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Abstract:
Dietary fiber has been proposed to protect against colorectal cancer.
Butyrate, a fiber metabolite that is produced by bacteria in the colon is
known to inhibit cell proliferation and promote cell differentiation, while
also inducing apoptotic cell death in colorectal cancer cells at
physiologically relevant concentrations. Unlike the majority of cells in the
human body that prefer utilizing glucose, colonocytes use butyrate the
primary energy source. However, colorectal cancer cells shift away from
utilizing butyrate towards glucose (the Warburg effect) leading to the
accumulation of butyrate in the nucleus. Once in the nucleus, butyrate
can act as an inhibitor of histone deacetylases to decrease proliferation
and promote apoptosis. Here, we sought to determine how the oxidation
of butyrate is impacted by knocking down pyruvate kinase M2 (PKM2), a
key enzyme in glycolysis necessary for tumor growth, in colorectal
cancer cells.
In this study, we used a lentivirus approach to knockdown PKM2 in
HCT116 colorectal cancer cells the efficiency of knockdown was
confirmed by western blot. Then butyrate oxidation was accessed using
an XF24 Analyzer, which measures oxygen consumption in real time. We
found that PKM2 knocked down exhibit an increase in butyrate oxidation.
Additionally, PKM2 deficiency altered butyrate's ability to slow cell
proliferation. These data suggest the perturbation of pyruvate kinase
isozyme M2 in colorectal cancer cells may shift metabolism towards
butyrate oxidation and away from the Warburg Effect. This is clinically
important to understand the potential of dietary fiber in cancer treatment
and prevention.
Background and Significance:
The Human Microbiome Project has shed light upon the effects of the
100 trillion microbes colonizing our gastrointestinal tract and their
functional contributions to human health and disease [1]. The symbiotic
relationship between human and microbial genomes have provided us
with traits that evolution would not have given rise to alone, allowing us
to view ourselves as more of a ‘human supraorganism’ [1]. For example,
the short chain fatty acid (SCFA), butyrate, is one of the metabolites
produced by the fermentation of dietary fiber by bacteria within the colon
[2]. Unlike other cells in the human body that utilize glucose as the
primary energy source, colonocytes prefer using butyrate [1]. Normal
colonocytes use butyrate through β-oxidation undergo and normal
growth and differentiation [3]. However, some studies have shown in
colorectal cancer patients that there is a decrease in butyrate producing
bacteria [4]. This effect may contribute to a decrease in butyrate
oxidation, growth regulation and differentiation. When cancerous
colonocytes are treated with butyrate, they often undergo apoptosis
instead of regulated growth as compared to their healthy counterparts.
This dichotomy is known as the butyrate paradox. The anti-cancer effects
through butyrate have been shown to regulate gene expression,
including p21 a cell cycle regulating protein, by inhibition of histone
deacetylases leading to hyperacetylation of histones [5-7]. This finding is
of great significance given that dietary fiber’s metabolite butyrate has
been proposed to suppress colorectal cancer (CRC), which is the third
most diagnosed and deadly cancer in the United States [8].
During tumorigenic process, it is understood that the colonocyte
changes from utilizing butyrate to glucose, and adopts a type of
metabolism known as the Warburg Effect. The Warburg effect causes
cancerous colonocytes to undergo high levels of aerobic glycolysis and
glucose uptake, while shifting away from mitochondrial butyrate
metabolism. This shifts metabolism away from butyrate and its histone
deactylase activity along with its ability to regulate apoptosis of the
cancerous colonocyte [2, 9-11]. The metabolic shift favoring glycolysis in
cancerous colonocytes provides necessary building blocks for cell
proliferation such as reducing agents (NADPH and NADH) as well as
ribose-5-phosphate for new DNA formation. Recent evidence has shown
that the overexpression of pyruvate kinase M2 (PKM2), a key enzyme in
glycolysis in cancerous colonocytes that is essential for tumor growth
and cancer metabolism, provides a pathway for pyruvate to be converted
to lactate which can be reconverted to glucose to be re-used by
cancerous colonocytes [12]. Given the difference of PKM2 expression in
normal colonocytes versus cancerous colonocytes, it has been
suggested by many to be a potential therapeutic target for various types
of cancer.
Contact Information:
Haley Porter
Department of Nutrition
1215 W. Cumberland Ave
229 Jessie Harris Building
Knoxville, TN 37917
hporter4@vols.utk.edu
How Colonocyte Metabolism Shifts in Colorectal Cancer
Haley Porter, Megan Johnstone, Natalie Bennett, Ahmed Bettaieb, and Dallas R. Donohoe. Nutrition, University of Tennessee, Knoxville, TN
Conclusions:
 Compared to control cells PKM2 knock
down cells exhibit no difference in
glucose oxidation, but show increased
butyrate oxidation.
 Increased capacity to oxidize butyrate
may lead to decreased histone
deactylase activity and increased
histone acetylation. This
hyperacetylation allows the p21
promotor site to be active and therefore
increase its transcription which can lead
to increased apoptosis in this PKM2 KD
cell line as compared to wild type
HCT116s
 Our findings confirm the importance of
the pyruvate kinase isozyme M2 in
cancer cell metabolism of tumerogenic
colonocytes. And suggest that the knock
down of PKM2 impacts butyrate
oxidation and shifts cell metabolism
away from a glycolytic (Warburg Effect)
phenotype.
 Further studies are warranted to confirm
downstream targets of PKM2 and
butyrate’s effects on cell cycle and
apoptosis.
References:
1) Turnbaugh, PJ, et al, The human microbiome project: exploring the microbial part of
ourselves in a changing world. Nature, 2007. 449(7164): p. 804-810.
2) Donohoe, DR, et al., Microbial Oncotarget: Bacterial-Produced Butyrate,
Chemoprevention and Warburg Effect. Oncotarget, 2013. 4(2): p. 182-183.
3) Roediger, W.E., Role of anaerobic bacteria in the metabolic welfare of the colonic
mucosa in man. Gut, 1980. 21(9): p. 793-8.
4) Louis, P., G.L. Hold, and H.J. Flint, The gut microbiota, bacterial metabolites and
colorectal cancer. Nat Rev Micro, 2014. 12(10): p. 661-672
5) Boffa, LC, et al., Supression of histone deacetylation in vivo and in vitro by sodium
butyrate. J Biol Chem, 1978. 253(10): p. 3364-6.
6) Candidio, EP, R Reeves, and JR Davie, Sodium butyrate inhibits histone deacetylation
in cultured cells. Cell, 1978. 14(1): p. 105-13.
7) Davie, JR, Inhibition of histone deacetylase activity by butyrate. J Nutr, 2003. 133(7
Suppl): p. 2485S-2493S.
8) Myzak, MC, E. Ho, and RH Dashwood, Dietary agents as histone deacetylase
inhibitors. Mol Carcinog, 2006. 45 (6): p. 443-6.
9) Seigel, R., D. Naishadham, and A. Jemal, Cancer Statistics, 2013. CA Cancer J Clin,
2013. 63(1): p. 11-30.
10) Donohoe, DR, et al., The Warburg effect dictates the mechanism of butyrate-mediated
histone acetylation and cell proliferation. Mol Cell, 2012. 48(4): p. 612-26.
11) Andriamihaja, M., et al., Butyrate metabolism in human colon carcinoma cells:
implications concerning its growth-inhibitory effect. J Cell Physiol, 2009. 218 (1): p. 58-
65.
12) Jahns, F., et al., Impact of butyrate on PKM2 and HSP90β expression in human colon
tissues of different transformation stages: a comparison of gene and protein data.
Genes and Nutrition, 2012. 7(2): 235-246.
Figure 1: PKM2 Knockdown confirmation. (A.): Hoescht
DNA staining of wild type HCT 116 cells. (B.) Hoescht DNA
staining of PKM2 KD cells with green fluorescent protein
(GFP) confirming the RNA interferent mutant PKM2 isozyme
presence. (C.) Western Blot quantification and confirmation
of PKM2 knockdown. (D.) Western blot of PKM2 confirmation
of PKM2 knockdown through RNAi (top) along with beta-actin
loading control (bottom).
Methodology:
Lentivirus mediated Knockdown of PKM2.
PKM2 silencing in HCT116 cells was achieved by testing five different hairpins (Open Biosystems). Two scramble non-silencing shRNA
were used as control. Packaging (psPAX2) and envelope (pMD2.G) vectors were obtained from Addgene (Boston, MA). Lentiviruses
were generated by co-transfection of vectors in HEK293FT cells using Lipofectamine 2000 (Invitrogen) following the guidelines of the
manufacturer and then used to infect HCT116 cells. Cells were selected using puromycin (2 μg/ml), and drug-resistant pools were
propagated.
Western Blot
Cells were lysed in radioimmunoprecipitation assay buffer (Cell signaling, #9806) supplemented with 1mM PMSF (Cell signaling,
#8553) and phosphatase inhibitor cocktail (Cell signaling, #5872). Lysates were clarified by centrifugation at 13,000 rpm for 10 min,
and protein concentrations were determined using Bradford assay. Proteins (30μg) were resolved by SDS-PAGE and transferred to
PVDF membranes.. Immunoblotting of lysates was performed using PKM2 (Cell Signaling #4053), β-actin (Sigma, A1978)., and after
incubation with secondary antibodies, proteins were visualized using enhanced chemiluminescence (Source). Pixel intensities of
immunoreactive bands were quantified with Quantity One software. The bar graph represents normalized data for PKM2/β-actin
Microscopy
HCT116s (WT) and PKM2 Knockdown were seeded on microscope slides and grown to 25% confluency. Hoechst 33342 was added
according to manufacturer instructions. Briefly, cells were incubated for ten minutes and then washed 3x with warm PBS. Warmed
phenol free media was then used to image cells.
Flux experiment
XF24 Analyzer (Seahorse Bioscience) was used to measure oxygen consumption rates (% OCR) in HCT116. Experiments were
conducted following manufacturer’s guidelines. HCT116s were split into XF24 cell culture microplate (Seahorse Bioscience, 100777-
004) at equal densities and grown to 90% confluency. Prior to the assay, cell plates were incubated with 1X KHB (50mM carnitine) in a-
CO2 incubator at 37℃ for 45 minutes; 15 minutes prior to running the seahorse cells are incubated with fresh 1X KHB (2.5mM glucose,
50mM carnitine) in non-CO2 incubator at 37℃. Butyrate (Sigma B5887) concentrations (5mM) were injected first, then 2-deoxy-D-
glucose (5 mM) (Sigma, D8375) was injected to block glycolysis. Finally, 10% sodium azide was injected to block oxidation capacity of
cells.
Figure 2: Diagram showing experimental strategy that
will be used to measure butyrate oxidation in cells with
XF24 Analyzer.
The XF24 Analyzer allows real time measurement of
oxygen and protons released into the extracellular
space. The system is capable of measuring 20 wells at
the same time and equipped with four injection ports.
The rate of oxygen consumption (OCR) is indicative of
oxidative phosphorylation.
Using the injection ports, concentrated treatments are
injected into each well. Our treatments include
butyrate with a final concentration of 5mM. 2-deoxy-D-
glucose (2DG) is an inhibitor of glycolysis is used at a
final concentration of 5mM. With glycolysis inhibited,
the majority of cellular metabolism will be shunted
towards mitochondrial respiration and therefore
butyrate represents the sole exogenous energetic
substrate. Finally, a 10% solution of sodium azide is
injected as a potent biocide and used as a control.
C.A.
Through knocking down pyruvate kinase M2 (PKM2), butyrate oxidation will increase in the cancerous
colonocyte and diminish the ability of butyrate to slow cell proliferation
Hypothesis:
Results:
Figure 5: PKM2 knockdown increases butyrate oxidation. (A): In the transition of normal colonocytes to cancerous
colonocytes, transcription of PKM2 is increased leading to an excess production of lactate (Warburg Effect) through
aerobic glycolysis. Since butyrate is no longer being oxidized for energy, it accumulates in the nucleus acting as an HDAC
inhibitor. In this gene regulatory role, butyrate decreases cell proliferation and induces cancer cell apoptosis.
Figure 3 (B): When PKM2 is knocked down, glucose metabolism is inhibited and shifts away from the Warburg Effect
towards butyrate oxidation. This shows the important role PKM2 plays in cancer cell metabolism.
A B
Figure 3. The knock down of
PKM2 increases Butyrate
Oxidation in Cancerous
Colonocytes. The % change in
the oxygen consumption rate
after addition/injection of
butyrate at a final concentration
of 5 mM in HCT116 colorectal
cancer cells pretreated for 45
min. Area under the curve
analysis from OCR
measurements 7 through 11
(after 2DG injection, but before
Sodium Azide injection). These
measurements represent the
butyrate oxidation (arbitrary
units). The glucose
concentration in the fatty acid
oxidation media was kept
constant at 2.5 mM. Data points
represent the average OCR (%)
over 3-5 replicates per
experimental condition.
A.
B. D.
Figure 4: The knock down of
PKM2 significantly increases
Butyrate Oxidation, but not
glucose oxidation in Cancerous
Colonocytes. (A.) There was no
significant difference in glucose
oxidation between groups. (B.) After
2DG inject, butyrate oxidation was
measured. There is a significant
different between PKM2 KD and all
other groups. p < 0.05.
A.
0.00
2000.00
4000.00
6000.00
8000.00
PKM2 WT PKM2 KD PKM2 WT
+BUTYRATE
PKM2 KD
+BUTYRATE
AUCAnovaOCR(%)
Butyrate Oxidation
(Measurements 7 - 11)B.
*
PKM2
β-Actin
0
0.2
0.4
0.6
0.8
WT
PKM2
SCR.
PKM2
SCR.
PKM2
KD
PKM2
KD
PKM2
KD
PKM2
KD
PKM2
KD
PKM2
PKM2/ β-Actin
p21