Professional Documents
Culture Documents
Abstract
During oncogenesis, cells must make key decisions about their metabolic status. The switch from a
normal to pathological oncogenic metabolic state was first described over eighty-five years ago by Otto
Warburg. It has now become clear that this alteration, known as the Warburg effect, is an integral
component of a much larger metabolic switch controlled by the mammalian target of rapamycin
(mTOR) signaling cascade. In recent years the mechanism that turns on the Warburg effect has been
revealed. This involves a key alternative splicing switch from the embryonic to adult isoform of the pyruvate kinase mRNA, which somehow rewires cell metabolism so that glucose is used as a building block
for the production of biomass rather than for energy consumption. Although much of this pathway remains to be elucidated, it is clear that this research has refocused much of cancer research back to the
regulation of metabolism. In this review we will give an account of this intriguing switch and how it
relates to the transformation that takes place during oncogenesis.
Key words: Oncogenesis, Warburg effect, metabolic switch, splicing, mRNA
Accepted October 15 2011
Abbreviations: HIF (Hypoxia Inducible Factor); iPS (Induced Pluripotent Stem); mTOR (mammalian target of rapamycin); PEP (Phosphoenolpyruvate); PET (Positron Emission Tomography); PGAM1 (Phosphoglycerate mutase 1);
PKM1 (Pyruvate Kinase type M1); PKM2 (Pyruvate Kinase type M2); PTB (Polypyrimidine Tract Binding protein)
Introduction
Oncogenesis is a multistep process in which normal human cells gradually accumulate genetic alterations that
drive the transformation to fully fledged malignant forms.
It is thought that these genetic changes provide a selective
growth advantage that allows normal cells to progressively transform into invading cancer cells- a process
analogous to Darwins theory of natural selection [1,2]. In
their landmark commentary, Hanahan and Weinberg describe six major hallmarks of tumourigenic cells [3, 4].
They are 1) self-sufficiency in growth signals, 2) evasion
of apoptosis, 3) insensitivity to anti-growth signals, 4)
limitless replicative potential, 5) sustained angiogenesis
and 6) tissue invasion and metastasis. However, our present understanding of cancer points to the existence of at
least one additional hallmark that accompanies this transformation, the alteration of cellular metabolism.
25
Palazzo/Mahadevan
work on oxidative metabolism. Since then, the ability of
tumour cells to convert glucose to lactic acid in the presence of oxygen has come to be known as aerobic glycolysis or the Warburg effect. Paradoxically, despite the
fact that glycolysis results in the production of fewer
ATPs per sugar, the rate of ATP production is much
greater in cancer cells than in normal tissue. This is due to
the fact that once it is uncoupled, glycolysis can operate at
much higher rates than what oxidative phosphorylation
typically allows. In addition, this alteration appears to
enable a greater fraction of carbon atoms in glucose metabolites to be incorporated into macromolecule synthesis
rather than CO2 [12]. As a result, the activation of this
metabolic switch is beneficial to rapidly growing tissues,
where the production of biomass is much more critical
than energy generation.
In order to replenish the rapid utilization of sugars, cancer
cells tend to increase their uptake of glucose. Clinical
practice has taken advantage of this differential
uptake
by introducing a radiolabelled glucose derivative (18F-fluorodeoxyglucose) into patients and visualizing primary and metastatic human cancers by positron
emission tomography [13-15]. Indeed, many tumour cells
show an upregulation in the glucose transporter GLUT-1,
in response to hypoxia [16]. In addition, the upregulation
of glycolytic enzymes has also been associated with various tumours and cancer cells [17-19]. These include
Hexokinase II (which also plays a role in anti-apoptosis
[20, 21]), phosphoglucose isomerase (which was also
identified as a secreted factor that positively regulates the
motility of tumour cells [22]), the 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase isoform PFKBP3
(which favors the synthesis of fructose-2,6-bisphosphate,
a co-activator of phosphofructokinase 1, and a stimulator
of glycolytic flux [23, 24]), and PKM2 (the embryonic
form of pyruvate kinase [25-29]). This last finding was
originally made by the late Erich Eigenbrodt who in 1981
observed that cancer cells did not express the normal form
of pyruvate kinase (PKM1), but instead had an alternatively spliced form of this enzyme (PKM2).
Pyruvate Kinase, a Switch for Metabolic Rewiring
Pyruvate kinase is the key enzyme responsible for shunting the products of glycolysis into the citric acid cycle. It
catalyzes the dephosphorylation of phosphoenolpyruvate
(PEP) to pyruvate, yielding one molecule of ATP independent of oxygen supply. Four pyruvate kinase isoforms
exist in mammals, 1) Pyruvate Kinase type L (expressed
in the liver, one of the principal sites for gluconeogenesis
in the body); 2) Pyruvate Kinase type R (expressed in red
blood cells); 3) Pyruvate Kinase type M1 (expressed in
most adult tissues, named after its expression in muscle);
and 4) Pyruvate Kinase type M2 (expressed in lung tissues, adult stem cells, embryonic tissues and tumours)
26
[29]. PKM1 and PKM2 result from the alternative splicing of the same transcript that contains 12 exons in total,
two of which (exon 9 and 10) being mutually exclusive
[30]. While PKM1 contains only exon 9, PKM2 contains
exon 10. These two exons are identical in length (166
nucleotides, encoding 56 amino acids), and highly similar
(61% identical, 82% similar), suggesting that they probably arose from an ancient duplication event [31].
According to Eigenbrodt, these various Pyruvate Kinase
isoforms dictated the cells metabolic wiring [26, 29].
This resetting of the metabolic state was critical in
allowing the cells to fulfill their jobs or to help meet their
needs. By this reasoning, adipose cells, which synthesize
lipids, need a lipogenic metabolic state and they accomplish this in part by expressing a particular pyruvate
kinase that is responsive to the lipid biosynthetic pathway.
In contrast, embryonic and cancer cells need to make
nucleic acids (DNA) and thus have a metabolism and a
pyruvate kinase isoform that favors the nucleogenic state.
According to Eigenbrodts theory, pyruvate kinases acted
as a valve that allowed sugar into the aerobic energy
production pathway (i.e., the Krebs cycle) as opposed to
other metabolic pathways, such as lipid, glycogen or
nucleic acid production. By making pyruvate kinase
responsive to the particular needs of the cell, glucosederived carbons could be efficiently allocated to the
production of different macromolecules. Thus Eigenbrodt
argued that oncogenic transformation was always accompanied by a shift in the isoenzyme composition of
pyruvate kinase from the adult to the embryonic form.
While the adult form of this enzyme is responsive to the
cells redox potential, the embryonic form would be
sensitive to other cues, such as growth factors, lipogenesis
and nucleogenesis requirements. In support of his theory,
Eigenbrodt demonstrated that PKM2 was almost
universally expressed across a wide spectrum of cancerous tissues [29]. Eventually Eigenbrodt patented the testing of urine and stool for embryonic pyruvate kinase as a
cancer diagnostic [32]. Sadly, Eigenbrodt passed away on
June 11, 2004 at the age of 55. Four years later, in a series
of landmark papers by Lewis Cantleys laboratory, at
Harvard Medical School, it was demonstrated that the
Warburg effect could be turned on by forcing cells to
switch expression from PKM1 to PKM2 [25, 27].
Flicking the Switch: a Splicing Decision
Two questions naturally arose from these landmark studies. First, what regulates the alternative splicing of the
PKM transcript? Second, how does the switch in isozyme
production rewire the cells metabolism?
This first question was recently answered by two key
studies [33, 34]. In the first, Manleys group at Columbia
University used several fragments of the PKM pre-mRNA
27
Palazzo/Mahadevan
glycolytic pathway? One intriguing outcome of this
switch is an alteration in the steady state levels of the
various intermediates of the glycolytic pathway. Indeed,
the switch of PKM1 to PKM2 appears to trigger an increase in the levels of PEP in response to growth factor
signaling [56]. This makes sense in light of the fact that
PKM1 and 2 convert PEP to pyruvate. Curiously, PEP
is also used as the phosphate donor in histidine phosphorylation
reactions, which are an integral part
of several bacterial signal transduction cascades [57,
58]. In an interesting twist, when the fate of the PEP
phosphate was
monitored in PMK2-expressing
mammalian cells,
it appeared to be transferred to
the active site histidine
of the phosphoglycerate
mutase 1 (PGAM1) protein [56]. This enzyme participates in a step of glycolysis (the conversion of 3phosphoglycerate to 2-phospho-glycerate), upstream
from the production of PEP [59]. In fact, as part of the
PGAM1 catalytic cycle, 2,3-bisphosphoglycerate donates a phosphate to the active site histidine to form 2phosphoglycerate. During the next catalytic round,
PGAM1 transfers the phosphate to the second hydroxyl
group of a new 3-phosphogly-cerate molecule. Interestingly purified PGAM1 could not acquire a PEP-derived
phosphate on its own [56]. Currently it is unclear
which enzyme catalyzes this phosphate transfer, however we do know that it is neither PKM2 nor enolase
(which can reversibly convert 2-phosphoglycerate to
PEP) [56].
It should be noted that the transfer of phosphate from
PEP to PGAM1 has two consequences. First, this reaction creates pyruvate in a pyruvate kinase independent
manner. Second, this influx of phosphate into the
phosphoglycerate pool is likely to increase the level of
the 2, 3-bisphosphoglycerate intermediate, as seen experimentally after PKM2 inhibition [56]. Paradoxically
it has been noted that the levels of 2, 3-bisphosphoglycerate are inversely linked to cancer [60]. It is also
uncertain whether any of these changes are related to
the decoupling of glycolysis from oxidative phosphorylation, although it is likely that these alterations reflect
an entirely new metabolic wiring that either accompanies or allows for the Warburg effect. PKM2 may also
have other roles unconnected to its enzymatic activity.
In a very recent report, PKM2 (but not PKM1) has
been shown to associate with HIF1 and promote the
transactivation of HIF-1 target genes [53]. Since HIF1
mediates transcriptional changes in response to redox
changes and has been linked to transformation, PKM2
may play a role in regulating the expression of genes
that manage oxidative load and/ or contribute to oncogenic transformation. Obviously much more work
needs to be done.
28
Acknowledgements
We would like to thank J. Wan and C. Mazurek for feedback on the manuscript. This work was supported by a
grant from the Canadian Institutes of Health Research to
A.F.P. (FRN 102725).
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Foulds L. The experimental study of tumor progression: A review. Cancer Res. 1954; 14(5):327-39.
Nowell PC. The clonal evolution of tumor cell
populations. Science. 1976; 194 (4260): 23-28.
Hanahan D, Weinberg RA. The hallmarks of cancer.
Cell. 2000; 100 (1): 57-70.
Hanahan D, Weinberg RA. Hallmarks of cancer: the
next generation. Cell. 2011; 144(5): 646-574.
Koppenol WH, Bounds PL, Dang CV. Otto Warburg's
contributions to current concepts of cancer metabolism.
Nat Rev Cancer. 2011; 11(5): 325-337.
Warburg O. "Otto Warburg-Nobel Lecture", The
oxygen-transferring ferment of respiartion Nobel
Lectures. 1931:254-68. Elsevier Publishing Company,
Amsterdam, 1965.
Bechtel W. Discovering cell mechanisms: The creation
of modern cell biology. 2006: 94-115. Cambridge
university press.
Racker E. History of the Pasteur effect and its
pathobiology. Mol Cell Biochem. 1974; 5 (1-2): 17-23.
Warburg O. On the origin of cancer cells. Science.
1956; 123 (3191): 309-314.
Warburg O. On respiratory impairment in cancer cells.
Science. 1956; 124 (3215): 269-270.
Warburg O. Ueber den stoffwechsel der tumoren.
Constable. 1930.
Lunt SY, Vander Heiden MG. Aerobic glycolysis:
meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol. 2011; 27: 441-64.
Weber WA, Avril N, Schwaiger M. Relevance of
positron emission tomography (PET) in oncology.
Strahlenther Onkol. 1999; 175 (8): 356-373.
Gatenby RA, Gillies RJ. Why do cancers have high
aerobic glycolysis? Nat Rev Cancer. 2004; 4(11):891-9.
Hawkins RA, Phelps ME. PET in clinical oncology.
Cancer Metastasis Rev. 1988; 7 (2):119-142.
Macheda ML, Rogers S, Best JD. Molecular and
cellular regulation of glucose transporter (GLUT)
proteins in cancer. J Cell Physiol. 2005; 202 (3): 654662.
31. Letunic I, Copley RR, Bork P. Common exon duplication in animals and its role in alternative splicing. Hum
Mol Genet. 2002; 11 (13):1561-1567.
32. Hardt PD, Mazurek S, Toepler M, Schlierbach P,
Bretzel RG, Eigenbrodt E, et al. Faecal tumour M2
pyruvate kinase: a new, sensitive screening tool for
colorectal cancer. Br J Cancer. 2004; 91 (5): 980-984.
33. David CJ, Chen M, Assanah M, Canoll P, Manley JL.
HnRNP proteins controlled by c-Myc deregulate
pyruvate kinase mRNA splicing in cancer. Nature.
2010; 463 (7279): 364-368.
34. Clower CV, Chatterjee D, Wang Z, Cantley LC,
Vander Heiden MG, Krainer AR. The alternative
splicing repressors hnRNP A1/A2 and PTB influence
pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A. 2010; 107 (5): 18941899.
35. Burd CG, Dreyfuss G. RNA binding specificity of
hnRNP A1: significance of hnRNP A1 high-affinity
binding sites in pre-mRNA splicing. EMBO J. 1994;
13 (5): 1197-1204.
36. Del Gatto-Konczak F, Olive M, Gesnel MC,
Breathnach R. hnRNP A1 recruited to an exon in vivo
can function as an exon splicing silencer. Mol Cell
Biol. 1999; 19 (1): 251-260.
37. Chen M, Manley JL. Mechanisms of alternative
splicing regulation: insights from molecular and
genomics approaches. Nat Rev Mol Cell Biol. 2009;
10 (11): 741-754.
38. Grosso AR, Martins S, Carmo-Fonseca M. The
emerging role of splicing factors in cancer. EMBO
Rep. 2008; 9 (11): 1087-1093.
39. Zhou J, Allred DC, Avis I, Martinez A, Vos MD, Smith
L, et al. Differential expression of the early lung cancer
detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and
neoplastic breast cancer. Breast cancer research and
treatment. 2001; 66 (3): 217-224.
40. Hanamura A, Caceres JF, Mayeda A, Franza BR, Jr.,
Krainer AR. Regulated tissue-specific expression of
antagonistic pre-mRNA splicing factors. RNA. 1998;
4(4): 430-444.
41. Zerbe LK, Pino I, Pio R, Cosper PF, Dwyer-Nield LD,
Meyer AM, et al. Relative amounts of antagonistic
splicing factors, hnRNP A1 and ASF/SF2, change
during neoplastic lung growth: implications for premRNA processing. Molecular carcinogenesis. 2004;
41 (4): 187-196.
42. Spellman R, Smith CW. Novel modes of splicing
repression by PTB. Trends Biochem Sci. 2006; 31 (2):
73-76.
43. Singh R, Valcarcel J, Green MR. Distinct binding
specificities and functions of higher eukaryotic
polypyrimidine tract-binding proteins. Science. 1995;
268 (5214): 1173-1176.
44. Sauliere J, Sureau A, Expert-Bezancon A, Marie J. The
polypyrimidine tract binding protein (PTB) represses
splicing of exon 6B from the beta-tropomyosin pre-
29
Palazzo/Mahadevan
45. mRNA by directly interfering with the binding of the
U2AF65 subunit. Mol Cell Biol. 2006; 26 (23): 87558769.
46. Valcarcel J, Singh R, Zamore PD, Green MR. The
protein Sex-lethal antagonizes the splicing factor U2AF
to regulate alternative splicing of transformer premRNA. Nature. 1993; 362 (6416): 171-175.
47. Shibayama M, Ohno S, Osaka T, Sakamoto R, Tokunaga A, Nakatake Y, et al. Polypyrimidine tract-binding
protein is essential for early mouse development and
embryonic stem cell proliferation. FEBS J. 2009; 276
(22): 6658-6668.
48. He X, Pool M, Darcy KM, Lim SB, Auersperg N, Coon
JS, et al. Knockdown of polypyrimidine tract-binding
protein suppresses ovarian tumor cell growth and
invasiveness in vitro. Oncogene. 2007; 26 (34): 4961498.
49. Takenaka M, Yamada K, Lu T, Kang R, Tanaka T,
Noguchi T. Alternative splicing of the pyruvate kinase
M gene in a minigene system. Eur J Biochem. 1996;
235 (1-2): 366-371.
50. Takahashi K, Yamanaka S. Induction of pluripotent
stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell. 2006; 126 (4): 663676.
51. Chen M, Zhang J, Manley JL. Turning on a fuel switch
of cancer: hnRNP proteins regulate alternative splicing
of pyruvate kinase mRNA. Cancer Res. 2010; 70 (22):
8977-8980.
52. Sun Q, Chen X, Ma J, Peng H, Wang F, Zha X, et al.
Mammalian target of rapamycin up-regulation of
pyruvate kinase isoenzyme type M2 is critical for
aerobic glycolysis and tumor growth. Proc Natl Acad
Sci U S A. 2011; 108 (10): 4129-4134.
53. Levine AJ, Puzio-Kuter AM. The control of the metabolic switch in cancers by oncogenes and tumor
suppressor genes. Science. 2010; 330 (6009): 13401344.
54. Luo W, Hu H, Chang R, Zhong J, Knabel M, O'Meally
R, et al. Pyruvate kinase M2 is a PHD3-stimulated
coactivator for hypoxia-inducible factor 1. Cell. 2011;
145 (5): 732-744.
55. Mazurek S. Pyruvate kinase type M2: a key regulator
of the metabolic budget system in tumor cells. The
international journal of biochemistry & cell biology.
2011; 43 (7): 969-980.
56. Hitosugi T, Kang S, Vander Heiden MG, Chung TW,
Elf S, Lythgoe K, et al. Tyrosine phosphorylation
inhibits PKM2 to promote the Warburg effect and
tumor growth. Science signaling. 2009; 2 (97): ra73.
57. Vander Heiden MG, Locasale JW, Swanson KD, Sharfi
H, Heffron GJ, Amador-Noguez D, et al. Evidence for
an alternative glycolytic pathway in rapidly proliferating cells. Science. 2010; 329 (5998): 1492-1499.
30
58. Postma PW, Lengeler JW, Jacobson GR. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of
bacteria. Microbiol Rev. 1993; 57 (3): 543-594.
59. Saier MH, Jr., Ye JJ, Klinke S, Nino E. Identification
of an anaerobically induced phosphoenolpyruvatedependent fructose-specific phosphotransferase system
and evidence for the Embden-Meyerhof glycolytic
pathway in the heterofermentative bacterium
Lactobacillus brevis. J Bacteriol. 1996; 178 (1): 314316.
60. Fothergill-Gilmore
LA,
Watson
HC.
The
phosphoglycerate mutases. Adv Enzymol Relat Areas
Mol Biol. 1989; 62: 227-313.
61. Engel M, Mazurek S, Eigenbrodt E, Welter C.
Phosphoglycerate mutase-derived polypeptide inhibits
glycolytic flux and induces cell growth arrest in tumor
cell lines. The Journal of biological chemistry. 2004;
279 (34): 35803-35812.
Correspondence to:
Alexander F. Palazzo
Department of Biochemistry
University of Toronto
Medical Sciences Building, Room 5242
Toronto, Ontario
Canada M5S 1A8