Post on 10-Dec-2018
Targeting a glycolysis HIF-1 feed-forward mechanism
1
Definition of a novel feed-forward mechanism for glycolysis-HIF1α signaling in hypoxic tumors
highlights adolase A as a therapeutic target
Geoffrey Grandjean 1, 2, 4, Petrus De Jong2,4, Brian James2, Mei Yee Koh2, Robert Lemos2, John
Kingston1, Alexander Aleshin2, Laurie A. Bankston2, Claudia P. Miller2, Eun Jeong Cho3,
Ramakrishna Edupuganti3, Ashwini Devkota3, Gabriel Stancu3, Robert C. Liddington2, Kevin
Dalby3 and Garth Powis2
1 Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center.
Houston, TX.
2 Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA
3 Department of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin,
Austin, TX.
4 G Grandjean and P De Jong contributed equally to this article.
Type of manuscript: Research Article
Running title: Targeting a glycolysis HIF-1 feed-forward mechanism
Keywords: glycolysis, HIF-1, feed-forward, ALDOA, structure.
Communicating Author:
Garth Powis, D. Phil. Director, NCI-Designated Cancer Center Sanford Burnham Prebys Medical Discovery Institute 10901 North Torrey Pines Road La Jolla, CA 92037 Email: gpowis@sbpdiscovery.org Tel: 858.795.5195 Fax: 858.795.5490
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
2
Word counts:
Abstract: 164
Text: (including figure legends, excluding references) 5,532
Figures: 7
Tables: 0
References: 30
Supplemental information file: 3 methods, 5 tables, 10 figures,
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
3
Abstract:
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes.
Here we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and
stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α
signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP
elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the
HIF1α transcriptional co-activator p300. Notably, of the HIF1α activated glycolytic enzymes we
evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease
in glycolysis, HIF-1 activity and cancer cell proliferation. Furthermore, either RNAi-mediated
silencing of ALDOA or systemic treatment with a specific small molecule inhibitor of aldolase A
was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In
establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cell, our
results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized
strategy to treat intractable hypoxic cancer cells found widely in most solid tumors.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
4
Introduction
Glycolysis is defined as the sequence of 10 enzymatic reactions converting glucose to
pyruvate, which is accompanied by release of energy in the form of ATP. In normal cells,
pyruvate then enters the mitochondrial carboxylic acid cycle in the presence of oxygen, or is
converted to lactic acid in its absence. Glycolysis is critical for providing rapidly-dividing normal
and cancer cells with energy and metabolic intermediates to synthesize cellular biomass (1,2).
Malignant transformation greatly increases aerobic glycolysis (the Warburg effect) (3), which
favors production of additional ATP and metabolites for biomass synthesis, and enables
uncontrolled proliferation. Solid tumors also have an abnormal vasculature that leads to poor
blood perfusion and hypoxia. A cancer cell’s response to hypoxia is mediated by the hypoxia-
inducible transcription factors HIF-1 and 2 (4), which increase the expression of numerous
survival factors, including genes that encode vascular endothelial growth factor (VEGF) (5,6).
HIF-1 also upregulates the transcription of several glycolytic enzymes (7). Both glycolysis and
HIF activity are critical for cancer cell survival, and have been proposed as therapeutic targets
for agents that inhibit tumor growth (8-11).
While HIF-1 is critical for tumor growth, in part by inducing glycolytic enzymes, our findings
suggest that glycolysis is necessary for maintaining HIF-1 activity. This constitutes a feed-
forward loop that promotes increased HIF-1 activity and glycolysis, which we show is mediated
by inhibition of the AMPK1/EA1 binding protein p300 pathway. HIF-1 is often studied in terms of
its protein levels, but we show here that HIF-1 may not be functionally active if glycolysis is
limiting. Inhibiting the glycolysis–HIF-1 feed-forward loop therefore offers a novel target for
blocking tumor energy and biomass production and the HIF-1 survival response. Although HIF-1
and glycolysis have previously been proposed as targets for cancer treatment, efforts to develop
inhibitors have been unsuccessful. Here, we used an unbiased approach to identify the
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
5
glycolytic enzyme, fructose-biphosphatase aldolase A (ALDOA) as a key target for inhibiting
both glycolysis and HIF1 activity. By using an inhibitor that targets ALDOA, we found that
inhibition of ALDOA does indeed break the feed-forward loop, blocking both glycolysis and HIF-
1 activity in cells, with the prospect of inhibiting tumor growth in vivo.
Materials and Methods:
Creation of Stable and Inducible Cell Lines
MIA PaCa-2 and PANC-1 pancreatic, MDA-MB-231 metastatic breast, HT-29 colon and 786-
O renal cell carcinoma cancer cell lines were obtained in 2012 from ATCC (Manassas, VA). The
identity of each line was authenticated on arrival, for each frozen stock and at two month
intervals during culture by the Molecular Cytogenetics Facility, University of Texas MD
Anderson Cancer Center. Each cell line was stably transfected a pGL3 plasmid (Promega)
containing a 5x repeat of the hypoxia transcriptional response element (HRE) flanking a
luciferase reporter and a G418 selection marker (HRE-luc). The reporter plasmid was a gift of
Dr. R. Gillies (Moffitt Cancer Center, Tampa, FL). Following selection, pools of stably
transfected cells were generated and stored frozen for later use.
For conditional Aldolase A (ALDOA) knockdown in an in vivo murine model, four sequences
predicted to target ALDOA gene expression were selected from the Thermo Scientific
Dharmacon shRNA library and each was inserted in a TRIPZ lentiviral vector (Open
Biosystems, Huntsville, Alabama). The HRE luciferase MDA-MB-231 line described above was
transduced with shALDOA-expressing lentivirus, and stable lines were selected in puromycin in
96-well plates with one cell per well to generate clonal populations. Sequence identification for
use in both in vitro and in vivo experiments was determined by relative ALDOA by Western blot.
After puromycin- and G418-resistant clones were selected, shALDOA expression in cells was
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
6
induced using 400 ng/ml doxycycline in both normoxia and hypoxia (1% O2) and for in vivo
tumors by feeding mice chow containing 625 mg/kg doxycycline (Harlan Laboratories,
Indianapolis, IN) to achieve ALDOA knockdown.
Cell Transfection
Transient siRNA reverse transfections were carried out for global siRNA screening using
XTremeGene (Roche, Basel, Switzerland) according to the manufacturer’s instructions with the
genome-wide SmartPool siRNA library from Dharmacon using the MIA PaCa-2 HRE luciferase
line. After identifying initial glycolysis genetic hits, follow-up work in each of the 3 additional cell
lines listed used Lipofectamine RNAiMax (Qiagen, Valencia, CA) and Dharmacon SMARTpool
siRNAs for HIF-1α, Aldolase A, AMPK, p300, PCAF, FIH, PLK-1 or the On-Target-Plus non-
targeting pool #4 (OTP4). Total siRNA concentration was kept at 40 nM for single or multiple
siRNA combinations. Knockdown efficiency was determined by Western blotting of cell lysates
96 hours post transfection.
Chemical Compounds
Synthesis of naphthalene-2,6-diyl bis(dihydrogen phosphate) is described in Supplementary
Material and Methods S1.
Western Blotting
Primary antibodies for Western blotting were: HIF-1α (BD Biosciences, San Diego,CA),
Aldolase A (Thermo Scientific, Waltham, Massachusetts), β-actin, p300, phospho-p300 (all from
Santa Cruz Biotechnology Inc.), and AMPK/phosphoAMPK (Cell Signaling Technologies,
Danvers, MA).
Cell Viability and HIF-1α Activity Assays
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
7
Viability of cell populations was quantified photometrically at 475 nm using the XTT Cell
Viability Assay (Biotium, Hayward, CA), according to the manufacturer’s instructions. HIF-1α
activity was measured using a Dual-Glo Luciferase Assay System (Promega, Fitchburg, WI)
according to the manufacturer’s protocol. Relative Luciferase activity (% control) were
calculated to correlate HIF-1 expression with cell viability data for each gene knockdown.
Determination of ATP Concentration
Cellular ATP was measured using an ATP Assay Kit (Abcam, Cambridge, United Kingdom)
according to the manufacturer’s protocol and quantified 96 hours post siRNA-transfection by
both colorimetric (OD 570 nm) and fluorometric (Ex/Em = 535/587 nm) methods,.
Measurement of Cellular Glycolysis
Glycolysis was measured as the rate of extracellular acidification (ECAR) using the Seahorse
Bioscience XF96e platform (Seahorse Bioscience, North Billerica, MA) and the XF Glycolysis
Stress Test Assay according to the manufacturer’s protocol. To measure glycolysis under
hypoxia, a modified hanging drop tissue culture method was used to evaluate 3-dimensional
spheroids of PANC-1 HRE cells transduced with shALDOA constructs. Three days after
seeding cells and 24 hours before measuring glycolysis spheroid shALDOA expression was
induced with 400 ng/ml doxycycline. A final volume of 175 μl of pre-conditioned assay medium
containing 18 spheroids was added to each well of a test plate and incubated at 37ºC in a CO2-
free incubator until the experiment was initiated. Spheroids exhibited a hypoxic core based on
analysis with a fluorescent hypoxia probe LOX-1 (SCIVAX USA, Inc, Woburn, MA) without the
need for hypoxic gassing conditions.
ALDOA kinetic assays.
ALDOA kinetic assays are described in Supplementary Materials and Methods S2.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
8
Crystallization and structure solution.
Protein crystallization and structure solution are described in Supplementary Materials and
Methods S3, and data collection and refinement statistics in Tables S1 and S2.
Xenografts
Approximately 107 MDA-MB-231 HRE cells, MDA-MB-231 cells harboring shALDOA clones
8.8 and 9.7, and MDA-MB-231 HRE empty vector cells, all in log cell growth, were suspended
each in 0.2 mL PBS and injected subcutaneously into the mammary fat pads of female NOD-
SCID mice. Groups contained five mice. When the tumors reached 250 mm3, chow containing
doxycycline was substituted for control feed (Harlan Laboratories) in test groups. Mice were
euthanized when they became clinically moribund, associated with the metastatic spread of the
MDA-MB-231 tumor to liver and lung (12). Animal studies were approved by SBPMRI’s Animal
Care and Use Committee.
Statistical Analysis
Data are shown as mean ± SD unless indicated otherwise. Student t test assuming two-tailed
distributions was used to calculate statistical significance between groups. Animal survival was
determined by the Kaplan–Meier analysis with P < 0.05 considered statistically significant.
Results:
Glycolytic enzymes regulate HIF-1α activity
To identify genes that regulate HIF activity, we conducted a genome-wide siRNA screen using
MIA PaCa-2 pancreatic cancer cells with a stably integrated 5 x HRE/promoter-luciferase (luc)
HIF reporter to identify genes that when knocked down inhibited HIF activity under hypoxic
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
9
conditions (1% O2). It should be noted that the reporter cannot distinguish between HIF-1 and
HIF-2 activity, although hypoxic MIA PaCa-2 cells express predominantly HIF-1, and HIF-2 has
been reported not to up-regulate glycolysis genes (5). Unexpectedly, the screen identified
several glycolysis-related genes whose knockdown inhibited HIF-1 activity (Table S3). The
expression of glycolysis genes has been reported to be increased by HIF-1 during hypoxia
(5,13). We confirmed this using RNAseq in MIA PaCa-2 cells, and found that 16 glycolysis
genes were up-regulated in hypoxia, 15 of which showed HIF-1 dependence (Table S4).
To test the possibility that a glycolysis–HIF-1 feed-forward loop existed we used a panel of
siRNAs to knockdown 30 glycolysis genes and their isoforms (including pyruvate
dehydrogenase, which is responsible for linking glycolysis to the citric acid cycle in the
mitochondria) modification in mitochondria), and then measured HRE-luciferase activity (a
surrogate of HIF-1 activity) and cell proliferation. Using the same MIA PaCa-2 pancreatic cancer
cells stably transfected with the HRE-luc reporter, we found compelling evidence that glycolytic
enzyme activity is indeed critical for the normal functionality of HIF-1 (Figure 1A). Similar results
were obtained using HRE-luc PANC-1 pancreatic, MDA-MB-231 metastatic breast, and HT-29
colon cancer cell lines (Figure S1, Table S5). The greatest decrease in HRE-luc activity in all
lines, normalized to cell number, was observed when ALDOA was knocked down. Similar
inhibitory effects were observed in the 786-O renal adenocarcinoma cell line that expresses
HIF-2 exclusively, suggesting that regulation of HIF activity by glycolytic enzymes is not limited
to HIF-1 (Figure 1B). Dual knockdown of PGK1 and PGK2 suggest they have additive activities
on HRE-luc inhibition, although this was not accompanied by an additive decrease in cell
proliferation (Figure 1C, and Figure S2). Knockdown of ALDOA, or PGK1 or PGK2 in
combination, inhibited glycolysis in all cell lines (Figure 2A and 2B), which was accompanied by
lower cellular ATP levels (Figure 2C and Figure S3).
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
10
HIF-1α activity is mediated by AMPK activation and p300 inactivation
We first established that, in all tumor cells tested, decreased HIF-1 activity caused by ALDOA
or PGK1 or 2 knockdown occurred without changes in HIF-1 protein levels, and was similar in
normoxia or hypoxia, although a greater overall effect was seen in hypoxia, where HIF-1 levels
are elevated (Figure 3A and Figure S4). In hypoxia the hydroxylation of key HIF-1 proline
residues by oxygen-sensitive dioxygenases is inhibited, thus preventing HIF-1 binding to the
von Hippel-Lindau protein (pVHL) which normaly leads to ubiquitination of HIF-1 and its
proteasomal degradation (4). In order to evaluate possible mechanisms underlying this change
in HIF-1 activity, we co-transfected glycolysis-related siRNAs together with siRNAs targeting
genes that are known to regulate HIF-1 activity. These included AMP-activated protein kinase
(AMPK) (14,15); E1A-associated cellular p300 transcriptional co-activator (p300) (16,17); PCAF
(p300/CBP-associated factor) (18,19); and FIH (Factor Inhibiting HIF-1), which interacts with
HIF-1α and VHL to repress HIF-1 transcriptional activity (20,21). We found that AMPK
knockdown rescued HIF-1 inhibition caused by ALDOA or PGK2 knockdown, but observed little
effect when either p300 or PCAF was knocked down (Figure 3B). FIH knockdown also negated
the effects of ALDOA and PGK1 knockdown on HIF-1 activity, likely due to loss of negative
regulation of HIF-1. Western blotting showed that ALDOA knockdown significantly increased
phosphorylation of AMPK on Thr172, a marker of AMPK activation in response to cellular stress
such as ATP depletion (22,23), in both normoxia and hypoxia (Figure 4A). Further evidence
that AMPK mediates the effects of ALDOA (or PGK1 or PGK2) knockdown on HIF-1 activity was
the rescue of HIF-1 activity by the AMPK inhibitor, dorsomorphin (Figure 4B). AMPK activation
can lead to phosphorylation of p300 at Ser89 that attenuates the interaction of p300 with a
variety of transcription factors in vitro and in vivo, including Hif-1 (24). We observed that
knockdown of ALDOA or PGK2, although not PGK1, resulted in p300 Ser89 phosphorylation in
HT-29 and MiaPaCa-2 cells (Figure 4A). Taken together the results suggest that inhibition of
HIF-1 activity and a concomitant decrease in glycolysis are mediated by AMPK activation
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
11
(possibly in response to low cellular ATP levels), which in turn promotes p300 phosphorylation,
preventing p300 from co-activating HIF-1 transcriptional activity (Figure 4C).
Variable expression of aldolase isoforms suggest compensatory effects on glycolysis
To further understand the basis for variability in the effects of ALDOA knockdown on
proliferation among the 4 HRE luciferase lines, we next tested for possible compensatory
mechanisms when ALDOA is eliminated, by immunoblotting to analyze expression of aldolase B
and C isoforms in normoxia and hypoxia (Figure S5A and 5B). Interestingly, we found that HT-
29 cells (a human colorectal adenocarcinoma cell line with epithelial morphology), which were
the only cells found to be recalcitrant to ALDOA knockdown (Figure S1), were also the only
cells to exhibit significantly higher expression of ALDOC, suggesting a compensatory effect in
the absence of ALDOA at this step of glycolysis.
ALDOA knockdown extends median survival in an in vivo model of metastatic breast
cancer
To validate ALDOA as a potential therapeutic target, and because we have shown that
ALDOA knockdown is acutely toxic to cancer cells in vitro, we expressed a doxycycline-
inducible ALDOA shRNA in MDA-MB-231 breast cancer cells, and then used those cells to
establish an orthotopic model of metastatic breast cancer in female NOD-SCID mice (12). Two
clonal lines that showed complete (clone 8.8) or partial (clone 9.7) ALDOA knockdown,
glycolysis inhibition, and hypoxia response element (HRE) activity inhibition following
doxycycline treatment, were used (Figures 5A and S6). Mice fed doxycycline starting either
one week before implantation of cells or when the primary tumor reached approximately 250
mm3, showed an increased median lifespan. Mice fed doxycycline a week before implantation
showed increases from 37 days in parental, and 41 days in empty vector transfected cells, to 50
and 56 days in two clonal cells lines (p<0.001in both cases); mice treated after tumors were
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
12
established showed increases from 41 days (untreated) to 48 days (p < 0.001 compared to
control) (Figure 5B). Transduction with clone 8.8, which showed complete ALDOA knockdown
associated with glycolysis and HIF-1 inhibition, promoted longer median survival time than did
the partial knockdown clone 9.7. Postmortem analysis of mice indicated marked tumor
metastasis to the lung and liver. While knockdown of ALDOA extended lifespan, doxycycline
given either as pretreatment or when tumors were established had a similar effect. This
suggests that ALDOA knockdown does not affect tumor implantation; rather, that ALDOA
knockdown inhibits metastasis from the primary tumor to the lungs and liver, which occurs late
in the tumor development process, and is the most likely cause of death.
Identification and characterization of a small-molecule allosteric inhibitor of ALDOA,
TDZD-8.
In order to confirm that ALDOA is a target with potential for cancer therapy, we sought a
small-molecule probe inhibitor of human ALDOA. We carried out a chemical library screen for
inhibitors of ALDOA using a novel biochemical assay, which allowed us to identify small
molecule inhibitors that would have interfered with the classic ALDOA assay. We identified the
compound TDZD-8 as an inhibitor with time-dependent inhibition (Figure 6A and 6B). In order
to determine the mechanism of inhibition, we first determined the crystal structure of native
human ALDOA at a resolution of 2.4 Å (see Supplementary Materials and Methods S3 and
Table S1). In the native crystals, the C-terminal tail (“C-tail”; residues 345-363) lies across the
active site, with the C-terminal Tyr363 inserted into the active site (Figure S7B). There is
abundant evidence that the C-tail is highly mobile and that its conformation and dynamics are
critical for catalysis (25). Indeed, we observed a 10-fold reduction in kCAT in an ALDOA construct
truncated before the C-tail (not shown). We next soaked a preformed native crystal in a solution
containing 1 mM TDZD-8, and determined its structure at 2.65 Å resolution (Table S1). We
found unambiguous evidence for TDZD-8 binding covalently to a single site, Cys239, which lies
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
13
on an exposed loop distal to the catalytic site (Figure S7B). Computational docking studies
support our observation that the thiadiazole ring of TDZD-8 can bind to the sulfhydryl group of
Cys239 without the need for significant conformational changes in the protein.
In the crystalline form, however, lattice contacts reduce the mobility of the protein, which may
obscure ligand-binding sites that are available in solution. This may be especially true for
ALDOA, given the known mobility of the C-tail. In the course of our studies, we had co-
crystallized ALDOA bound to the active site substrate-mimetic, ND1, and solved its structure at
2.2 Å resolution (Table S2). The binding of ND1 sterically occludes the C-terminal tyrosine,
causing the entire C-tail to be ejected from its groove and become disordered, a phenomenon
that is typical of this class of inhibitor (Figure S7A). We therefore proceeded to soak preformed
ALDOA-ND1 crystals with TDZD-8, and solved its crystal structure at 2.4 Å resolution (Table
S2). In this case, we observed strong ligand-binding at C239 (as before) as well as at a second
site, C289, which is also distal to the active site but proximal to the C-tail (Figure 6C, 6D, 6E
and Figure S7C and S7D). C289 is partly buried in the native structure, and the binding of
TDZD-8 to C289 induces local conformational changes in loops that would contact the C-tail in
native crystals.
Our data suggest that the reactivity of Cys239 toward TDZD-8 is not significantly influenced by
the absence or presence of the C-tail; by contrast, the reactivity of Cys289 is strongly
influenced, since binding is only observed when the C-tail has been removed. It should
therefore follow that ligand-binding to C289 in solution should perturb the conformation of the C-
tail, and thereby modulate catalytic activity. Indeed, in solution, we observed a near-doubling of
the half-life of inactivation by TDZD-8 (from 120 to 214 min) when Cys289 was mutated to Ala
(Figure 6B). Thus, TDZD-8 appears to act as an allosteric inhibitor, via modulation of the
structure and/or dynamics of the C-tail, mediated principally through modification of Cys289, a
residue that lies within a well-defined, three-dimensional pocket.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
14
TZDZ-8 inhibits glycolysis, HIF-1 activity, and cancer cell proliferation,
TDZD-8 treatment of MDA-2B-231 breast cancer cells inhibited glycolysis and HIF-1 activity as
well as cancer cell proliferation in a dose dependent manner at low µM concentrations (Figure
6F and S8), and also decreased cellular ATP while increasing phospho-AMPK (Figure 6G and
S9). Treatment of MDA-MB-231 cells with TDZD-8 under hypoxic conditions (1% O2) for 6 h
resulted in an approximately 2-fold reduction of dihydroxy acetone phosphate (DHAP), the
product of the cleavage of fructose-1,6-bisphosphate substrate by ALDOA, and pyruvate, while
levels of the lower-abundance intermediates 3-phosphoglycerate and phosphoenolpyruvate
were not affected (Figure S10).
We therefore used TDZD-8 as a pharmacological probe to see if we could show an antitumor
effect. When administered intraperitoneally daily for 20 days at a dose of 12 mg/kg per day to
mice with MDA-MB-231 orthotopic breast cancer tumors, TDZD-8 caused significant slowing of
tumor growth, by about 60% by day 32 (Figure 6H). Pharmacodynamic studies after a single
dose of TDZD-8 showed a ~50% decrease in tumor lactate levels within 4 h, a 40% decrease in
DHAP, and a slower decrease with daily dosing for 5 days in downstream phosphoglycerate
(Figure 6I). Thus, TDZD-8 itself at low levels, or an active metabolite appears to reach the
tumor in sufficient amounts to inhibit tumor glycolysis, associated with antitumor activity.
Discussion:
We have shown that HIF-1–induced upregulation of glycolytic genes during anaerobic
(hypoxic) glycolysis in cancer cells is itself stimulated by the product of glycolysis, ATP, thereby
completing a glycolysis HIF-1 feed-forward loop that stimulates tumor growth, When anaerobic
glycolysis is inhibited (e.g. via inhibition of ALDOA), ATP levels are reduced, and the feed-
forward loop is broken via activation of the AMP-activated protein kinase-1 (AMPK1), which is
sensitive to the ratio of AMP/ATP in the cell (22,23). Activated AMPK1 inhibits the
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
15
transcriptional activity of HIF-1 by phosphorylating its transcriptional co-activator, the E1A-
associated, cellular p300 (p300), at Ser 89, which blocks formation of the p300–HIF-1 co-
activation complex.
HIF-1 is critical for cancer cell survival and tumor growth in the stressed hypoxic tumor
environment (4,5). However, HIF-1 inhibitors used as single agents for the treatment of human
cancer have not advanced in the clinic. Thus, we took an unbiased approach, utilizing a high-
throughput siRNA synthetic lethal screen, initially to identify genes that might be targets to
inhibit tumor growth, which could also be used in combination with HIF-1 inhibition.
Unexpectedly, several hits in the screen were glycolytic enzymes. While it was known that HIF-
1 increases glycolytic activity in tumors by inducing expression of glycolytic enzymes (5,7,26), it
had not previously been reported that glycolysis increases HIF-1 activity. We found that the
knockdown of 16 of 30 glycolytic enzymes and isoforms were associated with detectable
inhibition of HIF-1 transcriptional activity. Among them, ALDOA and PGK1/2 knockdown
resulted in robust inhibition of HIF-1 activity in all lines tested. Importantly, inhibitors that target
these proteins should have the ability to block two processes critical for tumor growth:
glycolysis, the source of energy and metabolic support (this could be tumor or stroma
glycolysis); and HIF-1, which promotes cancer cell survival and tumor growth through increased
angiogenesis.
We chose ALDOA as a proof-of-principle target for inhibitor development, in part because its
knockdown in cancer cells was associated with greater inhibition of cancer cell proliferation than
PGK1/2 knockdown. This could be because ALDOA has other roles, including “moonlighting’ as
a nuclear protein (27), or that ALDOA is more important as a driver of glycolysis, whereas
PGK1/2 is a driver of tumor angiogenesis, which would not be apparent from our cell-based
studies. ALDOA expression has been reported to be significantly elevated relative to other
glycolytic enzymes in a number of human tumor types (28,29). Another consideration is that
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
16
ALDOA is the major ALDO isoform driving glycolysis in cancer cells, sometimes aided by
ALDOC, whereas PGK1 and PGK2 appear to have redundant activities.
In order to demonstrate the potential of small molecule ALDOA inhibitors for cancer therapy,
we turned to a probe inhibitor of human ALDOA that we discovered through a chemical library
screen, utilizing a novel biochemical assay. The compound, TDZD-8, a 1,2,4-thiadiazole,
showed time-dependent inhibition of ALDOA that suggested a covalent interaction with the
protein. Our crystallographic studies showed that TDZD-8 bound to 2 Cys residues (Cys289 and
Cys239) on the surface of each ALDOA monomer. Although both residues lie distal to the active
site, one of them Cys289, lies in a well-defined pocket, and we found its reactivity toward TDZD-
8 to be strongly influenced by the presence or absence of the C-tail. Thus, we propose that
TDZD-8 binding to Cys289 in solution should allosterically perturb the conformation or flexibility
of the C-tail, thereby inhibiting catalytic activity. The electron density derived by crystallography
is consistent with disulfide bond formation and ring-opening of TDZD-8. Studies using Cys-
directed reagents to inhibit ALDO Cys residues have previously suggested that they are
involved in enzyme activity (30). Of ALDO’s 8 Cys residues, only 4 are accessible in the
absence of denaturing agents, and these include Cys-289 and Cys239. TDZ8-8 has allowed
us, for the first time, to demonstrate crystallographically an allosteric interaction between
ALDOA Cys289 and the catalytic site. Most importantly, although TDZD-8 is a simple chemical
probe without optimized drug like properties, it has nonetheless allowed us to demonstrate an
association between inhibition of glycolysis, HIF-1 activity and the proliferation of cancer cell
lines at low µM concentrations. The compound also exhibited in vivo antitumor against MDA-
MB-231 xenografts in mice and was associated with decreased levels of the glycolytic products
of ALDOA activity in tumors.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
17
In summary, we have shown that a feed-forward loop in tumors, simultaneously promoting
increased HIF-1 activity and increased glycolysis, offers a target, ALDOA, with which to block
tumor energy/metabolite production pathways and the HIF-1α survival response. Our HIF-1
activity-oriented RNAi screen and subsequent mechanism-based analysis expand our
understanding of known and novel regulators of the HIF-1 transcription factor, and point to a
previously uncharacterized regulation of HIF-1 activity by increased glycolytic enzyme activity.
Acknowledgements
Supported by NIH Grants CA163541, CA188260 (GP) and CCSG grant P30CA030199. The
help of SBPMDI Cancer Center Animal and Genomic Services is gratefully acknowledged.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
18
ABBREVIATIONS
ALDOA fructose-biphosphatase aldolase A
AMPK1 5-AMP-Activated protein kinase
DHAP dihydroxy acetone phosphate
DMEM Dulbecco’s modified Eagle’s medium
ECAR extracellular acidification rate
FIH factor inhibiting HIF-1
HIF-1 hypoxia inducible factor-1
HIF-2 hypoxia inducible factor-2
HRE hypoxia transcriptional response element
luc luciferase
NDA naphthalene-2,6-diylbis(dihydrogen phosphate)
Ni-NTA nickel nitrilotriacetic acid
p300 E1A binding protein
PDGF platelet derived growth factor
PCAF p300/CBP-associated factor
PGK phosphoglycerate kinase
PLK-1 polo-like kinase
pVHL von Hippel-Lindau protein
TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione
VEGF vascular endothelial growth factor
XTT 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
19
References
1. Garber, K. Energy deregulation: licensing tumors to grow. Science. 2006; 312: 1158-9.
2. DeBerardinis, R., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S, et al. Beyond
aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the
requirement for protein and nucleotide synthesis. Proc Natl Acad Sci. 2007; 104: 19345-50.
3 Warburg O, K. Posener, E. Negelein R: Ueber den Stoffwechsel der Tumoren; Biochemische
Zeitschrift, 1924. 152, 319-344. Reprinted in english in “On metabolism of tumors” by O.
Warburg, 1930. Publisher: Constable, London,
4. Semenza, G.L. HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic
mutations. J Clin Invest. 2013; 123, 3664-71.
5. Hu, C.J., Wang, L.Y., Chodosh, L.A., Keith, B., Simon, M.C. Differential roles of hypoxia-
inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol.
2003; 23: 9361-74.
6. Manalo, D.J., Rowan, A., Lavoie, T., Natarajan, L., Kelly, B.D., Ye, et al. Transcriptional
regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 2005; 105: 659-69.
7. Marin-Hernandez, A., Gallardo-Perez, J.C., Ralph, S.J., Rodriguez-Enriquez, S. Moreno-
Sanchez, R. HIF-1alpha modulates energy metabolism in cancer cells by inducing over-
expression of specific glycolytic isoforms. Mini Rev Med Chem. 200p; 9: 1084-101.
8. Granchi, C. F. Minutolo. Anticancer agents that counteract tumor glycolysis.
ChemMedChem. 2012; 7: 1318-50.
9. Zhao, Y., Butler, E.B., Tan, M. Targeting cellular metabolism to improve cancer
therapeutics. Cell Death Dis. 2013; 4: e532.
10. Hewitson, K.S. Schofield, C.J. The HIF pathway as a therapeutic target. Drug Discov
Today. 2004; 9: 704-11.
11. Scatena, R., Bottoni, P., Pontoglio, A., Mastrototaro, L Giardina, B. Glycolytic enzyme
inhibitors in cancer treatment. Expert Opin Investig Drugs. 2008; 17: 1533-45.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
20
12. Iorns, E., Drews-Elger, K., Ward, T.M., Dean, S., Clarke, J., Berry, D., et al. A new mouse
model for the study of human breast cancer metastasis. PLoS One 72012, e47995.
13. Lum, J.J., Bui, T., Gruber, M., Gordan, J.D., DeBerardinis, R.J., Covello, K.L, et al. The
transcription factor HIF-1alpha plays a critical role in the growth factor-dependent regulation of
both aerobic and anaerobic glycolysis. Genes Dev. 2007; 21: 1037-49.
14. Lee, M., Hwang, J.T., Lee, H.J., Jung, S.N., Kang, I., Chi, S.G., et al. AMP-activated
protein kinase activity is critical for hypoxia-inducible factor-1 transcriptional activity and its
target gene expression under hypoxic conditions in DU145 cells. J Biol Chem. 2003; 278:
39653-61.
15. Laderoute, K.R., Amin, K., Calaoagan, J.M., Knapp, M., Le, T., Orduna, J., et al. 5'-AMP-
activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions
found in solid-tumor microenvironments. Mol Cell Biol. 2006; 26: 5336-47.
16. Arany, Z., Huang, L.E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, M.A., et al. An
essential role for p300/CBP in the cellular response to hypoxia. Proc Natl Acad Sci U S A. 1996;
93: 12969-73.
17. Freedman, S.J., Sun, Z.Y., Poy, F., Kung, A.L., Livingston, D.M., Wagner, G., et al.
Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1 alpha. Proc Natl Acad
Sci U S A. 2002; 99: 5367-72.
18. Obacz, J., Pastorekova, S., Vojtesek, B., Hrstka, R. Cross-talk between HIF and p53 as
mediators of molecular responses to physiological and genotoxic stresses. Mol Cancer. 2013;
12: 93.
19. Xenaki, G., Ontikatze, T., Rajendran, R., Stratford, I.J., Dive, C., Krstic-Demonacos, M. et
al. PCAF is an HIF-1alpha cofactor that regulates p53 transcriptional activity in hypoxia.
Oncogene. 2008; 27: 5785-96.
20. Lando, D., Peet, D.J., Gorman, J.J., Whelan, D.A., Whitelaw, M.L. Bruick, R.K. FIH-1 is an
asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible
factor. Genes Dev. 2002; 16: 1466-71.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
21
21. Mahon, P.C., Hirota, K., Semenza, G.L. FIH-1: a novel protein that interacts with HIF-
1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001; 15:
2675-86.
22. Shaw, R.J., Kosmatka, M., Bardeesy, N., Hurley, R.L., Witters, L.A., DePinho, R.A.,et al.
The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates
apoptosis in response to energy stress. Proc Natl Acad Sci U S A.2004; 101: 3329-35.
23. Woods, A., Johnstone, S.R., Dickerson, K., Leiper, F.C., Fryer, L.G., Neumann, D., et al.
LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol. 2003; 13:
2004-8.
24. Yang, W., Hong, Y.H., Shen, X.Q., Frankowski, C., Camp, H.S., Leff, T. Regulation of
transcription by AMP-activated protein kinase: phosphorylation of p300 blocks its interaction
with nuclear receptors. J Biol Chem. 2001; 276: 38341-4.
25. St-Jean, M., Sygusch J. Stereospecific proton transfer by a mobile catalyst in mammalian
fructose-1,6-bisphosphate aldolase. J Biol Chem, 2007; 282: 31028-37.
26. Koh, M.Y. G. Powis. Passing the baton: the HIF switch. Trends Biochem Sci. 2012; 37:
364-72.
27. Ritterson Lew, C. Tolan, D.R. Targeting of several glycolytic enzymes using RNA
interference reveals aldolase affects cancer cell proliferation through a non-glycolytic
mechanism. J Biol Chem. 2012; 287: 42554-63.
28. Du, S., Guan, Z., Hao, L., Song, Y., Wang, L., Gong, L., et al. Fructose-bisphosphate
aldolase a is a potential metastasis-associated marker of lung squamous cell carcinoma and
promotes lung cell tumorigenesis and migration. PLoS One. 2014; 9: e85804.
29. Oparina, N.Y., Snezhkina, A.V., Sadritdinova, A.F., Veselovskii, V.A., Dmitriev, A.A.,
Senchenko, V.N., et al. Differential expression of genes that encode glycolysis enzymes in
kidney and lung cancer in humans. Genetika. 2013; 49: 814-23.
30. Steinman, H.M., Richards, F.M. Participation of cysteinyl residues in the structure and
function of muscle aldolase. Characterization of mixed disulfide derivatives. Biochemistry.
1970; 9: 4360-72.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
22
40. Szutowicz, A., Kwiatkowski, J., Angielski, S. Lipogenetic and glycolytic enzyme activities in
carcinoma and nonmalignant diseases of the human breast. Br J Cancer. 1979; 39: 681-7.
41. Hennipman, A., Smits, J., van Oirschot, B., van Houwelingen, J.C., Rijksen, G., Neyt, J.P.,
et al. Glycolytic enzymes in breast cancer, benign breast disease and normal breast tissue.
Tumour Biol. 1987; 8: 251-63.
FIGURE LEGENDS:
Figure 1. Changes in glycolytic enzyme expression alters HIF-1 activity. 30 genes
encoding glycolytic enzyme isoforms were selected for validation from a genome-wide siRNA
screen for inhibitors of cellular HIF-1 activity. A, HIF-1 activity was measured using MIA PaCa-2
pancreatic cancer cells harboring a constitutively expressed HRE-luciferase reporter 72 hr after
siRNA reverse transfection and after 24 h hypoxia (1% oxygen). siRNAs were from a different
manufacturer that in the original screen. Values were normalized to MIA PaCa-2 cell viability
under the same conditions as determined using a XTT viability assay and results expressed as
a % of non-treated cells. Closed bars are cell viability and open bars are HRE-luciferase (HIF-
1) activity. Bars represent S.D. of 3 determinations. Scrambled non-targeting (siSCR) and polo
like kinase-1 (PLK1) siRNAs served as a siRNA control and transfection control, respectively. B
Effect of targeted siRNAs for HIF-2α, ALDOA, PGK 1 and PGK2, and a non targeting siRNA
(siSCR) on HRE-luciferase reporter activity in 786-O renal cell carcinoma cells which express
HIF-2 but not HIF-1. The HRE-luciferase reporter responds to both HIF-1 and HIF-2. siRNA
reverse transfection was for 72 hr and hypoxia for 24 hr. Bars are S.D. of 3 determinations.
C, Effect of siRNA knockdown of ALDOA, and PGK1 and PGK2 alone and in combination, 72 hr
after siRNA reverse transfection and 24 hr hypoxia on HIF-1 activity in Panc-1 pancreatic
cancer and MDA-MB-231 triple-negative breast cancer cells harboring HRE-luciferase reporters.
Bars are S.D. of 3 studies. * p = 0.05 compared to siSCR transfected cells.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
23
Figure 2. Decreased glycolysis and ATP formation following knockdown of ALDO or
PGK1 and 2. Cellular aerobic glycolysis and glycolytic reserve (defined as excess glycolytic
capacity following consecutive addition of 10 mM glucose, 1.5 µM oligomycin to inhbit
mitochondrial oxidative phosphorylation, and 100 mM 2-deoxyglucose to inhibit glycolysis) was
measured using Seahorse® technology as illustrated in the data key at upper left, in PANC-1
and MIA PaCa-2 pancreatic, MDA-MB-231 metastatic breast and HT-29 colon cancer lines, 72
h after reverse-transfection with siRNA targeting ALDO isoforms. A, Cells transfected with
siRNA targeting ALDO A, B and C, alone and in combination. siSCR non-targeting siRNA
served as a control. ECAR is the extracellular acidification rate measured in mpH/min. B,
Transfection with siRNA targeting PGK1 and PGK2 isoforms alone and in combination. C,
Cellular ATP levels 72 hr after transfection with siRNA targeting HIF-1α, ALDOA, and PGK1 or
2, in PANC-1 and MDA-MB-231 cells. Bars represent S.D. from 3 separate determinations.
Figure 3. Knockdown of ALDOA or PGK1 or 2 inhibits HIF-1 activity without decreasing
HIF-1α protein levels. A, HIF-1 activity measured with a HRE-luciferase reporter in air
(Normoxia) or 1% O2 (Hypoxia) using Panc-1 pancreatic and MDA-MB-231 breast cancer cells
stably transfected with HRE-luciferase reporter 72 h after transfection with siRNA targeting
ALDOA or PGK1 or PGK2, and in hypoxia for the last 24 h. siSCR and HIF-1α siRNAs served
as controls. Bars are S.D. of 3 determinations. HIF-1α protein was measured by Western
blotting. There was no HIF-1 detectable MDA-MB-231 cells in air. B, Dual reverse transfection
of siRNAs targeting ALDOA or PGK, with or without siAMPK (protein kinase, AMP-activated,
alpha 2 catalytic subunit), siP300 (E1A-associated cellular p300 transcriptional co-activator
protein), siPCAF (p300/CBP-associated factor), and siFIH (Factor Inhibiting HIF1). Bars are
S.D. of 3 determinations. The results indicate that AMPK inhibition mediates the effects of loss
of ALDOA, PGK1 or PGK2 on HIF-1 activity, without effect on HIF-1α protein levels.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
24
Figure 4. p300 phosphorylation mediates AMPK effects on HIF-1 activity. A, Western blot
showing increased phosphorylation of AMPK (at Thr172) and p300 (at Ser89) following
knockdown of ALDOA, or PGK1 or PGK2 ,72 hr after siRNA transfection and 24 hr in hypoxia in
PANC-1 and MIA PaCa-2. B, HIF-1 activity measured with the HRE-luciferase reporter in MDA-
MB-231 cells relative to untreated cells following knockdown of ALDOA, PGK1, or PGK2 72 hr
after transfection and after 24 hr in hypoxia (filled boxes). Effects are rescued following
treatment with the AMPK inhibitor dorsomorphin at 5 µM, or with siRNA targeting AMPKα * p =
< 0.05 compared to siSCR control. C, Suggested mechanism of glycolytic control of cellular
HIF-1α activity through AMPK/p300 signaling. In normoxic conditions cancer cell aerobic
glycolysis maintains the cellular ATP/ADP ratio at levels sufficient to prevent phosphorylation of
AMPK, thus allowing the unrestricted formation of a p300/HIF1 complex that activates HIF-1.
When cellular ATP formation is decreased under hypoxic conditions, a decreased ratio of
ATP/ADP leads to phosphorylation of AMPK, which in turn phosphorylates p300 preventing its
association HIF-1, thus leading to decreased HIF-1 activity. Since this leads to decreased
synthesis of glycolytic enzymes, a feed-forward inhibition loop of decreased
glycolysis/decreased HIF-1 activity is established despite elevated HIF-1 protein levels due to
HIF-1 stabilization under hypoxic conditions.
Figure 5. Inducible ALDOA knockdown in MDA-MB-231 breast cancer tumors extends
survival of xenografted mice. A, Two lentiviral shALDOA doxycycline-inducible clones of
MDA-MB-231 metastatic breast cancer cells were established (clones 8.8 and 9.7). Both
showed doxycycline-induced ALDOA knockdown together with inhibition of hypoxic glycolysis
measured using Seahorse® technology, and decreased HIF-1α activity under hypoxic
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
25
conditions measured by HRE-luciferase reporter. B, Groups of 5 female immunodeficient SCID
mice were injected orthotopically in the breast fat pad with 106 MDA-MB-231 parental or vector-
only cells, or with clones 8.8 or 9.7. Animals received dietary doxycycline (625 mg/kg) starting 7
days before injection of cells (pre dox) or when tumors reached approximately 250 mm3 (dox).
When animals became clinically moribund they were euthanized. Median survival of mice with
parental MDA-MB-231 tumors was 37 days and with empty vector cells 41 days. Following
doxycycline treatment mice injected with clone 8.8 or clone 9.7 cells 7 days before cell injection
had a median survival of 56 days and 50 days, respectively (p<0.001 relative to combined
controls groups in both cases). Animals receiving doxycycline treatment when the tumors
reached ~ 250 mm3 had a median survival of 41 and 48 days (p<0.001 compared to combined
control groups in both cases). At the time of euthanization the lungs and liver of the mice
showed extensive metastatic nodules.
Figure 6 Antitumor activity of an ALDOA inhibitor. A, The small molecule TDZD-8 was
discovered as an inhibitor of recombinant human ALDOA activity through a chemical library high
throughput screen. Inhibition of ALDOA was time dependent suggesting that TDZD-8 could be
interacting directly with the protein. B. Time dependent inhibition of expressed ALDOA by
TDZD-8 at different concentrations with a 10 min or a 30 min preincubation. C, Crystallographic
analysis of TDZD-8 binding to ALDOA shown as a ribbon diagram of one monomer of the
tetrameric ALDOA co-crystallized with the active-site inhibitor, ND1, and soaked in 1 mM TDZD-
8. TDZD-8 binds specifically to 2 cysteine residues (239 and 289), as shown. The C-tail is not
visible in this structure, but its conformation derived from the native crystal structure is shown
schematically, colored spectrally with the C-terminus in red. Data collection and refinement
statistics are presented in Tables S3 and S4. D. Surface representation of the pocket occupied
by TDZD-8 bound to Cys289. E. Fit of the Cys289–TDZD-8 covalent adduct into the electron
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Targeting a glycolysis HIF-1 feed-forward mechanism
26
density map (2Fo-Fc), shown as blue chicken-wire. F, Panel showing TDZD-8 inhibition of MDA-
2B-231 breast cancer cell proliferation, medium acidification (lactate formation) measured by
Seahorse® technology, a decreased the ratio of lactate to glucose in the medium, and block of
cellular HIF-1 activity measured with a HRE-luciferase reporter, all at low micromolar IC50
concentrations. G, MDA-MB-231 cells were treated with TDZD-8 or 2-deoxyglucose (2-DG) for 4
hr in normoxia. Left panel: ATP in cell lysates. Values are mean ± S.E.. * p <0.05 compared to
control. Right panel: Western blot showing increased phospho-AMPKα. H, TDZD-8 was
administered at daily doses of 12 mg/kg by intraperitoneal injection daily for 20 days to
immunodeficient SCID mice with MDA-2B -231 tumor xenografts shows significant inhibition of
tumor growth. There were 10 mice per group and bars are S.E. of the mean. * p = < 0.05, and
** p = <0.01 compared to control mice. I , MDA-2B-231 xenografts (~ 250 mm3) were collected
at 1 and 4 hr after a single intraperitoneal dose of TDZD-8 of 12 mg/kg, or 4 hr after the last of 5
daily doses (Q5D), and tumor levels of lactic acid, dihydroxyacetone phosphate (DHAP) and 3-
phospoglycerate (3PG) measured. Values are the mean of 4 mice each and bars are S.E. * p =
< 0.5 compared to non treated control tumors.
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
0
20
40
60
80
100
120
Cells Only siSCR siHIF2α siALDOA siPGK1 siPGK2
0
20
40
60
80
100
120
siS
CR
siH
IF1α
siA
LD
OA
siP
GK
1
siP
GK
2
siP
GK
1/2
siS
CR
siH
IF1α
siA
LD
OA
siP
GK
1
siP
GK
2
siP
GK
1/2
PANC1 HRE-luc MDAMB231 HRE-luc
A
B
C
FIG 1
PANC-1 MDA-MB-231
*
*
*
*
*
*
* *
*
0
20
40
60
80
100
120
Cells
Only
siS
CR
siH
IF1a
siP
LK
1
siH
K1
siH
K2
siH
K3
siG
P1
siP
FK
FB
1
siP
FK
FB
2
siP
FK
FB
3
siP
FK
FB
4
siP
FK
L
siP
FK
M
siP
FK
P
siA
LD
OA
siA
LD
OB
siA
LD
OC
siT
PI1
siG
AP
DH
siP
GK
1
siP
GK
2
siP
GA
M1
siP
GA
M2
siB
PG
M
siE
NO
1
siE
NO
2
siE
NO
3
siP
KLR
siP
KM
2
siP
DK
1
siP
DK
2
siP
DK
3
siP
DK
4
rela
tive s
igna
l (%
of contr
ol)
MIA PaCa-2 cell proliferation HRE-luciferase
rela
tive s
igna
l (%
of co
ntr
ol)
786-O cell proliferation HRE-luciferase
rela
tive s
igna
l (%
of co
ntr
ol)
cell proliferation HRE-luciferase
*
*
*
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
FIG 2
A
B
Glycolytic Reserve
Glucose
Injection
Oligomycin
Injection
2-DG
Injection
Glycolysis
Glycolytic
Capacity
Non-glycolytic Acidification
PANC-1 MDA-MB-231
C
0
20
40
60
80
100
120
140
Cells
Only
siS
CR
siH
IF1a
siA
LD
OA
siP
GK
1
siP
GK
2
Re
lati
ve
[A
TP
]
0
20
40
60
80
100
120
140
Cells
Only
siS
CR
siH
IF
siA
LD
OA
siP
GK
1
siP
GK
2
Re
lati
ve
[A
TP
]
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
0
20
40
60
80
100
120
140
Cel
ls O
nly
siO
TP3
siH
IF1
a
siP
GK
1
siP
GK
2
siA
LDO
A
Cel
ls O
nly
siO
TP3
siH
IF1
a
siP
GK
1
siP
GK
2
siA
LDO
A
Normoxia Hypoxia
Re
lati
ve
Lu
cif
era
se
Un
its
(R
LU
)
0
20
40
60
80
100
120
140
Cells
Only
siO
TP
3
siH
IF1
a
siP
GK
1
siP
GK
2
siA
LD
OA
Cells
Only
siO
TP
3
siH
IF1
a
siP
GK
1
siP
GK
2
siA
LD
OA
Normoxia Hypoxia
Re
lati
ve
Lu
cif
era
se
Un
its
(R
LU
)
Cells
Only
siS
CR
siH
IF1α
siP
GK
1
siP
GK
2
siA
LD
OA
Cells
Only
siS
CR
siH
IF1α
siP
GK
1
siP
GK
2
siA
LD
OA
Cells
Only
siS
CR
siH
IF1α
siP
GK
1
siP
GK
2
siA
LD
OA
Cells
Only
siS
CR
siH
IF1α
siP
GK
1
siP
GK
2
siA
LD
OA
β-Actin
HIF-1α
PANC-1 MDA-MB-231
HIF-1α
β-Actin
FIG 3
A
B
Normoxia Hypoxia Normoxia Hypoxia
0
25
50
75
100
125
150
175
200
225
250
Cells
Only
siS
CR
siH
IF1a
siA
MP
K
siE
P300
siP
CA
F
siF
IH
siA
LD
OA
siA
LD
OA
+ s
iSC
R
siA
LD
OA
+ s
iAM
PK
siA
LD
OA
+ s
iEP
300
siA
LD
OA
+ s
iPC
AF
siA
LD
OA
+ s
iFIH
siP
GK
1
siP
GK
1 +
siS
CR
siP
GK
1 +
siA
MP
K
siP
GK
1 +
siE
P300
siP
GK
1 +
siP
CA
F
siP
GK
1 +
siF
IH
siP
GK
2
siP
GK
2 +
siS
CR
siP
GK
2 +
siA
MP
K
siP
GK
2 +
siE
P300
siP
GK
2 +
siP
CA
F
siP
GK
2 +
siF
IH
Re
lati
ve L
uci
fers
e
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
A
FIG 4
0
20
40
60
80
100
120
140
Ce
lls O
nly
siS
CR
siP
LK
1
siH
IF1α
siA
LD
OA
siP
GK
1
siP
GK
2
siA
MP
K
siA
LD
OA
+siA
MP
K
siP
GK
1+
siA
MP
K
siP
GK
2+
siA
MP
K
HR
E-l
ucife
rase
(p
er
ce
nt co
ntr
ol)
cell proliferation HRE-luciferase
B
* * *
dorsomorphin siAMPKα
C
ATP/ADP AMPK HIF-1α
p300 glycolysis
p
p
Normoxia Hypoxia
β-actin
total AMPK
p-AMPKα(Thr172)
total p300
p-p300(Ser89)
HIF1α
HT
-29 H
RE
β-actin
total AMPK
p-AMPKα(Thr172)
total p300
p-p300(Ser89)
HIF1α
MIA
PaC
a-2
HR
E
siP
GK
2
siP
GK
1
siA
LD
OA
siH
IF1α
siS
CR
siP
GK
2
siP
GK
1
siA
LD
OA
siH
IF-1α
siS
CR
cells
only
cells
only
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
FIG 5
A
B
perc
ent surv
ival
MDA-MB-231 shALDOA (clones 8.8 and 9.3) + pre dox
MDA-MB-231 parental + pre dox
MDA-MB-231 empty vector + pre dox
30 40 50 60 0
50
100
time (days)
p <.0001
shALDOA clone 8.8
30 40 50 60 0
50
100
time (days)
perc
ent surv
ival
p <.0001
shALDOA clone 9.7
MDA-MB-231 shALDOA (clones 8.8 and 9.3) + dox
HIF1α
ALDOA
β-actin
hypoxia + + +
doxycycline - 24h 48h
shALDOA Clone 9.7
doxycycline - 24h 48h
HIF-1α
ALDOA
β-actin
hypoxia + + +
shALDOA Clone 8.8
cells
cells + 24 hr dox
cells + 48 hr dox
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
FIG 6
TDZD-8
A
F
0
50
100
150
200
250
300
350
10 15 20 25 30 35
tum
or
volu
me (
mm
3)
days post cell injection
control
TDZD-8 12mg/kg QD ip
* *
**
H I
mM
/mg
0 1 2 3 4 Q5D 0
5
10 Lactate
*
μM
/mg
0 1 2 3 4 0 20 40 60 80
*
Q5D
DHAP
0 1 2 3 4 0
2
4
6
μM
/mg
* *
Q5D
3PG
time (hours)
cell
via
bili
ty (
% c
ontr
ol)
-7 -6 -5 -4 0
25
50
75
100
125
TDZD-8 (M)
IC50 19 μM
TDZD-8 (M)
HR
E –
luc (
% c
ontr
ol)
-7 -6 -5 -4 0
25
50
75
100
125
IC50 9.9 μM
TDZD-8 (M)
lacta
te/g
lucose r
atio)
-7 -6 -5 -4 0
25
50
75
100
125
IC50 9.5 μM
EC
AR
(m
ph/m
in)
TDZD-8 (M) -7 -6 -5 -4
12
16
0
4
8
IC50 22 μM
B
E
D
TDZD-8 (μM)
0 20 40 60 80
AL
DO
A a
ctivity
0
0.2
0.4
0.6
0.8
1.0
1.2
wt 30 min
C289A 10 min
wt 10 min
C289A 30 min
C
p-AMPKα
actin
TDZD-8 (µM) 0 5 10 20
AMPKα
AT
P (
nm
ol/m
g p
rote
in)
TDZD-8
(µM)
2-DG
(mM)
G
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401
Published OnlineFirst June 3, 2016.Cancer Res Geoffrey Grandjean, Petrus R De Jong, Brian P James, et al. adolase A as a therapeutic target
signaling in hypoxic tumors highlightsαglycolysis-HIF1Definition of a novel feed-forward mechanism for
Updated version
10.1158/0008-5472.CAN-16-0401doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2016/06/02/0008-5472.CAN-16-0401.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2016/06/03/0008-5472.CAN-16-0401To request permission to re-use all or part of this article, use this link
Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401