth) signals, 3—evasion of programmed cell death (apoptosis), 4—limitless replicative potential, 5—sustained angiogenesis, and 6—tissue invasion and metastases) should also include metabolic reprogramming, as the seventh hallmark of cancer. This amendment was already proposed by Tennant et al. in 2009 [27]. In 2006, the review Science published a debate on the controversial views of Warburg theory [28], in support of a more realistic description of cancer cell’s variable bioenergetic profile. The pros think that high glycolysis is an obligatory feature of human tumors, while the cons propose that high glycolysis is not exclusive and that tumors can use OXPHOS to derive energy. A unifying theory closer to reality might consider that OXPHOS and glycolysis cooperate to sustain energy needs along tumorigenesis [20]. The concept of oxidative tumors, against Warburg’s proposal, was introduced by Guppy and colleagues, based on the observation that breast cancer cells can generate 80% of their ATP by the mitochondrion [29]. The comparison of different cancer cell lines and excised tumors revealed a variety of cancer cell’s bioenergetic signatures which raised the question of the mechanisms underlying tumor cell metabolic reprogramming, and the relative contribution of oncogenesis and microenvironment in this process. It is now widely accepted that rapidly growing cancer cells within solid tumors suffer from a lack of oxygen and nutrients as tumor grows. In such situation of compromised energy substrate delivery, cancer cell’s metabolic reprogramming is further used to sustain anabolism (Fig. 1), through the deviation of glycolysis, Krebs cycle truncation and OXPHOS redirection toward lipid and protein synthesis, as needed to support uncontrolled tumor growth and survival [30] and [31]. Again, these features are not exclusive to all tumors, as Krebs cycle truncation was only observed in some cancer cells, while other studies indicated that tumor cells can maintain a complete Krebs cycle [13] in parallel with an active citrate efflux. Likewise, generalizations should be avoided to prevent over-interpretations.
Fig. 1. Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

The oncogene C-MYC participate to these changes via the stimulation of glutamine utilization through the coordinate expression of genes necessary for cells to engage in glutamine catabolism [30]. According to Newsholme EA and Board M [32] both glycolysis and glutaminolysis not only serve for ATP production, but also provide precious metabolic intermediates such as glucose-6-phosphate, ammonia and aspartate required for the synthesis of purine and pyrimidine nucleotides (Fig. 1). In this manner, the observed apparent excess in the rates of glycolysis and glutaminolysis as compared to the requirement for energy production could be explained by the need for biosynthetic processes. Yet, one should not reduce the shift from glycolysis to OXPHOS utilization to the sole activation of glutaminolysis, as several other energy substrates can be used by tumor mitochondria to generate ATP [21]. The contribution of these different fuels to ATP synthesis remains poorly investigated in human tumors.

  1. The metabolism of pre-cancer cells and its ongoing modulation by carcinogenesis

At the beginning of cancer, there might have been a cancer stem cell hit by an oncogenic event, such as alterations in mitogen signaling to extracellular growth factor receptors (EGFR), oncogenic activation of these receptors, or oncogenic alterations of downstream targets in the pathways that leads to cell proliferation (RAS–Raf–ERK and PI3K–AKT, both leading to m-TOR activation stimulating cell growth). Alterations of checkpoint genes controlling the cell cycle progression like Rb also participate in cell proliferation (Fig. 2) and this re-entry in the cell cycle implies three major needs to fill in: 1) supplying enough energy to grow and 2) synthesize building blocks de novo and 3) keep vital oxygen and nutrients available. However, the bioenergetic status of the pre-cancer cell could determine in part the evolution of carcinogenesis, as shown on mouse embryonic stem cells. In this study, Schieke et al. showed that mitochondrial energy metabolism modulates both the differentiation and tumor formation capacity of mouse embryonic stem cells [37]. The idea that cancer derives from a single cell, known as the cancer stem cell hypothesis, was introduced by observations performed on leukemia which appeared to be organized as origination from a primitive hematopoietic cell [38]. Nowadays cancer stem cells were discovered for all types of tumors [39][40][41] and [42], but little is known of their bioenergetic properties and their metabolic adaptation to the microenvironment. This question is crucial as regards the understanding of what determines the wide variety of cancer cell’s metabolic profile.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Fig. 2. Impact of different oncogenes on tumor progression and energy metabolism remodeling.

The analysis of the metabolic changes that occur during the transformation of adult mesenchymal stem cells revealed that these cells did not switch to aerobic glycolysis, but their dependency on OXPHOS was even increased [24]. Hence, mitochondrial energy metabolism could be critical for tumorigenesis, in contrast with Warburg’s hypothesis. As discussed above, the oncogene C-MYC also stimulates OXPHOS [30]. Furthermore, it was recently demonstrated that cells chronically treated with oligomycin repress OXPHOS and produce larger tumors with higher malignancy [19]. Likewise, alteration of OXPHOS by mutations in mtDNA increases tumorigenicity in different types of cancer cells [43][44] and [45].

Recently, it was proposed that mitochondrial energy metabolism is required to generate reactive oxygen species used for the carcinogenetic process induced by the K-RAS mutation [46]. This could explain the large number of mitochondrial DNA mutations found in several tumors. The analysis of mitochondria in human embryonic cells which derive energy exclusively from anaerobic glycolysis have demonstrated an immature mitochondrial network characterized by few organelles with poorly developed cristae and peri-nuclear distribution [47] and [48]. The generation of human induced pluripotent stem cell by the introduction of different oncogenes as C-MYC and Oct4 reproduced this reduction of mitochondrial OXPHOS capacity[49] and [50]. This indicates again the impact of oncogenes on the control of OXPHOS and might explain the existence of pre-cancer stem cells with different bioenergetic backgrounds, as modeled by variable sequences of oncogene activation. Accordingly, the inhibition of mitochondrial respiratory chain has been recently found associated with enhancement of hESC pluripotency [51].

Based on the experimental evidence discussed above, one can argue that 1) glycolysis is indeed a feature of several tumors and associates with faster growth in high glucose environment, but 2) active OXPHOS is also an important feature of (other) tumors taken at a particular stage of carcinogenesis which might be more advantageous than a “glycolysis-only” type of metabolism in conditions of intermittent shortage in glucose delivery. The metabolic apparatus of cancer cells is not fixed during carcinogenesis and might depend both on the nature of the oncogenes activated and the microenvironment. It was indeed shown that cancer cells with predominant glycolytic metabolism present a higher malignancy when submitted to carcinogenetic induction and analysed under fixed experimental conditions of high glucose [19]. Yet, if one grows these cells in a glucose-deprived medium they shift their metabolism toward predominant OXPHOS, as shown in HeLa cells and other cell types [9]. Therefore, one might conclude that glycolytic cells have a higher propensity to generate aggressive tumors when glucose availability is high. However, these cells can become OXPHOS during tumor progression [24] and [52]. All these observations indicate again the importance of maintaining an active OXPHOS metabolism to permit evolution of both embryogenesis and carcinogenesis, which emphasizes the importance of targeting mitochondria to alter this malignant process.

  1. Oncogenes and the modulation of energy metabolism

Several oncogenes and associated proteins such as HIF-1α, RAS, C-MYC, SRC, and p53 can influence energy substrate utilization by affecting cellular targets, leading to metabolic changes that favor cancer cell survival, independently of the control of cell proliferation. These oncogenes stimulate the enhancement of aerobic glycolysis, and an increasing number of studies demonstrate that at least some of them can also target directly the OXPHOS machinery, as discussed in this article (Fig. 2). For instance, C-MYC can concurrently drive aerobic glycolysis and/or OXPHOS according to the tumor cell microenvironment, via the expression of glycolytic genes or the activation of mitochondrial oxidation of glutamine [53]. The oncogene RAS has been shown to increase OXPHOS activity in early transformed cells [24][52] and [54] and p53 modulates OXPHOS capacity via the regulation of cytochrome c oxidase assembly [55]. Hence, carcinogenic p53 deficiency results in a decreased level of COX2 and triggers a shift toward anaerobic metabolism. In this case, lactate synthesis is increased, but cellular ATP levels remain stable [56]. The p53-inducible isoform of phosphofructokinase, termed TP53-induced glycolysis and apoptotic regulator, TIGAR, a predominant phosphatase activity isoform of PFK-2, has also been identified as an important regulator of energy metabolism in tumors [57].

  1. Tumor specific isoforms (or mutated forms) of energy genes

Tumors are generally characterized by a modification of the glycolytic system where the level of some glycolytic enzymes is increased, some fetal-like isozymes with different kinetic and regulatory properties are produced, and the reverse and back-reactions of the glycolysis are strongly reduced [60]. The GAPDH marker of the glycolytic pathway is also increased in breast, gastric, lung, kidney and colon tumors [18], and the expression of glucose transporter GLUT1 is elevated in most cancer cells. The group of Cuezva J.M. developed the concept of cancer bioenergetic signature and of bioenergetic index to describe the metabolic profile of cancer cells and tumors [18], [61], [64], [65]. This signature describes the changes in the expression level of proteins involved in glycolysis and OXPHOS, while the BEC index gives a ratio of OXPHOS protein content to glycolytic protein content, in good correlation with cancer prognostic[61]. Recently, this group showed that the beta-subunit of the mitochondrial F1F0-ATP synthase is downregulated in a large number of tumors, thus contributing to the Warburg effect [64] and [65]. It was also shown that IF1 expression levels were increased in hepatocellular carcinomas, possibly to prevent the hydrolysis of glytolytic ATP [66]. Numerous changes occur at the level of OXPHOS and mitochondrial biogenesis in human tumors, as we reviewed previously [67]. Yet the actual impact of these changes in OXPHOS protein expression level or catalytic activities remains to be evaluated on the overall fluxes of respiration and ATP synthesis. Indeed, the metabolic control analysis and its extension indicate that it is often required to inhibit activity beyond a threshold of 70–85% to affect the metabolic fluxes [68] and [69]. Another important feature of cancer cells is the higher level of hexokinase II bound to mitochondrial membrane (50% in tumor cells). A study performed on human gliomas (brain) estimated the mitochondrial bound HK fraction (mHK) at 69% of total, as compared to 9% for normal brain [70]. This is consistent with the 5-fold amplification of the type II HK gene observed by Rempel et al. in the rapidly growing rat AS-30D hepatoma cell line, relative to normal hepatocytes [71]. HKII subcellular fractionation in cancer cells was described in several studies [72][73] and [74]. The group led by Pete Pedersen explained that mHK contributes to (i) the high glycolytic capacity by utilizing mitochondrially regenerated ATP rather than cytosolic ATP (nucleotide channelling) and (ii) the lowering of OXPHOS capacity by limiting Pi and ADP delivery to the organelle [75] and [76].

All these observations are consistent with the increased rate of FDG uptake observed by PET in living tumors which could result from both an increase in glucose transport, and/or an increase in hexokinase activity. However, FDG is not a complete substrate for glycolysis (it is only transformed into FDG-6P by hexokinase before to be eliminated) and cannot be used to evidence a general increase in the glycolytic flux. Moreover, FDG-PET scan also gives false positive and false negative results, indicating that some tumors do not depend on, or do not have, an increased glycolytic capacity. The fast glycolytic system described above is further accommodated in cancer cells by an increase in the lactate dehydrogenase isoform A (LDH-A) expression level. This isoform presents a higher Vmax useful to prevent the inhibition of high glycolysis by its end product (pyruvate) accumulation. Recently, Fantin et al. showed that inhibition of LDH-A in tumors diminishes tumorigenicity and was associated with the stimulation of mitochondrial respiration [79]. The preferential expression of the glycolytic pyruvate kinase isoenzyme M2 (PKM2) in tumor cells, determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form) [80]. In the last five years, mutations in proteins of the respiratory system (SDH, FH) and of the TCA cycle (IDH1,2) leading to the accumulation of metabolite and the subsequent activation of HIF-1α were reported in a variety of human tumors [81], [82] and [83].

  1. Tumor microenvironment modulates cancer cell’s bioenergetics

It was extensively described how hypoxia activates HIF-1α which stimulates in turn the expression of several glycolytic enzymes such as HK2, PFK, PGM, enolase, PK, LDH-A, MCT4 and glucose transporters Glut 1 and Glut 3. It was also shown that HIF-1α can reduce OXPHOS capacity by inhibiting mitochondrial biogenesis [14] and [15], PDH activity [87] and respiratory chain activity [88]. The low efficiency and uneven distribution of the vascular system surrounding solid tumors can lead to abrupt changes in oxygen (intermittent hypoxia) but also energy substrate delivery. .. The removal of glucose, or the inhibition of glycolysis by iodoacetate led to a switch toward glutamine utilization without delay followed by a rapid decrease in acid release. This illustrates once again how tumors and human cancer cell lines can utilize alternative energy pathway such as glutaminolysis to deal with glucose limitation, provided the presence of oxygen. It was also observed that in situations of glucose limitation, tumor derived-cells can adapt to survive by using exclusively an oxidative energy substrate [9] and [10]. This is typically associated with an enhancement of the OXPHOS system. … In summary, cancer cells can survive by using exclusively OXPHOS for ATP production, by altering significantly mitochondrial composition and form to facilitate optimal use of the available substrate (Fig. 3). Yet, glucose is needed to feed the pentose phosphate pathway and generate ribose essential for nucleotide biosynthesis. This raises the question of how cancer cells can survive in the growth medium which do not contain glucose (so-called “galactose medium” with dialysed serum [9]). In the OXPHOS mode, pyruvate, glutamate and aspartate can be derived from glutamine, as glutaminolysis can replenish Krebs cycle metabolic pool and support the synthesis of alanine and NADPH [31]. Glutamine is a major source for oxaloacetate (OAA) essential for citrate synthesis. Moreover, the conversion of glutamine to pyruvate is associated with the reduction of NADP+ to NADPH by malic enzyme. Such NADPH is a required electron donor for reductive steps in lipid synthesis, nucleotide metabolism and GSH reduction. In glioblastoma cells the malic enzyme flux was estimated to be high enough to supply all of the reductive power needed for lipid synthesis [31].

Fig. 3. Interplay between energy metabolism, oncogenes and tumor microenvironment during tumorigenesis (the “metabolic wave model”).

Interplay between energy metabolism, oncogenes and tumor microenvironment

Interplay between energy metabolism, oncogenes and tumor microenvironment

While the mechanisms leading to the enhancement of glycolytic capacity in tumors are well documented, less is known about the parallel OXPHOS changes. Both phenomena could result from a selection of pre-malignant cells forced to survive under hypoxia and limited glucose delivery, followed by an adaptation to intermittent hypoxia, pseudo-hypoxia, substrate limitation and acidic environment. This hypothesis was first proposed by Gatenby and Gillies to explain the high glycolytic phenotype of tumors [91], [92] and [93], but several lines of evidence suggest that it could also be used to explain the mitochondrial modifications observed in cancer cells.

  1. Aerobic glycolysis and mitochondria cooperate during cancer progression

Metabolic flexibility considers the possibility for a given cell to alternate between glycolysis and OXPHOS in response to physiological needs. Louis Pasteur found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen (Pasteur effect). Moreover, energy metabolism of normal cell can vary widely according to the tissue of origin, as we showed with the comparison of five rat tissues[94]. During stem cell differentiation, cell proliferation induces a switch from OXPHOS to aerobic glycolysis which might generate ATP more rapidly, as demonstrated in HepG2 cells [95] or in non-cancer cells[96] and [97]. Thus, normal cellular energy metabolism can adapt widely according to the activity of the cell and its surrounding microenvironment (energy substrate availability and diversity). Support for this view came from numerous studies showing that in vitro growth conditions can alter energy metabolism contributing to a dependency on glycolysis for ATP production [98].

Yet, Zu and Guppy analysed numerous studies and showed that aerobic glycolysis is not inherent to cancer but more a consequence of hypoxia[99].

Table 1. Impact of different oncogenes on energy metabolism

Impact of different oncogenes on energy metabolism.

Impact of different oncogenes on energy metabolism. Mitohormesis

Yun J, Finkel T
Cell Metab May 2014; 19(5):757–766

For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.

Figure 1. The Basis of Mitohormesis. Any of a number of endogenous or exogenous stresses can perturb mitochondrial function. These perturbations are relayed to the cytosol through, at present, poorly understood mechanisms that may involve mitochondrial ROS as well as other mediators. These cytoplasmic signaling pathways and subsequent nuclear transcriptional changes induce various long-lasting cytoprotective pathways. This augmented stress resistance allows for protection from a wide array of subsequent stresses.

Figure 2. Potential Parallels between the Mitochondrial Unfolded Protein Response and Quorum Sensing in Gram-Positive Bacteria. In the C. elegans UPRmt response, mitochondrial proteins (indicated by blue swirls) are degraded by matrix proteases, and the oligopeptides that are generated are then exported through the ABC transporter family member HAF-1. Once in the cytosol, these peptides can influence the subcellular localization of the transcription factor ATFS-1. Nuclear ATFS-1 is capable of orchestrating a broad transcriptional response to mitochondrial stress. As such, this pathway establishes a method for mitochondrial and nuclear genomes to communicate. In some gram-positive bacteria, intracellularly generated peptides can be similarly exported through an ABC transporter protein. These peptides can be detected in the environment by a membrane-bound histidine kinases (HK) sensor. The activation of the HK sensor leads to phosphorylation of a response regulator (RR) protein that, in turn, can alter gene expression. This program allows communication between dispersed gram-positive bacteria and thus coordinated behavior of widely dispersed bacterial genomes.

Figure 3. The Complexity of Mitochondrial Stresses and Responses. A wide array of extrinsic and intrinsic mitochondrial perturbations can elicit cellular responses. As detailed in the text, genetic or pharmacological disruption of electron transport, incorrect folding of mitochondrial proteins, stalled mitochondrial ribosomes, alterations in signaling pathways, or exposure to toxins all appear to elicit specific cytoprotective programs within the cell. These adaptive responses include increased mitochondrial number (biogenesis), alterations in metabolism, increased antioxidant defenses, and augmented protein chaperone expression. The cumulative effect of these adaptive mechanisms might be an extension of lifespan and a decreased incidence of age-related pathologies. Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives

Mayevsky A
Mitochondrion. 2009 Jun; 9(3):165-79

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.

 Energy metabolism in mammalian cells

Fig. 1. Schematic representation of cellular energy metabolism and its relationship to microcirculatory blood flow and hemoglobin oxygenation.

Fig. 2. Schematic representation of the central role of the mitochondrion in the various processes involved in the pathology of cancer cells and tumors. Six issues marked as 1–6 are discussed in details in the text.

In vivo monitoring of tissue energy metabolism in mammalian cells

Fig. 3. Schematic presentation of the six parameters that could be monitored for the evaluation of tissue energy metabolism (see text for details).

Optical spectroscopy of tissue energy metabolism in vivo

Multiparametric monitoring system

Fig. 4. (A) Schematic representation of the Time Sharing Fluorometer Reflectometer (TSFR) combined with the laser Doppler flowmeter (D) for blood flow monitoring. The time sharing system includes a wheel that rotates at a speed of3000 rpm wit height filters: four for the measurements of mitochondrial NADH(366 nm and 450 nm)and four for oxy-hemoglobin measurements (585 nm and 577 nm) as seen in (C). The source of light is a mercury lamp. The probe includes optical fibers for NADH excitation (Ex) and emission (Em), laser Doppler excitation (LD in), laser Doppler emission (LD out) as seen in part E The absorption spectrum of Oxy- and Deoxy- Hemoglobin indicating the two wave length used (C).

Fig. 7. Comparison between mitochondrial metabolic states in vitro and the typical tissue metabolic states in vivo evaluated by NADH redox state, tissue blood flow and hemoglobin oxygenation as could be measured by the suggested monitoring system.

(very important) Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate

Ward PS, Thompson CB.
Cancer Cell 2012; 21(3):297-308

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer.

The propensity for proliferating cells to secrete a significant fraction of glucose carbon through fermentation was first elucidated in yeast. Otto Warburg extended these observations to mammalian cells, finding that proliferating ascites tumor cells converted the majority of their glucose carbon to lactate, even in oxygen-rich conditions. Warburg hypothesized that this altered metabolism was specific to cancer cells, and that it arose from mitochondrial defects that inhibited their ability to effectively oxidize glucose carbon to CO2. An extension of this hypothesis was that dysfunctional mitochondria caused cancer (Koppenol et al., 2011). Warburg’s seminal finding has been observed in a wide variety of cancers. These observations have been exploited clinically using 18F-deoxyglucose positron emission tomography (FDG-PET). However, in contrast to Warburg’s original hypothesis, damaged mitochondria are not at the root of the aerobic glycolysis exhibited by most tumor cells. Most tumor mitochondria are not defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells mitochondrial metabolism is reprogrammed to meet the challenges of macromolecular synthesis. This possibility was never considered by Warburg and his contemporaries.

Advances in cancer metabolism research over the last decade have enhanced our understanding of how aerobic glycolysis and other metabolic alterations observed in cancer cells support the anabolic requirements associated with cell growth and proliferation. It has become clear that anabolic metabolism is under complex regulatory control directed by growth factor signal transduction in non-transformed cells. Yet despite these advances, the repeated refrain from traditional biochemists is that altered metabolism is merely an indirect phenomenon in cancer, a secondary effect that pales in importance to the activation of primary proliferation and survival signals (Hanahan and Weinberg, 2011). Most proto-oncogenes and tumor suppressor genes encode components of signal transduction pathways. Their roles in carcinogenesis have traditionally been attributed to their ability to regulate the cell cycle and sustain proliferative signaling while also helping cells evade growth suppression and/or cell death (Hanahan and Weinberg, 2011). But evidence for an alternative concept, that the primary functions of activated oncogenes and inactivated tumor suppressors are to reprogram cellular metabolism, has continued to build over the past several years. Evidence is also developing for the proposal that proto-oncogenes and tumor suppressors primarily evolved to regulate metabolism.

We begin this review by discussing how proliferative cell metabolism differs from quiescent cell metabolism on the basis of active metabolic reprogramming by oncogenes and tumor suppressors. Much of this reprogramming depends on utilizing mitochondria as functional biosynthetic organelles. We then further develop the idea that altered metabolism is a primary feature selected for during tumorigenesis. Recent advances have demonstrated that altered metabolism in cancer extends beyond adaptations to meet the increased anabolic requirements of a growing and dividing cell. Changes in cancer cell metabolism can also influence cellular differentiation status, and in some cases these changes arise from oncogenic alterations in metabolic enzymes themselves.

Metabolism in quiescent vs. proliferating cells nihms-360138-f0001

Metabolism in quiescent vs. proliferating cells: both use mitochondria.
(A) In the absence of instructional growth factor signaling, cells in multicellular organisms lack the ability to take up sufficient nutrients to maintain themselves. Neglected cells will undergo autophagy and catabolize amino acids and lipids through the TCA cycle, assuming sufficient oxygen is available. This oxidative metabolism maximizes ATP production. (B) Cells that receive instructional growth factor signaling are directed to increase their uptake of nutrients, most notably glucose and glutamine. The increased nutrient uptake can then support the anabolic requirements of cell growth: mainly lipid, protein, and nucleotide synthesis (biomass). Excess carbon is secreted as lactate. Proliferating cells may also use strategies to decrease their ATP production while increasing their ATP consumption. These strategies maintain the ADP:ATP ratio necessary to maintain glycolytic flux. Green arrows represent metabolic pathways, while black arrows represent signaling.

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling.
(A) The traditional demand-based model of how metabolism is altered in proliferating cells. In response to growth factor signaling, increased transcription and translation consume free energy and decrease the ADP:ATP ratio. This leads to enhanced flux of glucose carbon through glycolysis and the TCA cycle for the purpose of producing more ATP. (B) Supply-based model of how metabolism changes in proliferating cells. Growth factor signaling directly reprograms nutrient uptake and metabolism. Increased nutrient flux through glycolysis and the mitochondria in response to growth factor signaling is used for biomass production. Metabolism also impacts transcription and translation through mechanisms independent of ATP availability.

Alterations in classic oncogenes directly reprogram cell metabolism to increase nutrient uptake and biosynthesis. PI3K/Akt signaling downstream of receptor tyrosine kinase (RTK) activation increases glucose uptake through the transporter GLUT1, and increases flux through glycolysis. Branches of glycolytic metabolism contribute to nucleotide and amino acid synthesis. Akt also activates ATP-citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis. Mitochondrial citrate can be synthesized when glucose-derived acetyl-CoA, generated by pyruvate dehydrogenase (PDH), condenses with glutamine-derived oxaloacetate (OAA) via the activity of citrate synthase (CS). mTORC1 promotes protein synthesis and mitochondrial metabolism. Myc increases glutamine uptake and the conversion of glutamine into a mitochondrial carbon source by promoting the expression of the enzyme glutaminase (GLS). Myc also promotes mitochondrial biogenesis. In addition, Myc promotes nucleotide and amino acid synthesis, both through direct transcriptional regulation and through increasing the synthesis of mitochondrial metabolite precursors.

Pyruvate kinase M2 (PKM2) expression in proliferating cells is regulated by signaling and mitochondrial metabolism to facilitate macromolecular synthesis. PKM2 is a less active isoform of the terminal glycolytic enzyme pyruvate kinase. It is also uniquely inhibited downstream of tyrosine kinase signaling. The decreased enzymatic activity of PKM2 in the cytoplasm promotes the accumulation of upstream glycolytic intermediates and their shunting into anabolic pathways. These pathways include the serine synthetic pathway that contributes to nucleotide and amino acid production. When mitochondrial metabolism is excessive, reactive oxygen species (ROS) from the mitochondria can feedback to inhibit PKM2 activity. Acetylation of PKM2, dependent on acetyl-CoA availability, may also promote PKM2 degradation and further contribute to increased flux through anabolic synthesis pathways branching off glycolysis.

IDH1 and IDH2 mutants convert glutamine carbon to the oncometabolite 2-hydroxyglutarate to dysregulate epigenetics and cell differentiation. (A) α-ketoglutarate, produced in part by wild-type isocitrate dehydrogenase (IDH), can enter the nucleus and be used as a substrate for dioxygenase enzymes that modify epigenetic marks. These enzymes include the TET2 DNA hydroxylase enzyme which converts 5-methylcytosine to 5-hydroxymethylcytosine, typically at CpG dinucleotides. 5-hydroxymethylcytosine may be an intermediate in either active or passive DNA demethylation. α-ketoglutarate is also a substrate for JmjC domain histone demethylase enzymes that demethylate lysine residues on histone tails. (B) The common feature of cancer-associated mutations in cytosolic IDH1 and mitochondrial IDH2 is the acquisition of a neomorphic enzymatic activity. This activity converts glutamine-derived α-ketoglutarate to the oncometabolite 2HG. 2HG can competitively inhibit α-ketoglutarate-dependent enzymes like TET2 and the JmjC histone demethylases, thereby impairing normal epigenetic regulation. This results in altered histone methylation marks, in some cases DNA hypermethylation at CpG islands, and dysregulated cellular differentiation.

Hypoxia and HIF-1 activation promote an alternative pathway for citrate synthesis through reductive metabolism of glutamine. (A) In proliferating cells under normoxic conditions, citrate is synthesized from both glucose and glutamine. Glucose carbon provides acetyl-CoA through the activity of PDH. Glutamine carbon provides oxaloacetate through oxidative mitochondrial metabolism dependent on NAD+. Glucose-derived acetyl-CoA and glutamine-derived oxaloacetate condense to form citrate via the activity of citrate synthase (CS). Citrate can be exported to the cytosol for lipid synthesis. (B) In cells proliferating in hypoxia and/or with HIF-1 activation, glucose is diverted away from mitochondrial acetyl-CoA and citrate production. Citrate can be maintained through an alternative pathway of reductive carboxylation, which we propose to rely on reverse flux of glutamine-derived α-ketoglutarate through IDH2. This reverse flux in the mitochondria would promote electron export from the mitochondria when the activity of the electron transport chain is inhibited because of the lack of oxygen as an electron acceptor. Mitochondrial reverse flux can be accomplished by NADH conversion to NADPH by mitochondrial transhydrogenase and the resulting NADPH use in α-ketoglutarate carboxylation. When citrate/isocitrate is exported to the cytosol, some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH.

A major paradox remaining with PKM2 is that cells expressing PKM2 produce more glucose-derived pyruvate than PKM1-expressing cells, despite having a form of the pyruvate kinase enzyme that is less active and more sensitive to inhibition. One way to get around the PKM2 bottleneck and maintain/enhance pyruvate production may be through an proposed alternative glycolytic pathway, involving an enzymatic activity not yet purified, that dephosphorylates PEP to pyruvate without the generation of ATP (Vander Heiden et al., 2010). Another answer to this paradox may emanate from the serine synthetic pathway. The decreased enzymatic activity of PKM2 can promote the accumulation of the 3-phosphoglycerate glycolytic intermediate that serves as the entry point for the serine synthetic pathway branch off glycolysis. The little studied enzyme serine dehydratase can then directly convert serine to pyruvate. A third explanation may lie in the oscillatory activity of PKM2 from the inactive dimer to active tetramer form. Regulatory inputs into PKM2 like tyrosine phosphorylation and ROS destabilize the tetrameric form of PKM2 (Anastasiou et al., 2011; Christofk et al., 2008b; Hitosugi et al., 2009), but other inputs present in glycolytic cancer cells like fructose-1,6-bisphosphate and serine can continually allosterically activate and/or promote reformation of the PKM2 tetramer (Ashizawa et al., 1991; Eigenbrodt et al., 1983). Thus, PKM2 may be continually switching from inactive to active forms in cells, resulting in an apparent upregulation of flux through anabolic glycolytic branching pathways while also maintaining reasonable net flux of glucose carbon through PEP to pyruvate. With such an oscillatory system, small changes in the levels of any of the above-mentioned PKM2 regulatory inputs can cause exquisite, rapid, adjustments to glycolytic flux. This would be predicted to be advantageous for proliferating cells in the setting of variable extracellular nutrient availability. The capability for oscillatory regulation of PKM2 could also provide an explanation for why tumor cells do not select for altered glycolytic metabolism upstream of PKM2 through deletions and/or loss of function mutations of other glycolytic enzymes.

IDH1 mutations at R132 are not simply loss-of-function for isocitrate and α-ketoglutarate interconversion, but also acquire a novel reductive activity to convert α-ketoglutarate to 2-hydroxyglutarate (2HG), a rare metabolite found at only trace amounts in mammalian cells under normal conditions (Dang et al., 2009). However, it still remained unclear if 2HG was truly a pathogenic “oncometabolite” resulting from IDH1 mutation, or if it was just the byproduct of a loss of function mutation. Whether 2HG production or the loss of IDH1 normal function played a more important role in tumorigenesis remained uncertain.

A potential answer to whether 2HG production was relevant to tumorigenesis arrived with the study of mutations in IDH2, the mitochondrial homolog of IDH1. Up to this point a small fraction of gliomas lacking IDH1 mutations were known to harbor mutations at IDH2 R172, the analogous residue to IDH1 R132 (Yan et al., 2009). However, given the rarity of these IDH2 mutations, they had not been characterized for 2HG production. The discovery of IDH2 R172 mutations in AML as well as glioma samples prompted the study of whether these mutations also conferred the reductive enzymatic activity to produce 2HG. Enzymatic assays and measurement of 2HG levels in primary AML samples confirmed that these IDH2 R172 mutations result in 2HG elevation (Gross et al., 2010; Ward et al., 2010).

It was then investigated if the measurement of 2HG levels in primary tumor samples with unknown IDH mutation status could serve as a metabolite screening test for both cytosolic IDH1 and mitochondrial IDH2 mutations. AML samples with low to undetectable 2HG were subsequently sequenced and determined to be IDH1 and IDH2 wild-type, and several samples with elevated 2HG were found to have neomorphic mutations at either IDH1 R132 or IDH2 R172 (Gross et al., 2010). However, some 2HG-elevated AML samples lacked IDH1 R132 or IDH2 R172 mutations. When more comprehensive sequencing of IDH1 and IDH2 was performed, it was found that the common feature of this remaining subset of 2HG-elevated AMLs was another mutation in IDH2, occurring at R140 (Ward et al., 2010). This discovery provided additional evidence that 2HG production was the primary feature being selected for in tumors.

In addition to intensifying efforts to find the cellular targets of 2HG, the discovery of the 2HG-producing IDH1 and IDH2 mutations suggested that 2HG measurement might have clinical utility in diagnosis and disease monitoring. While much work is still needed in this area, serum 2HG levels have successfully correlated with IDH1 R132 mutations in AML, and recent data have suggested that 1H magnetic resonance spectroscopy can be applied for 2HG detection in vivo for glioma (Andronesi et al., 2012; Choi et al., 2012; Gross et al., 2010; Pope et al., 2012). These methods may have advantages over relying on invasive solid tumor biopsies or isolating leukemic blast cells to obtain material for sequencing of IDH1 and IDH2. Screening tumors and body fluids by 2HG status also has potentially increased applicability given the recent report that additional IDH mutations can produce 2HG (Ward et al., 2011). These additional alleles may account for the recently described subset of 2HG-elevated chondrosarcoma samples that lacked the most common IDH1 or IDH2 mutations but were not examined for other IDH alterations (Amary et al., 2011). Metabolite screening approaches can also distinguish neomorphic IDH mutations from SNPs and sequencing artifacts with no effect on IDH enzyme activity, as well as from an apparently rare subset of loss-of-function, non 2HG-producing IDH mutations that may play a secondary tumorigenic role in altering cellular redox (Ward et al., 2011).

Will we find other novel oncometabolites like 2HG? We should consider basing the search for new oncometabolites on those metabolites already known to cause disease in pediatric inborn errors of metabolism (IEMs). 2HG exemplifies how advances in research on IEMs can inform research on cancer metabolism, and vice versa. Methods developed by those studying 2HG aciduria were used to demonstrate that R(-)-2HG (also known as D-2HG) is the exclusive 2HG stereoisomer produced by IDH1 and IDH2 mutants (Dang et al., 2009; Ward et al., 2010). Likewise, following the discovery of 2HG-producing IDH2 R140 mutations in leukemia, researchers looked for and successfully found germline IDH2 R140 mutations in D-2HG aciduria. IDH2 R140 mutations now account for nearly half of all cases of this devastating disease (Kranendijk et al., 2010). While interest has surrounded 2HG due to its apparent novelty as a metabolite not found in normal non-diseased cells, there are situations where 2HG appears in the absence of metabolic enzyme mutations. For example, in human cells proliferating in hypoxia, α-ketoglutarate can accumulate and be metabolized through an enhanced reductive activity of wild-type IDH2 in the mitochondria, leading to 2HG accumulation in the absence of IDH mutation (Wise et al., 2011). The ability of 2HG to alter epigenetics may reflect its evolutionary ancient status as a signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency.

With this broadened view of what constitutes an oncometabolite, one could argue that the discoveries of two other oncometabolites, succinate and fumarate, preceded that of 2HG. Loss of function mutations in the TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) have been known for several years to occur in pheochromocytoma, paraganglioma, leiomoyoma, and renal carcinoma. It was initially hypothesized that these mutations contribute to cancer through mitochondrial damage producing elevated ROS (Eng et al., 2003). However, potential tumorigenic effects were soon linked to the elevated levels of succinate and fumarate arising from loss of SDH and FH function, respectively. Succinate was initially found to impair PHD2, the α-ketoglutarate-dependent enzyme regulating HIF stability, through product inhibition (Selak et al., 2005). Subsequent work confirmed that fumarate could inhibit PHD2 (Isaacs et al., 2005), and that succinate could also inhibit the related enzyme PHD3 (Lee et al., 2005). These observations linked the elevated HIF levels observed in SDH and FH deficient tumors to the activity of the succinate and fumarate metabolites. Recent work has suggested that fumarate may have other important roles that predominate in FH deficiency. For example, fumarate can modify cysteine residues to inhibit a negative regulator of the Nrf2 transcription factor. This post-translational modification leads to the upregulation of antioxidant response genes (Adam et al., 2011; Ooi et al., 2011).

There are still many unanswered questions regarding the biology of SDH and FH deficient tumors. In light of the emerging epigenetic effects of 2HG, it is intriguing that succinate has been shown to alter histone demethylase activity in yeast (Smith et al., 2007). Perhaps elevated succinate and fumarate resulting from SDH and FH mutations can promote tumorigenesis in part through epigenetic modulation.

Despite rapid technological advances in studying cell metabolism, we remain unable to reliably distinguish cytosolic metabolites from those in the mitochondria and other compartments. Current fractionation methods often lead to metabolite leakage. Even within one subcellular compartment, there may be distinct pools of metabolites resulting from channeling between metabolic enzymes. A related challenge lies in the quantitative measurement of metabolic flux; i.e., measuring the movement of carbon, nitrogen, and other atoms through metabolic pathways rather than simply measuring the steady-state levels of individual metabolites. While critical fluxes have been quantified in cultured cancer cells and methods for these analyses continue to improve (DeBerardinis et al., 2007; Mancuso et al., 2004; Yuan et al., 2008), many obstacles remain such as cellular compartmentalization and the reliance of most cell culture on complex, incompletely defined media.

Over the past decade, the study of metabolism has returned to its rightful place at the forefront of cancer research. Although Warburg was wrong about mitochondria, he was prescient in his focus on metabolism. Data now support the concepts that altered metabolism results from active reprogramming by altered oncogenes and tumor suppressors, and that metabolic adaptations can be clonally selected during tumorigenesis. Altered metabolism should now be considered a core hallmark of cancer. There is much work to be done. A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

Schell JC, Olson KA, …, Xie J, Egnatchik RA, Earl EG, DeBerardinis RJ, Rutter J.
Mol Cell. 2014 Nov 6; 56(3):400-13

Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.

Figure 2. Re-Expressed MPC1 and MPC2 Form a Mitochondrial Complex (A and B) (A) Western blot and (B) qRT-PCR analysis of the indicated colon cancer cell lines with retroviral expression of MPC1 (or MPC1-R97W) and/or MPC2. (C) Western blots of human heart tissue, hematologic cancer cells, and colon cancer cell lines with and without MPC1 and MPC2 re-expression. (D) Fluorescence microscopy of MPC1-GFP and MPC2-GFP overlaid with Mitotracker Red in HCT15 cells. Scale bar: 10 mm. (E) Blue-native PAGE analysis of mitochondria from control and MPC1/2-expressing cells. (F) Western blots of metabolic and mitochondrial proteins across four colon cancer cell lines with or without MPC1/2 expression

Figure 3. MPC Re-Expression Alters Mitochondrial Pyruvate Metabolism (A) OCR at baseline and maximal respiration in HCT15 (n = 7) and HT29 (n = 13) with pyruvate as the sole carbon source (mean ± SEM). (B and C) Schematic and citrate mass isotopomer quantification in cells cultured with D-[U-13C]glucose and unlabeled glutamine for 6 hr (mean ± SD, n = 2). (D) Glucose uptake and lactate secretion normalized to protein concentration (mean ± SD, n = 3). (E–G) (E) Western blots of PDH, phospho-PDH, and PDK1; (F) PDH activity assay and (G) CS activity assay with or without MPC1 and MPC2 expression (mean ± SD, n = 4). (H and I) Effects of MPC1/2 re-expression on mitochondrial membrane potential and ROS production (mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. MPC Re-Expression Alters Growth under Low-Attachment Conditions (A) Cell number of control and MPC1/2 re-expressing cell lines in adherent culture (mean ± SD, n = 7). (B) Cell viability determined by trypan blue exclusion and Annexin V/PI staining (mean ± SD, n = 3). (C–F) (C) EdU incorporation of MPC re-expressing cell lines at 3 hr post EdU pulse. Growth in 3D culture evaluated by (D) soft agar colony formation (mean ± SD, n = 12, see also Table S1) and by ([E] and [F]) spheroid formation ± MPC inhibitor UK5099 (mean ± SEM, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7. MPC Re-Expression Alters the Cancer Initiating Cell Population (A) Western blot quantification of ALDHA and Lin28A from control or MPC re-expressing HT29 xenografts (mean ± SEM, n = 10). (B and C) Percentage of ALDHhi (n = 3) and CD44hi (n = 5) cells as determined by flow cytometry (mean ± SEM). (D) Western blot analysis of stem cell markers in control and MPC re-expressing cell lines. (E) Relative MPC1 and MPC2 mRNA levels in ALDH sorted HCT15 cells (n = 4,mean ± SEM). 2D growth of (F) whole-population HCT15 cells and (G) ALDH sorted cells. Area determined by ImageJ after crystal violet staining (mean ± SD, n = 6). (H and I) (H) Adherent and (I) spheroid growth of main population (MP) versus side population (SP) HCT15 cells. (mean ± SD, n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Our demonstration that the MPC is lost or underexpressed in many cancers might provide clarifying context for earlier attempts to exploit metabolic regulation for cancer therapeutics. The PDH kinase inhibitor dichloroacetate, which impairs PDH phosphorylation and increases pyruvate oxidation, has been explored extensively as a cancer therapy (Bonnet et al., 2007; Olszewski et al., 2010). It has met with mixed results, however, and has typically failed to dramatically decrease tumor burden as a monotherapy (Garon et al., 2014;
Sanchez-Arago et al., 2010; Shahrzadetal.,2010). Is one possible reason for these failures that the MPC has been lost or inactivated, thereby limiting the metabolic effects of PDH activity? The inclusion of the MPC adds additional complexity to targeting cancer metabolism for therapy but has the potential to explain why treatments may be more effective in some studies than in others (Fulda et al., 2010; Hamanaka and Chandel, 2012; Tennant et al., 2010; Vander Heiden, 2011). The redundant measures to limit pyruvate oxidation make it easy to understand why expression of the MPC leads to relatively modest metabolic changes in cells grown in adherent culture conditions. While subtle, we observed a number of changes in metabolic parameters, all of which are consistent with enhanced mitochondrial pyruvate entry and oxidation. There are at least two possible explanations for the discrepancy that we observed between the impact on adherent and nonadherent cell proliferation. One hypothesis is that the stress of nutrient deprivation and detachment combines with these subtle metabolic effects to impair survival and proliferation.  ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

Lee KM, Nam K, Oh S, Lim J, Lee T, Shin I.
Cell Signal. 2015 Feb; 27(2):228-35

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

Fig. 1.ECM1 induces a metabolic shift toward promoting Warburg effect. (A) The levels of glucose uptake were examined with a cell-based assay. (B) Levels of lactate production were measured using a lactate assay kit. (C) Cellular ATP content was determined with a Cell Titer-Glo luminescent cell viability assay. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, ***p b 0.0005).

Fig.2. ECM1 up-regulates expression of gene sassociated with the Warburg effect. (A) Cell lysates were analyzed by western blotting using antibodies specific for ECM1, LDHA, GLUT1,and actin (as a loading control). The intensities of the bands were quantified using 1D Scan software and plotted. (BandC) mRNA levels of each gene were determined by real-time PCR using specific primers. (D) HIF-1α-dependent transcriptional activities were examined using a hypoxia response element (HRE) reporter indual luciferase assays. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, **p b 0.005, ***p b 0.0005).

Fig.3. ECM1-dependent upregulation of gene expression is not mediated byEgr-1.

Fig.4. ECM1 activates PKM2 via EGF-mediated ERK activation

Fig. 5. TheWarburg effect is attenuated by silencing of PKM2 in breast cancer cells

Recently, a non-glycolytic function of PKM2 was reported. Phosphorylated PKM2 at Ser37 is translocated into the nucleus after EGFR and ERK activation and regulates the expression of cyclin D1, c-Myc, LDHA, and GLUT1[19,37]. Here, we showed that ECM1 regulates the phosphorylation level and translocation of PKM2 via the EGFR/ ERK pathway. As we previously showed that ECM1 enhances the EGF response and increases EGFR expression through MUC1-dependent stabilization [17], it seemed likely that activation of the EGFR/ERK pathway by ECM1 is linked to PKM2 phosphorylation. Indeed, we show here that ECM1 regulates the phosphorylation of PKM2 at Ser37 and enhances the Warburg effect through the EGFR/ERK pathway. HIF-1α is known to be responsible for alterations in cancer cell metabolism [38] and our current studies showed that the expression level of HIF-1α is up-regulated by ECM1 (Fig. 2C and D). To determine the mechanism by which ECM1 upregulated HIF-1α expression, we focused on the induction of Egr-1 by EGFR/ERK signaling [39]. However, although Egr-1 expression was regulated by ECM1 we failed to find evidence that Egr-1 affected the expression of genes involved in the Warburg effect (Fig. 3C). Moreover, ERK-dependent PKM2 activation did not regulate HIF-1α expression in BT-474 cells (Fig. 4D and5B). These results suggested that the upregulation of HIF-1α by ECM1 is not mediated by the EGFR/ERK pathway.


In the current study we showed that ECM1 altered metabolic phenotypes of breast cancer cells toward promoting the Warburg effect.

Phosphorylation and nuclear translocation of PKM2 were induced by ECM1 through the EGFR/ERK pathway. Moreover, phosphorylated PKM2 increased the expression of metabolic genes such as LDHA and GLUT1, and promoted glucose uptake and lactate production. These findings provide a new perspective on the distinct functions of ECM1 in cancer cell metabolism. Supplementary data to this article can be found online at


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Chendong Yang, B Ko, CT. Hensley,…, J Rutter, ME. Merritt, RJ. DeBerardinis
Molec Cell  6 Nov 2014; 56(3):414–424


  • Mitochondria produce acetyl-CoA from glutamine during MPC inhibition
    •Alanine synthesis is suppressed during MPC inhibition
    •MPC inhibition activates GDH to supply pools of TCA cycle intermediates
    •GDH supports cell survival during periods of MPC inhibition


Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Yang et al, Graphical Abstract

Yang et al, Graphical Abstract

Graphical abstract

Figure 1. Pyruvate Depletion Redirects Glutamine Metabolism to Produce AcetylCoA and Citrate (A) Top: Anaplerosis supplied by [U-13C]glutamine. Glutamine supplies OAA via a-KG, while acetylCoA is predominantly supplied by other nutrients, particularly glucose. Bottom: Glutamine is converted to acetyl-CoA in the absence of glucosederived pyruvate. Red circles represent carbons arising from [U-13C]glutamine, and gray circles are unlabeled. Reductive carboxylation is indicated by the green dashed line. (B) Fraction of succinate, fumarate, malate, and aspartate containing four 13C carbons after culture of SFxL cells for 6 hr with [U-13C]glutamine in the presence or absence of 10 mM unlabeled glucose (Glc). (C) Mass isotopologues of citrate after culture of SFxL cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate (Pyr). (D) Citrate m+5 and m+6 after culture of HeLa or Huh-7 cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Isolated Mitochondria Convert Glutamine to Citrate (A) Western blot of whole-cell lysates (Cell) and preparations of isolated mitochondria (Mito) or cytosol from SFxL cells. (B) Oxygen consumption in a representative mitochondrial sample. Rates before and after addition of ADP/GDP are indicated. (C) Mass isotopologues of citrate produced by mitochondria cultured for 30 min with [U-13C] glutamine and with or without pyruvate.

Figure 3. Blockade of Mitochondrial Pyruvate Transport Activates Glutamine-Dependent Citrate Formation (A) Dose-dependent effects of UK5099 on citrate labeling from [U-13C]glucose and [U-13C]glutamine in SFxL cells. (B) Time course of citrate labeling from [U-13C] glutamine with or without 200 mM UK5099. (C) Abundance of total citrate and citrate m+6 in cells cultured in [U-13C]glutamine with or without 200 mM UK5099. (D) Mass isotopologues of citrate in cells cultured for 6 hr in [U-13C]glutamine with or without 10 mM CHC or 200 mM UK5099. (E) Effect of silencing ME2 on citrate m+6 after 6 hr of culture in [U-13C]glutamine. Relative abundances of citrate isotopologues were determined by normalizing total citrate abundance measured by mass spectrometry against cellular protein for each sample then multiplying by the fractional abundance of each isotopologue. (F) Effect of silencing MPC1 or MPC2 on formation of citrate m+6 after 6 hr of culture in [U-13C]glutamine. (G) Citrate isotopologues in primary human fibroblasts of varying MPC1 genotypes after culture in [U-13C]glutamine. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 4. Kinetic Analysis of the Metabolic Effects of Blocking Mitochondrial Pyruvate Transport (A) Summation of 13C spectra acquired over 2 min of exposure of SFxL cells to hyperpolarized [1-13C] pyruvate. Resonances are indicated for [1-13C] pyruvate (Pyr1), the hydrate of [1-13C]pyruvate (Pyr1-Hydr), [1-13C]lactate (Lac1), [1-13C]alanine (Ala1), and H[13C]O3 (Bicarbonate). (B) Time evolution of appearance of Lac1, Ala1, and bicarbonate in control and UK5099-treated cells. (C) Relative 13C NMR signals for Lac1, Ala1, and bicarbonate. Each signal is summed over the entire acquisition and expressed as a fraction of total 13C signal. (D) Quantity of intracellular and secreted alanine in control and UK5099-treated cells. Data are the average and SD of three independent cultures. *p < 0.05; ***p < 0.001. See also Figure S2.

Figure 5. Inhibiting Mitochondrial Pyruvate Transport Enhances the Contribution of Glutamine to Fatty Acid Synthesis (A) Mass isotopologues of palmitate extracted from cells cultured with [U-13C] glucose or [U-13C]glutamine, with or without 200 mM UK5099. For simplicity, only even-labeled isotopologues (m+2, m+4, etc.) are shown. (B) Fraction of lipogenic acetyl-CoA derived from glucose or glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. ***p < 0.001. See also Figure S3.

Figure 6. Blockade of Mitochondrial Pyruvate Transport Induces GDH (A) Two routes by which glutamate can be converted to AKG. Blue and green symbols are the amide (g) and amino (a) nitrogens of glutamine, respectively. (B) Utilization and secretion of glutamine (Gln), glutamate (Glu), and ammonia (NH4+) by SFxL cells with and without 200 mM UK5099. (C) Secretion of 15N-alanine and 15NH4+ derived from [a-15N]glutamine in SFxL cells expressing a control shRNA (shCtrl) or either of two shRNAs directed against GLUD1 (shGLUD1-A and shGLUD1-B). (D) Left: Phosphorylation of AMPK (T172) and acetyl-CoA carboxylase (ACC, S79) during treatment with 200 mM UK5099. Right: Steady-state levels of ATP 24 hr after addition of vehicle or 200 mM UK5099. (E) Fractional contribution of the m+6 isotopologue to total citrate in shCtrl, shGLUD1-A, and shGLUD1-B SFxL cells cultured in [U-13C]glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 7. GDH Sustains Growth and Viability during Suppression of Mitochondrial Pyruvate Transport (A) Relative growth inhibition of shCtrl, shGLUD1A, and shGLUD1-B SFxL cells treated with 50 mM UK5099 for 3 days. (B) Relative growth inhibition of SFxL cells treated with combinations of 50 mM of the GDH inhibitor EGCG, 10 mM of the GLS inhibitor BPTES, and 200 mM UK5099 for 3 days. (C) Relative cell death assessed by trypan blue staining in SFxL cells treated as in (B). (D) Relative cell death assessed by trypan blue staining in SF188 cells treated as in (B) for 2 days. (E) (Left) Growth of A549-derived subcutaneous xenografts treated with vehicle (saline), EGCG, CHC, or EGCG plus CHC (n = 4 for each group). Data are the average and SEM. Right: Lactate abundance in extracts of each tumor harvested at the end of the experiment. Data in (A)–(D) are the average and SD of three independent cultures. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.

Mitochondrial metabolism complements glycolysis as a source of energy and biosynthetic precursors. Precursors for lipids, proteins, and nucleic acids are derived from the TCA cycle. Maintaining pools of these intermediates is essential, even under circumstances of nutrient limitation or impaired supply of glucose-derived pyruvate to the mitochondria. Glutamine’s ability to produce both acetyl-CoA and OAA allows it to support TCA cycle activity as a sole carbon source and imposes a greater cellular dependence on glutamine metabolism when MPC function or pyruvate supply is impaired. Other anaplerotic amino acids could also supply both OAA and acetyl-CoA, providing flexible support for the TCA cycle when glucose is limiting. Although fatty acids are an important fuel in some cancer cells (Caro et al., 2012), and fatty acid oxidation is induced upon MPC inhibition, this pathway produces acetyl-CoA but not OAA. Thus, fatty acids would need to be oxidized along with an anaplerotic nutrient in order to enable the cycle to function as a biosynthetic hub. Notably, enforced MPC overexpression also impairs growth of some tumors (Schell et al., 2014), suggesting that maximal growth may require MPC activity to be maintained within a narrow window. After decades of research on mitochondrial pyruvate transport, molecular components of the MPC were recently reported (Halestrap, 2012; Schell and Rutter, 2013). MPC1 and MPC2 form a heterocomplex in the inner mitochondrial membrane, and loss of either component impairs pyruvate import, leading to citrate depletion (Bricker et al., 2012; Herzig et al., 2012). Mammalian cells lacking functional MPC1 display normal glutamine-supported respiration (Bricker et al., 2012), consistent with our observation that glutamine supplies the TCA cycle in absence of pyruvate import. We also observed that isolated mitochondria produce fully labeled citrate from glutamine, indicating that this pathway operates as a self-contained mechanism to maintain TCA cycle function. Recently, two well-known classes of drugs have unexpectedly been shown to inhibit MPC. First, thiazolidinediones, commonly used as insulin sensitizers, impair MPC function in myoblasts (Divakaruni et al.,2013). Second, the phosphodiesterase inhibitor Zaprinast inhibits MPC in the retina and brain (Du et al., 2013b). Zaprinast also induced accumulation of aspartate, suggesting that depletion of acetyl-CoA impaired the ability of a new turn of the TCA cycle to be initiated from OAA; as a consequence, OAA was transaminated to aspartate. We noted a similar phenomenon in cancer cells, suggesting that UK5099 elicits a state in which acetyl-CoA supply is insufficient to avoid OAA accumulation. Unlike UK5099, Zaprinast did not induce glutamine-dependent acetyl-CoA formation. This may be related to the reliance of isolated retinas on glucose rather than glutamine to supply TCA cycle intermediates or the exquisite system used by retinas to protect glutamate from oxidation (Du et al., 2013a). Zaprinast was also recently shown to inhibit glutaminase (Elhammali et al., 2014), which would further reduce the contribution of glutamine to the acetyl-CoA pool.

Comment by reader –

The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
2001). The vaccine is delivered in the adjuvant setting
and induces T helper type cells.

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Introduction to Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP


Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence.  It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.

There are essentially two major sources of fuel, mainly, carbohydrate and fat.  A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.

Carbohydrates are a major fuel utilized by way of either of two pathways.  They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle.  Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.

Carbohydrate metabolism has either of two routes of utilization.  This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle.  In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate.  In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital.  Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld,  who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.

This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.

The subchapters are as follows:

1.1      Carbohydrate Metabolism

1.2      Studies of Respiration Lead to Acetyl CoA

1.3      Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

1.4      The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy

Complex I or NADH-Q oxidoreductase

Complex I or NADH-Q oxidoreductase

Fatty acid oxidation and ETC

Fatty acid oxidation and ETC

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Introduction to Metabolomics

Introduction to Metabolomics

Author: Larry H. Bernstein, MD, FCAP


This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time bachalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career.

In the Preface, I failed to disclose that the term Metabolomics applies to plants, animals, bacteria, and both prokaryotes and eukaryotes.  The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence.  Was it William Faulkner who said in his Nobel Prize acceptance that mankind shall not merely exist, but survive? That seems to be the overlying theme for all of life. If life cannot persist, a surviving “remnant” might continue. The history of life may well be etched into the genetic code, some of which is not expressed.

This work is apportioned into chapters in a sequence that is first directed at the major sources for the energy and the structure of life, in the carbohydrates, lipids, and fats, which are sourced from both plants and animals, and depending on their balance, results in an equilibrium, and a disequilibrium we refer to as disease.  There is also a need to consider the nonorganic essentials which are derived from the soil, from water, and from the energy of the sun and the air we breathe, or in the case of water-bound metabolomes, dissolved gases.

In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways.  In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators.This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substartes, and are related to the tertiary structure of a protein.  The framework for diseases in a separate chapter.  Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded. Neutraceuticals and plant based nutrition are covered in Chapter 8.

Chapter 1: Metabolic Pathways

Chapter 2. Lipid Metabolism

Chapter 3. Cell Signaling

Chapter 4. Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8. Impairments in Pathological States: Endocrine Disorders; Stress Hypermetabolism and Cancer

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