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Special Issue: Cancer Metabolism

In memory of Erich Eigenbrodt

Biomedical Research 2012; 23: SI 25-30

Editor-in-Chief: M.A. Qayyum

Alternative splicing rewires cellular metabolism to turn on the

Warburg effect.
Alexander F. Palazzo1 and Kohila Mahadevan
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

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)

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.

The Warburg Effect

In work that began more than one hundred years ago, Otto
Warburg investigated oxidative pathways in various bio-

logical systems (for an overview of Warburgs life, see

[5-7]). His research eventually expanded to an investigation of the metabolic pathways of normal and tumour tissues. When cells need to convert sugars into energy, they
process glucose into pyruvate using glycolysis. Under
aerobic conditions, normal adult human cells then oxidize
the pyruvate to CO2 and H2O in the mitochondria (via the
Krebs cycle) to generate a proton gradient across the mitochondrial inner membrane. This chemical gradient is
then used to regenerate ATP from ADP and inorganic
phosphate in a process commonly referred to as oxidative
phosphorylation. When oxygen levels are low, glycolysis
is decoupled from oxidative phosphorylation and pyruvate is further metabolized to lactic acid. Thus the limited
use of glycolysis in adult tissues is mediated by the presence of oxygen (the Pasteur effect [8]). However, Warburg observed that tumour cells convert glucose to lactic
acid even in the presence of oxygen [9-12]. Based on
these observations, he hypothesized that cancer results
from a dysfunction in mitochondrial metabolism. Later it
was shown that mitochondrial function is not impaired in
most cancer cells, suggesting an alternative origin for the
switch to aerobic glycolysis [12]. In 1931, Warburg
won the Nobel Prize in Physiology or Medicine for his

Biomedical Research 2012 Volume 23 (Special Issue Cancer Metabolism)


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
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)

[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

Biomedical Research 2012 Volume 23 (Special Issue Cancer Metabolism)

Alternative Splicing Rewires the Metabolism of the Cell

to isolate RNA-binding factors from HeLa cell nuclear
extract. Using an RNA that contained the exon 9-intron 9
junction sequence they identified two proteins, hnRNPA1
and hnRNPA2, which recognized a UAGGGC sequence
in intron 9 immediately downstream of the junction [33].
Consistent with this observation, the consensus hnRNPA1
recognition motif was previously described as
UAGGG(A/U) [35]. Furthermore these two RNA-binding
proteins previously were characterized as splice site repressors [36, 37], and found to be up-regulated in embryonic and cancer tissues [38-41]. Indeed, Manleys group
found a tight correlation between their expression and the
exclusion of exon 9 from the mature PKM transcript [33].
Then to determine whether there were additional factors
that regulated the intron 8-exon 9 junction, Manleys
group used this RNA fragment to identify a third splicing
regulator, the polypyrimidine tract binding protein (PTB),
which bound to two UCUUC motifs upstream of the junction [33]. This RNA-binding protein had also been implicated in the repression of splicing, often by binding to
polypyrimidine tracts and suppressing the utilization of a
proximal 3splice site [42-44]. Interestingly, the suppression of 3 splice site selection by a related polypyrimidine
binding protein, Sex-lethal, is one of the most well-known
alternative splicing events that ultimately determines sexspecification in fruit flies [45]. As with the other two
PKM regulators, PTB is expressed in embryonic [46] and
cancer cells [47] and this expression pattern correlated
with exon 9 skipping [33]. Indeed, the simultaneous depletion of hnRNPA1, hnRNPA2, and PTB in cancer and
embryonic cell lines reverted expression from PKM2 to
PKM1 [33, 34], and inhibited the Warburg effect [34].
Despite all this progress, some outstanding issues remain.
One is the mechanism that regulates the inclusion/exclusion of exon 10. Since the junctions surrounding this exon are efficiently utilized during in vitro
splicing reactions [48], it appears that exon 10 skipping
should be actively promoted in adult tissues. Given that
the codepletion of hnRNPA1, hnRNPA2, and PTB, was
sufficient to promote the switch in splicing, these proteins
may indirectly downregulate factors that promote exon 10
skipping. Another issue is how the expression of these
splicing regulators is turned on in proliferating cells. Part
of the answer appears to be that the production of all three
factors is promoted by the proto-oncogenic transcription
factor c-myc [33]. Indeed, the depletion of c-myc in
NIH3T3 mouse fibroblasts (derived from mouse embryos
and thus expressing both c-myc and PKM2) resulted in a
decrease in PTB, hnRNPA1, and hnRNPA2 and a switch
from PKM2 to PKM1 [33]. Interestingly, c-myc is not
only over-expressed in many tumours but has also been
shown to promote induced pluripotent stem (iPS) cell
reprogramming [49]. This agrees with the notion that the
reprogramming of adult to embryonic-like cells must be
accompanied by a switch in metabolic states. However, it

should be noted that the depletion of c-myc from HeLa

cells (which also express c-myc and PKM2) had no effect
on the expression of these splicing factors and on PKM
isoform switching [33]. This latter finding indicates that
there are probably several transcription factors that can
change the level of these critical regulators of alternative
splicing. In support of this idea, other transcription factors
that are used in iPS cell generation, such as Nanog and
Klf4, have also been implicated in regulating PTB,
hnRNPA1, and hnRNPA2 levels [50]. Another key regulator of cellular activity in response to metabolic state is
the mTOR signaling cascade; thus, it was no surprise that
this pathway, through the activation of the hypoxiainducible factor 1 (HIF1) transcription factor and the
up-regulation of c-myc, can also promote PKM1 to
PKM2 switching [51-53].
Turning Off PKM2 to Get More Out of Glycolysis
The second question, how the switch in PKM isozyme
production rewires the cells metabolism, has become
more interesting and puzzling with each new development. The answer to this question hinges on differences
between the two alternative PKM exons. So what exactly
changes? First, PKM2 is much less active than PKM1
[54]. Although both isoforms can exist in an active
tetrameric state, PKM2 also exists in a dimeric state that
is characterized by a low PEP affinity [19, 26, 29]. Unlike
PKM1, PKM2 tetramerization (and thus activation) is
enhanced by the association of fructose-1,6-bisphosphate.
Binding of this cofactor can in turn be disrupted by the
association of phosphotyrosine peptides with the fructose1,6-bisphosphate-binding site on PKM2 [27]. As to the
identity of the phospho-tyrosine protein, it appears that
this might be PKM2 itself [55]. In support of this idea, a
phosphopeptide derived from PKM2 (residues 98-112)
disrupts the interaction of fructose-1, 6-biphosphate and
full-length PKM2. Critically, this peptide only disrupts
association when its single tyrosine (corresponding to
position 105 in full length PKM2), is phosphorylated
[55]. Indeed, PKM2 is phosphorylated on this residue by
the fibroblast growth factor receptor [55]. Additionally, it
is likely that PKM2 is the target of several other tyrosine
kinases, one potential candidate being Akt, a key modulator of mTOR signaling [51, 55]. Thus it appears that in
response to growth factor activation, a minority of PKM2
gets phosphorylated, stimulating the inactivation of bulk
unphosphorylated pool of PKM2. Moreover, all of the
data collected thus far indicates that this inactivation cascade stimulates the Warburg effect [27, 55].
While all these developments have been interesting, they
pose a serious conundrum: how can PKM2 inhibition,
which should decrease glucose consumption and lactate
production, cause the Warburg effect, which is typically
accompanied by an increase of glucose flux through the

Biomedical Research 2012 Volume 23 (Special Issue Cancer Metabolism)


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.


Although much about this switch remains mysterious, it

has opened a new door into understanding how cells must
rewire their metabolism in order to accomplish different
tasks. Moreover, PKM2 has become a novel target for
cancer therapeutics. Hopefully the further elucidation of
this pathway will lead to more insights and other potential
targets for drug discovery.

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).







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Correspondence to:
Alexander F. Palazzo
Department of Biochemistry
University of Toronto
Medical Sciences Building, Room 5242
Toronto, Ontario
Canada M5S 1A8

Biomedical Research 2012 Volume 23 (Special Issue Cancer Metabolism)