Accepted Manuscript

Title: Deciphering the metabolic role of AMPK in cancer

multi-drug resistance<!–<query id="Q2">Your article is
registered as belonging to the Special Issue/Collection entitled
T̈arget Enzymes.̈ If this is NOT correct and your article is a
regular item or belongs to a different Special Issue please
contact r.nair@elsevier.com immediately prior to returning
your corrections.</query>–>

Authors: Wen Tan, Zhangfeng Zhong, Randy P. Carney,

Yongfan Men, Jiannan Li, Tingrui Pan, Yitao Wang

PII: S1044-579X(18)30027-0
DOI: https://doi.org/10.1016/j.semcancer.2018.09.005
Reference: YSCBI 1513

To appear in: Seminars in Cancer Biology

Received date: 15-2-2018

Revised date: 2-9-2018
Accepted date: 18-9-2018

Please cite this article as: Tan W, Zhong Z, Carney RP, Men Y, Li J, Pan T, Wang Y,
Deciphering the metabolic role of AMPK in cancer multi-drug resistance, Seminars in
Cancer Biology (2018), https://doi.org/10.1016/j.semcancer.2018.09.005

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 Deciphering the Metabolic Role of AMPK in Cancer Multi-Drug Resistance

Wen Tana, b#, Zhangfeng Zhongc, d#, Randy P. Carneye, Yongfan Menb, Jiannan Lib, Tingrui

Panb*, Yitao Wangd*

T
R IP
a.
School of Pharmacy, Lanzhou University, Lanzhou, Gansu province 730000,

SC
China
b.
Micro-Nano Innovations (MiNI) Laboratory, Biomedical Engineering, University

of California, Davis, California 95616, United StatesU

N
c.
Center for Developmental Therapeutics, Chemistry of Life Processes Institute,
A
Northwestern University, Evanston, Illinois 60202, United States
M

d.
Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research

in Chinese Medicine, University of Macau, Macau SAR 999078, China

D

e.
Department of Biomedical Engineering, University of California Davis, Davis,
TE

California 95616, United States

# The authors equally contribute this work.

EP
CC

*Corresponding author

Prof. Yitao Wang, the State Key Laboratory of Quality Research in Chinese Medicine,
A

Institute of Chinese Medical Sciences, University of Macau, Macau SAR 999078, China.

Email: ytwang@umac.mo

Prof. Tingrui Pan, Department of Biomechanical Engineering, University of California,

Davis, 95616, USA. Email: tingrui@ucdavis.edu.

1
Abstract

Multi-drug resistance (MDR) is a curious bottleneck in cancer research and chemotherapy,

T
whereby some cells rapidly adapt to the tumor microenvironment via a myriad of

IP
heterogeneous metabolic activities. Despite being a major impediment to treatment, there is a

R
silver lining: control over metabolic regulation could be an effective approach to overcome or

SC
correct resistance pathways. In this critical review, we comprehensively and carefully curated

and analyzed large networks of previously identified proteins associated with metabolic

U
adaptation in MDR. We employed data and text mining to study and categorize more than
N
600 studies in PubMed, with particular focus on AMPK, a central and fundamental modulator
A
in the energy metabolism network that has been specifically implicated in cancer MDR

pathways. We have identified one protein set of metabolic adaptations with 137 members
M

closely related to cancer MDR processes, and a second protein set with 165 members derived
D

from AMPK-based networks, with 28 proteins found at the intersection between the two sets.
TE

Furthermore, according to genomics analysis of the cancer genome atlas (TCGA) provisional

data, the highest alteration frequency (80.0%) of the genes encoding the intersected proteins
EP

(28 proteins), ranked three cancer types with quite remarkable significance across 166 studies.

The hierarchical relationships of the entire identified gene and protein networks indicate
CC

broad correlations in AMPK-mediated metabolic regulation pathways, which we use decipher

and depict the metabolic roles of AMPK and demonstrate the potential of metabolic control
A

for therapeutic intervention in MDR.

Keywords: AMPK; metabolic reprogramming; multi-drug resistance; cancer

2
1. Introduction

Multi-drug resistance (MDR) is one of the major obstacles in chemotherapy and occurs in

the majority of cancer patients receiving chemotherapeutic treatment, eventually leading to

T
final failure. The magnitude of MDR incidence is frightening, and the impact of MDR on

IP
cancer mortality is significant and worldwide. Overcoming MDR has been regarded as one of

the main promises of the era of personalized medicine, and different strategies have been

R
conducted to reduce clinical burden [1, 2]. First, it is important to understand that MDR is an

SC
evolving paradigm, featuring a series of mechanisms including drug-related factors (drug

transport and metabolism, alterations in drug targets) and cell response-related factors (DNA

U
damage repair, intrinsic adaptive responses promoting survival and resistance, a wider
N
chemical view of the tumor microenvironment) [3, 4]. Despite the heterogenous character of
A
MDR, most of the focus on intervention has involved improving drug transport, for example
M

by developing new delivery systems to overcome low intracellular drug concentrations that

occur via active drug expulsion by MDR cancer cells. Yet these methods treat the symptoms
D

of MDR, not the underlying causes. More recently, a novel approach of reprogramming
TE

metabolism of the cancer microenvironment has begun to be applied to proactively change

the underlying dynamic cellular processes causing MDR [5].

EP

1.1 Metabolic Adaptation in Resistant Tumor Tissue

CC

Tumor tissue exhibits vast cellular heterogeneity, dictated by a dynamic and complex

spatial and chemical system known as the tumor microenvironment. Various regions of the
A

tumor are characterized in part by cellular changes in energy metabolism in response to

chemical gradients of oxygen, sugar, and other biomolecules. Acquisition of cancer resistance

and tumor microenvironment support are often achieved by metabolic reprogramming,

comprising a variety of adaptive mechanisms that occur naturally and spontaneously to

downregulate or upregulate biomolecules acting in key energy metabolism pathways [6]. For

3
example, cancer cells have the capacity to adapt to hypoxic and hypo-nutrient conditions,

typically via a relatively inefficient metabolic process of glycolysis, permitting rapid

malignant proliferation [7]. Cancer-associated fibroblasts (CAFs), for example, respond to

such conditions by active glycolysis and autophagy, altered lactate production, and altered

uptake due to heterogeneity of monocarboxylate transporter expression [8]. Compared with

K562 cells or NCI-H460 cells, their MDR counterparts (K562Dox cells or NCI-H460/R) have

T
increasing rates of glycolysis and methylation capacity, decreasing rates of pentose phosphate

IP
pathway (PPP) and oxidative phosphorylation (OXPHOS), and altered glutathione

metabolism [9]. In highly resistant multiple myeloma cells (AMO-BTZ and AMO-CFZ), a

R
large group (>600) of aberrantly-expressed proteins were identified and further found to be

SC
involved with metabolic control and redox homeostasis pathways, including ones related to

the respiratory chain, metabolite generation, glycolysis, and metabolism of amino acids,

U
lipids, and nucleic acids. It is clear from previous reports that there is a deep relationship
N
between metabolic reprogramming and cancer resistance [10].
A

1.2 Metabolic Control for Cancer Treatment

M

Given that metabolic reprogramming majorly drives MDR progression, it is likely that
D

metabolic control could be an effective approach to overcome MDR. This potential has been
TE

confirmed by the novel perspective of omics-related findings in recent years, which have

helped to identify personalized anticancer treatments and new therapeutic targets [11-13].
EP

Many flavors of therapeutic intervention to metabolic-associated cancer progression have

been attempted. For example, metabolic adaptation in cancer stem cells is regarded as a major
CC

potential cause of chemotherapy failure [14]. In one study, following chemotherapy failure in

melanoma, a subpopulation of slow-cycling melanocytes was characterized by high levels of

A

histone demethylase JARID1B and elevated mitochondrial metabolism [15]. Subsequent

blocking of the related mitochondrial respiratory chain was found to overcome MDR [15].

Metabolic suppression of the drug-resistant subpopulation of cancer spheroid cells in a 3D

model has also been confirmed to be a viable approach to treat MDR [16]. Regulation of

energy sources in the tumor microenvironment has been shown to restore chemo-sensitivity

4
[17, 18]. In that approach, bioenergetic modifiers work to increase sensitivity to conventional

chemotherapeutic agents by overcoming the extracellular acidification in breast cancer cells,

known as the “Warburg effect” [19]. Metformin, a first-line drug for type 2 diabetes, which

targets cell metabolism through a mimicry of calorie-energy restriction at multiple levels,

shows a promising potential in cancer prevention and treatment [20]. Combination of

metformin with 2-deoxylucose further aggravated metabolic stress on resistant cancer cells

T
and induced a series of cell cycle arrest and apoptosis, increasing doxorubicin accumulation

IP
[21, 22]. It is clear that metabolic control is a high-potential therapeutic strategy capable of

proactive management of energy metabolism in cancer cells, bringing along a new path to

R
overcome MDR for lower morbidity and mortality rates.

SC
In Section 2, we review the major players associated with the AMPK-mediated energy

U
metabolism network, focusing on key signaling pathways related to MDR, while highlighting
N
successful metabolic targets that have been reported in the literature. In Section 3, we
A
perform critical text-mining on those reports to reveal a highly interconnected and complex

regulatory network that largely dictates MDR in cancer. It is our hope that this global view
M

will aid researchers in developing more effective therapeutic interventions targeting metabolic
D

pathways at the root of MDR.

TE

2. AMPK and the Energy Metabolism Network

The AMPK enzyme is regarded as a sensor of energy and nutrients in cells. It is comprised
EP

of a catalytic subunit 𝛼 (5'-AMP-activated protein kinase subunit 1, PRKAA1), two

regulatory subunits 𝛽 (5'-AMP-activated protein kinase subunit beta-1 and 2, PRKAB1 and
CC

PRKAB2), and three regulatory subunits 𝛾 (5'-AMP-activated protein kinase subunit gamma-

1, 2 and 3, or PRKAG1, PRKAG2, and PRKAG3). AMPK is activated by phosphorylation,

A

and phosphorylated-AMPK was used as an independent prognostic marker for patients with

resected gastric cancer, where it significantly correlated with longer survival (61.0% in 621

patients) [23]. AMPK activation mediates metabolic reprogramming in resistant cancer cells

through promoting both the Warburg effect and also mitochondrial biogenesis. It also

5
regulates the self-renewal ability of cancer stem cells and induces autophagy, a double punch

for chemotherapy resistance [24]. A complex network of energy metabolism surrounds the

central AMPK enzyme, illustrating both its ubiquitous role in energy balance and also the

substantial amount of crosstalk between AMPK and other signaling pathways. These

signaling pathways involve (i) some pro-drugs converting to AMP analogs in cells, (ii) some

compounds inhibiting mitochondrial function and increasing AMP/ADP, or (iii) some direct

T
activators of AMPK [25, 26]. Among them, the most concentrated field is glycolysis,

IP
mitochondrial-related, and fatty acid synthesis (FASN), but also include glutamine

consumption and others.

R
SC
2.1 An Indispensable Glycolysis and a Pentose Phosphate Pathway (PPP)

coordinator

U
Glycolysis is an indispensable and routine way cancer cells use to extract energy. The
N
pentose phosphate pathway (PPP) coordinates glycolysis, biosynthesis, and redox regulation
A
through an ingenious approach, producing ribose-5-phosphate (Ru-5-P), NADPH, and
M

balancing redox homeostasis. HK2 is the major mitochondrial-bound hexokinase isoform in

cancers with the “Warburg effect”. As the most pivotal player in the “Warburg effect”, HK2
D

drives the prominent phenotype (high aerobic glycolysis) and helps immortalize cancer cells
TE

[27, 28]. For human breast carcinoma, HK2 activity and intracellular distribution indicated

invasiveness and aggressiveness, among other major prognosis markers [29]. In glioblastoma
EP

multiform (GBM), it supported tumor growth in vitro and in vivo [30].

As the product of the first rate-limiting step of glycolysis, phosphofructokinase 1 (PFK-1,

CC

or PFKP) provides an alternative way to survival of cancer cells even under hypoxia

condition. O-linked 𝛽-N-acetylglucosamine (O-GlcNAcylation) of PFK at Ser529 site occurs

A

in response to hypoxia, thereby inhibiting PFK activity and redirecting glucose metabolism to

PPP. It was observed that blocking the O-GlcNAcylation of PFK at Ser529 site leads to a

reduction of cancer cell proliferation in vitro and impaired tumor formation in vivo [31].

Besides glycosylation, transcriptional repression of epithelial-mesenchymal transition (EMT)

6
and functional mutation can also switch the flux of glucose towards PPP for cancer cell

survival and metastasis. For example, Snail suppresses the platelet isoform of PFK-1 (PFKP)

and turns to PPP for cancer cell survival under metabolic stress and increasing metastasis in

vivo [32]. Functional significance of selective PFKP mutations also contribute to metabolic

states in certain cancers [33]. The up-regulated predominant isoform of PFK-1, PFKP, is

found in human clear cell renal cell carcinoma (RCC) and has been associated with p53

T
suppression and malignant proliferation [22]. PFKP is mainly regulated by 6-phosphofructo-

IP
2-kinase/fructose-2,6- bisphosphatase 3 (PFKFB3, or PFK-2), the latter is regulated by

AMPK in a phosphorylation-dependent manner. Characteristic prolonged mitotic arrest and

R
induced mitophagy in cancer cells is achieved by AMPK, and PFKFB3 mediates glycolysis

SC
for survival [34]. 6-phosphofructo-2-kinase/fructose- 2,6-biphosphatase 4 (PFKFB4) is an

isoform of PFKFB3 and exerts balancing glycolytic activity and antioxidant production for a

cellular redox balance in prostate cancer cells [35]. U

N
A
Pyruvate kinase 2 (PKM2) catalyzes the final step of glycolysis, from phosphoenolpyruvate

(PEP) to pyruvate. Inhibition of PKM2 might be mediated by AMPK activation, and applied
M

to breast cancer cells, resulted in apoptosis due to low levels of glucose or amino acid [36].
D

Pyruvate dehydrogenase (PDH) is the first component enzyme of a larger pyruvate

TE

dehydrogenase complex (PDC). Pyruvate dehydrogenase kinase (PDK) can phosphorylate

PDH at its Ser294 site, which inactivates the PDC and leads to lactate production instead of
EP

the conversion of pyruvate to acetyl-CoA typically catalyzed by PDC. There is some evidence

of an indirect relationship between PDK and AMPK, since silencing pyruvate dehydrogenase
CC

kinase isoform 4 (PDK4) attenuates the phosphorylation level of AMPK induced by casein

kinase 2 (CK2), a result that could be produced in vitro and in vivo [37]. In low-nutrient
A

conditions in HeLa cells, PDK activation inhibits PDH phosphorylation and eventually drives

survival of cancer cells in a AMPK-dependent manner [38]. Thus, there is evidence that in

certain situations, PDK and AMPK exhibit a synchronized pace. Conversely, PDK4 was

observed in cancer cells to bypasses AMPK activation and subsequently activate mTOR

complex 1 (mTORC1) for aerobic glycolysis and tumorigenesis [39]. Lactate dehydrogenase

7
A (LDHA) prefers to promote lactate production, and exhibits a similar pattern to AMPK

expression, especially in triple negative breast cancer (TNBC). Increased expressions of

LDHA and AMPK were consistent with TNBC stage, distant metastasis, Ki67 status, and

survival outcome of patients [40]. Meanwhile, LDHA may be linked with AMPK activation

to support a glycolytic phenotype through the repression of mTORC1 inhibition and the

subsequent increase of HIF-1𝛼, which could bind to specific sites on LDHA promoter in

T
cancer cells [41]. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first

IP
committed step of PPP, converting glucose-6-phosphate to 6-phosphogluconolactone and

producing most reducing equivalents of NADPH for nucleotide synthesis. G6PDH is

R
inhibited by AMPK activation in arachidonic acid-treated hepatocytes [42]. In cancer cells, 6-

SC
phosphogluconate dehydrogenase (6PGDH), the third enzyme in PPP, stimulates the

production of ribulose-5-phosphate (Ru-5-P), which inhibits AMPK activation through

U
disruption of the active LKB1 complex [43]. NADPH production from PPP is impaired when
N
cancer cells face energy stress, for instance, during glucose limitation, anchorage-independent
A
growth, or solid formation in vivo. AMPK activation occurs to decrease NADPH
M

consumption in fatty acid synthesis and increase its production in fatty acid oxidation to

maintain NADPH and prolong cell survival [44].

D

2.2 TCA Cycle in Mitochondria

TE

Citrate synthase (CS), the first enzyme of the tricarboxylic acid (TCA) cycle, is highly
EP

expressed in malignant ovarian tumors and ovarian cancer cells. CS knockdown enhances

sensitivity to cisplatin in SKOV3 and A2780 cells and decreases phosphorylated AMPK [45].
CC

Overexpression of tumor suppressor succinate dehydrogenase subunit B (SDHB) inhibits

proliferation, invasion, and migration, and promotes apoptosis in ovarian cancer cell lines
A

SKOV3 and A2780, accompanied by a reduction of phosphate AMPKα [46]. Notably, a loss

of another tumor suppressor, fumarase (FH), induces fumarate accumulation that activates

AMPK, which in turn confers protection from apoptosis in hereditary leiomyomatosis and

renal cell cancer syndrome (HLRCC) [47]. Malate dehydrogenase 2 (MDH2) inhibition

induces AMPK activation in human colorectal cancer HCT116 cells [48]. This mechanism is

8
embodied in a novel MDH2 inhibitor that exhibits significant growth inhibition of HCT116

cells in vitro and tumor growth suppression of HCT116 xenograft mouse model in vivo [49].

2.3 Fatty Acid Synthesis (FASN)

It is well known that phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79 by

AMPK causes ACC inhibition. Besides ACC inhibition, ATP-citrate lyase (ACLY) is another

potential target. ACLY inhibition in castration-resistant prostate cancer (CRPC) cells

T
promotes energetic stress and AMPK activation, which supports androgen receptor (AR) level

IP
maintaining, inhibition of proliferation, and apoptosis induction. An ACLY-AMPK-AR

R
feedback loop might be applied to sensitize CRPC cells to AR antagonism for preventing

SC
inappropriate reactivation of AR after androgen deprivation therapy [50]. In colon cancer,

phosphate AMPK levels correlated with cellular ROS levels in vitro and in vivo, with lower

U
basal levels found to be critical for the anticancer effect of ACLY depletion [51]. From a co-
N
immunoprecipitation assay, an in vitro glutathione S-transferase (GST) pulldown assay, and
A
an in vitro kinase assay, the inhibition of ACLY on AMPK activation was confirmed to be
M

due to a physical interaction with the catalytic subunit 𝛼2 [52]. Stearoyl-CoA desaturase 1

(SCD1) is a rate-limiting enzyme that catalyzes the conversion of saturated fatty acids (SFA)
D

into monounsaturated fatty acids (MUFA), the main building blocks for phospholipids,
TE

triacylglycerols, and cholesterol esters. A stable knockdown of SCD1 in cancer cells

downregulated lipogenesis and reduced proliferation through AMPK-mediated inactivation of

EP

ACC, accompanied by in vitro invasion suppression and tumor growth inhibition [53].

2.4 Glutamine Consumption and related players

CC

Glutamine is an important source of carbon and nitrogen for malignant cancer cells [54].
A

Glutaminase (GLS) catalyzes the generation of glutamate from glutamine. Glutamate

dehydrogenase 1 (GLUD) is responsible for glutamine anaplerosis, by means of converting L-

glutamate into alpha-ketoglutarate. GLS2 inhibition or silencing could suppress proliferation

and anchorage-independent colony formation of cancer cells and induce autophagy through

AMPK-mediated mTORC1 inhibition [45]. Alpha-ketoglutarate and aspartate are catalyzed

9
by glutamic-oxaloacetic transaminase 1 (GOT1) to produce glutamate. In addition to

glutamate metabolism, pH regulation and serine synthesis are also viable targets. Solute

carrier family 9 (SLC9A1) regulates pH homeostasis by eliminating acid generated either by

active metabolism or in response to adverse environmental conditions. Carbonic anhydrase 9

(CA9) catalyzes reversible hydration of carbon dioxide to regulate pH homeostasis.

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first step of serine biosynthesis.

T
2.5 AMPK-mediated approaches overcoming MDR in experimental research

IP
There are some clinical drugs, small molecular inhibitors and bioactive compounds derived

R
from natural products exerting MDR improvement by means of AMPK-mediated metabolic

SC
modulation. Here the Table 1 illustrates these compounds targeting AMPK-mediated

pathways in MDR with in vitro or in vivo investigations briefly and concludes the effects and

U
the potential mechanisms. In majority of the involved pathways, AMPK is activated and
N
amplified by the downstream effectors, such as mTOR inhibition. Besides, multidrug
A
resistance protein 1 (MDR1) inhibition at gene and/or protein level, down-regulations on
M

multidrug resistance-associated protein 1 (MRP1), NF-𝜅B signaling suppression, are the most

common pathways against MDR. Autophagic or apoptotic cell death, the enhanced
D

cytotoxicity and proliferation inhibition, the cell cycle arrest, and metastasis or invasion
TE

attenuation are found. Among the energy metabolism network, only a limited amount of

proteins (e.g. ACACA [55], ACLY [56], HK2 [57] and LDHA [58] in Table 1) are focused
EP

with further study for in-deep research, and majority of them have not been explored yet,

especially on the specific metabolic network of AMPK-mediated. It might also be noted that
CC

the role of AMPK might be context dependent and the exact mechanism in specific cancer

type or tumor microenvironment should be viewed with some caution. Since many of the
A

compounds in Table 1 have multiple actions, in our present study we focus on the specific

effect and the corresponding mechanisms of MDR improvement by means of metabolic

control.

10
 R I
SC
Table 1 List of compounds targeting AMPK-mediated pathways in MDR

U
Compo Classific Refere

N
Resistant
und ation/Ta Pathway Effect nce
Model

A
Name rget (Year)

M
In vitro
Cisplatin-
ED Enhancing Inducing autophagic
resistant [59]
phosphorylation of cell death and
human oral (2017)
AMPK apoptotic death
cavity
PT

cancer cell
Chemo-
Reducing Increasing oxaliplatin
E

Resver preventive
In vitro transcriptional toxicity in
atrol phytochem
CC

Oxaliplatin- activities of NF-𝜅B HCT116/L-OHP

ical
resistant and MDR1; cells, not affecting
[60]
colorectal Activating AMPK; cytotoxicity of
A

(2015)
cancer cell Down-regulating oxaliplatin in
HCT116/L- MDR1 expression by HCT116 cells;
OHP inhibiting degradation Increasing
and phosphorylation accumulation of

11
 R I
SC
of I𝜅B𝛼 and p65 Rh123 in resistant

U
translocation, and cells;

N
through CRE
transcriptional

A
activity decrease

M
mediated by

ED phosphorylation of
AMPK;
The reduction in
In vitro Reducing phosphorylation of
PT

5-FU- phosphorylation of AMPK might be

AMPK [61]
AICAR resistant AMPK; related to 5-FU
E

activator (2016)
gastric Reducing mdr1 gene resistance in 5-FU-
CC

cancer cell expression; resistant gastric

cancer cell
A

In vivo Inhibition of HK-2 Inducing intrinsic

HK-2 pten-/-p53- phosphorylates apoptosis while [57]
2-DG
inhibitor deficiency- AMPK, which simultaneously (2016)
driven suppresses mTORC1- inducing ULK-1-

12
 R I
SC
CRPC p70S6K-Mcl-1 axis dependent pro-

U
mouse and induces ULK-1 survival autophagy

N
model signaling
In vitro

A
Having a synergistic
Human Activating AMPK and
anti-proliferation

M
hepatocellul inhibiting mTOR
effect with 5-Fu;
ar phosphorylation;
ED Promoting apoptosis
carcinoma Decreasing
and inducing cell
Bel-7402/5- expressions of HIF- [62]
G0/G1 cycle arrest;
Anti- Fu cell; 1𝛼, MDR1 and (2014)
PT

Resensitizing MCF-
Metfor diabetes Acquired MRP1; [63]
7/5-Fu cell and
min biguanide drug- Inhibiting MDR1 (2014)
E

innate drug-resistant
drug resistant expression by [64]
CC

MDA-MB-231 cell
MCF-7/5- blocking MDR1 gene (2010)
and promoting
Fu cell and transcription;
apoptosis;
A

innate drug- Increasing

Reversing EMT
resistant intracellular drug
phenotype and
MDA-MB- accumulation;
decreasing invasion;
231 cell;

13
 R I
SC
Human Reducing NF-𝜅B Increasing

U
breast activity and I𝜅B adriamycin toxicity

N
cancer degradation; in MCF-7/adr cell;
adriamycin Inhibiting

A
resistant phosphorylation of

M
cell line CREB;

ED(MCF-
7/adr);
Convergent activation
of pathways Imposing a great
PT

In vitro
underlying tumor selective pressure for
Human
microenvironment cellular states;
E

breast
interactions leads to a Triggering a [65]
CC

cancer
notable transcriptome (2014)
metformin-
downregulation of the reprogramming
resistant
A

G2/M DNA damage toward a metastatic

MCF-7 cell
checkpoint regulators stem-like profile;
that maintain genome

14
 R I
SC
stability and pro-

U
autophagic features;

N
Increasing the
potential of metastatic

A
dissemination by

M
amplifying the

ED number of pro-
migratory and
stemness inputs via
the activation of a
PT

significant number of
proteases and EMT
E

regulators;
CC

In vitro Activating AMPK;

Enhancing the
A Hepatocellu Reducing intracellular
cytotoxicity of
A

Retinoi metabolite lar ATP level; [58]

Sorafenib in
c acid of vitamin carcinoma Decreasing gene (2015)
hepatocellular
A cell lines expressions of GLUT-
carcinoma cells;
(HepG2, 1 and LDHA;

15
 R I
SC
PLC/PRF/5, Enhancing apoptosis

U
HuH6, induced by sorafenib,

N
HLE, HLF, accompanied by
Hep3B) activation of p38

A
MAPK and JNK,

M
upregulation and

ED translocation of Bax,
and activation of
caspase-3;
Active
PT

sesquiterpe Regulating
ne isolated In vitro Activation AMPK; mitochondrial
E

Furano from Doxorubici Decreasing phosphate function and [56]

CC

diene Curcuma n-resistant ACLY and phosphate reducing ATP level; (2016)
wenyujin MCF-7 cell GSK-3𝛽; Inducing apoptotic
A

Y. H. Chen cell death;

& C. Ling
21𝛼- Active Inhibiting expression, Regaining paclitaxel [66]
In vitro
methyl triterpenoi function and sensitivity (2015)

16
 R I
SC
melian d Paclitaxel- transcription of

U
odiol derivative resistant MDR1;

N
of A549 cell Regulating
Poncirus PI3K/mTOR

A
trifoliate signaling pathway;

M
In vitro

EDHuman
colon Inducing necrosis
cancer and apoptosis when
HCT-116 Activating AMPK and combined with
PT

Short chain cell, human p53; vincristine in vitro; [60]

C6
cell- pancreatic Inactivating Sensitizing (2015)
E

cerami
permeable cancer mTORC1; doxorubicin-induced [67]
CC

de
ceramide PANC-1 Down-regulating Bcl- apoptosis in vitro; (2010)
cell and 2/HIF-1𝛼; Enhancing anti-
A

L3.6 cell, proliferative activity

human of vincristine in vivo;
ovarian
cancer

17
 I
R
SC
A2780 cell

U
and

N
CaOV3,
human

A
breast

M
cancer
MCF-7 cell,
ED non-
Hodgkin’s
lymphoma
PT

KARPAS-
422 cell and
E

Hodgkin’s
CC

lymphoma
L428 cell,
A

melanoma
cancer
WM-115
cell;
In vivo

18
 R I
SC
HCT-116 or

U
A2780

N
xenografts
In vitro

A
Activating AMPK and Promoting AMPK-
Bortezomib
Bortez Proteasom promoting ULK1/2 to dependent [68]

M
-resistant
omib e inhibitor form a complex with autophagosome (2014)
myeloma
ED ATG13; formation;
cell
Increasing cell
In vitro response to
PT

Human doxorubicin in the

Small [69]
C19 melanoma Activating AMPK; presence of
E

molecular (2014)
WM266 conditioned medium
CC

cell from cells treated

with belinostat;
A

Bioactive In vitro Activating AMPK and

Inducing growth
Cordyc compound Gallbladder inhibiting mTORC1; [70]
inhibition and
epin from genus cancer Down-regulating (2014)
apoptosis;
Cordyceps GBC-SD MDR expression;

19
 R I
SC
cell, Mz- Enhancing sensitivity

U
ChA-1 cell, of GBC-SD cell to

N
QBC939 gemcitabine and 5-
cell Fu;

A
Down-regulating P-gp

M
In vitro expression;
Bioactive
Human Activating AMPK;
compound ED breast Inhibiting NF-𝜅B Reversing P-gp-
Mollug from roots [71]
cancer signaling pathway and mediated multidrug
in of Rubica (2013)
MCF- COX-2 expression resistance
PT

cordifolia
7/Adriamyc and attenuating CRE
L.
in cell transcriptional
E

activity;
CC

In vitro Activating AMPK;

Multidrug- Inhibiting Raf-1 and Inhibiting
Raf-
A

resistant
Sorafen B-Raf kinase activity; proliferation; [72](20
targeting Ras-NIH
ib Abolishing both ERK Inducing autophagy 13)
drug 3T3/Mdr and MER and apoptosis;
cell phosphorylation;

20
 R I
SC
Inhibiting LKB1 Reversing the

U
expression and resistance to

N
inducing a sustained paclitaxel;
activation of AMPK

A
at a concentration of 5

M
𝜇M;

ED Inducing decreases of
mTOR
phosphorylation at
Ser-2448, and
PT

phosphorylation of
p70S6K and 4E-BP1;
E

Negatively regulating
CC

P-gp function;

In vitro Inducing cell cycle

A

A selective Multidrug- arrest at G1/S; Inducing

[73]
PP2 SFK resistant v- Inducing hyper proliferation
(2011)
inhibitor Ha-ras- activation of AMPK inhibition;
transformed and mTOR inhibition;

21
 R I
SC
NIH 3T3 Delay Raf activation;

U
cell

N
A
M
Inhibiting MDR1
ED expression and
suppressing MDR1
In vitro
mRNA and MDR1
PT

Bioactive Human
promoter activity;
compound breast
Puerari Increasing drug Reversing cancer [55]
E

from cancer
n accumulation of drug resistance; (2010)
Pueraria MDR cell
CC

doxorubicin;
lobata line (MCF-
Inhibiting NF-𝜅B
7/adr cell)
A

activity and I𝜅B

degradation;
Activating AMPK;

22
 R I
SC
Inducing

U
phosphorylation of

N
ACC and GSK-3𝛽;
Decreasing CREB

A
phosphorylation;

M
ED
EPT
CC
A

23
3. Analysis of Mined Protein Alterations Related to MDR and Cancer Metabolism

3.1 Text Mining Criteria and Protein Inclusion

In the present study, we employed text and data mining to identify and map all reported

altered proteins associated with metabolic adaptation in MDR, by retrieving relevant

pharmacological studies, including quantitative research and qualitative research. Publications

were searched in PubMed with the terms (“Drug Resistance, Neoplasm [MeSH])” and

T
(“Energy Metabolism [MeSH]”). By 18 September 2017, a total of 636 publications were

IP
retrieved and reviewed. First, abstracts of the selected publications were used to screen for

R
pharmacological studies associated with the alterations of key proteins known to be involved

SC
in energy metabolic pathways. Next, to ensure the applicability of each study to our present

review, the full reports of the selected publications were reviewed rigorously. Our inclusion

U
criteria were proteins that (i) exhibited definite alterations in resistant cancer cells or
N
transformed cancer cells with the MDR phenotype and (ii) were reported in experimental data
A
across multiple pharmacological approaches. Studies performed with these criteria in cancer
M

stem cells were also included, given their well-known MDR characteristics [74-76]. Studies

involving MDR intervention induced by novel drug delivery systems were ruled out. Those
D

criteria identified one key protein subset composed of 137 metabolic adaptations with many
TE

related to reported cancer MDR processes. A detailed list of the altered proteins is shown in

Supplement 1.
EP

Primary among the 137 altered MDR-related proteins identified are glucose transportation

and metabolism related proteins (GLUT1, 2, 3, 4, 10, HK1, HK2, PFKFB2, PFKFB3, PGM1,
CC

PGM2), aerobic glycolysis related proteins (PFKP, ALDOA, PKM2, LDHA and B, GAPDH,

MLXIP, PGAM1), mitochondrial and energy metabolism related proteins (ATP5A1, ATP5B,
A

ATP5D, CYC1, DNM1L, IDH1, LKB1, MCU, MDH, ME1, MT-CO1, mTOR, NRF1,

PDHA1, PDK1, PDK2, PDK3, PDK4, PEPCK, PPARGC1A, PPIF, PRDX1, PYGB, SDHA,

SDHB, SIRT1, TFAM, TKT, UCP1, UCP2, UMPS, UQCRC1, UQCRC2), as well as lipid

metabolism process related proteins (FASN, ACACA, ACLY, ACSF2, ACSF3, PTEN).

24
Others include members of the aldo-keto reductase family, AKR1B10 (Aldose reductases),

AKR1C1 and AKR1C3 (Hydroxysteroid dehydrogenases), which are responsible for

catalyzing redox transformations in biosynthesis, intermediary metabolism, and

detoxification. Also identified were ALDH1 and ALDH3A1, detoxification-related proteins

that regulate lipid peroxidation and the metabolism of corticosteroids, biogenic amines, and

neurotransmitters. Monocarboxylate transportation proteins (MCT1 and MCT4),

T
NADP/NADPH related proteins (APOA1BP, BLVRA, NDUFB8, NOX2, NQO1),

IP
glutathione metabolism related enzymes (GCS, GGT, GOT1, GPX1, GSTM1, GSTM4,

MGST1, MGST3), and proteins responsible for glucose uptake regulation (AKT1 and 2) were

R
also selected. Still others include: the ESRRA/PGC1alpha complex, a regulator of energy

SC
metabolism; G6PD, the rate-limiting enzyme of pentose-phosphate pathway; GLS1 and

GLUL, glutamine metabolism related enzymes; and finally, HIF-1A, the hypoxia

U
conditioning protein closely related to glucose transporters, glycolytic enzymes, and
N
metabolic adaptation. The diversity of the proteins significantly associated with metabolic
A
adaptation in cancer MDR clearly indicate a highly complex and dynamic regulatory network.
M

3.2 AMPK related

D

Of the 137 altered proteins reported in pharmacological studies to be associated with the
TE

metabolic reprogramming adaptation of resistant cancer cells (as shown in Supplement 1),

and especially weighing the different roles of AMPK in these metabolic processes (as
EP

discussed above in 2.1.1-4). Finding a general consensus on metabolic activities in cancer

cells has also influenced our selection criteria [5, 77, 78]. For instance, in glucose
CC

transportation and glycolysis process, HK2, phosphofructokinase (L, M, P), PFKFB3,

PFKFB4, PHGDH, PKM2, LDHA, ME1 and monocarboxylate transporters (such as

A

SLC16A) were chosen as the initial objects to further explore deeper correlations. While in

mitochondrial related process, PDHX, PDK1, CS, ACO2, Isocitrate Dehydrogenase 1

(IDH1), IDH2, succinate dehydrogenase complex (A, B, C, D), FH, MDH, were selected as

the initial data. For fatty acid synthesis, ACLY, ACC, HMGCR, FASN and SCD were

25
selected. Finally, GLS, GLUD, GOT1, SLC9A1, CA9, G6PD and PHGDH were included to

investigate glutamine metabolism and related processes.

To assess and integrate protein and protein interaction (PPI) networks we utilized the

STRING biological database, which contains thousands of functional protein association

networks identified from many sources, including text mining (networks extracted from the

abstracts of scientific literature), experiments (networks detected by biochemical and

T
biophysical assays), other curated databases, co-expression (protein-encoding genes co-

IP
expressed in the same or in different species), neighborhood analysis (repeatedly occurring

R
spatially close networks of protein-encoding genes), gene fusion (gene fusion events per

SC
species for protein-encoding genes), and co-occurrence (the presence or absence information

on linked proteins across species) [79, 80]. In our present study, to comprehensively and

U
efficiently predict direct or indirect associations, PPI networks were characterized using
N
systematic co-expression data, database annotations, and automated text mining of the
A
scientific literature for each metabolic process. By including statistical and/or semantic links

between proteins derived from Medline abstracts and full-text articles, a widely
M

comprehensive network was built up.

D

The four panels of Figure 1 illustrate the specific interactions of AMPK (represented as a
TE

grouped circle comprised of seven key proteins) in each identified metabolic process, i.e.

glycolysis, mitochondrial-related, fatty acid synthesis, and glutamine-related as Fig 1A-D,

EP

respectively. The minimum required interaction score was set as medium confidence (0.400)

for a broad and comprehensive analysis. The max number of interactions included was no
CC

more than 50 interactors. Networks were clustered using a Markov cluster algorithm (MCL)

[81]. Key proteins with interaction value greater than 0.500 in automated text mining were
A

selected. Of those, 56.75% (668 PPIs among 1177 PPIs) were related to glycolysis (76

proteins), 41.97% (410 PPIs among 977 PPIs) to mitochondrial-related (59 proteins), 44.86%

(388 PPIs among 865 PPIs) to fatty acid synthesis (54 proteins), and 54.63% (336 PPIs

among 615 PPIs) to glutamine or related processes (55 proteins). Cytoscape, a software

platform that builds 2D depictions of molecular interaction networks [82], was applied to

26
visualize the identified key proteins and interactions as depicted in Fig 1. Nodes represent

proteins and edges (the lines between two nodes) represent protein-proteins associations. The

size of the nodes indicates the level of functional associations between proteins according to

number of database annotations. Node color was derived from co-expression data. Edge color

represents the variance of interactions parsed across primary databases during automated text

mining data. To better emphasize the main trends of the entire network in each metabolic

T
process, multiple edges between two nodes were bundled together, generating overlapping

IP
lines.

R
The hierarchical relationships of the entire network can be easily analyzed by considering

SC
together the main trends, node size, and node/edge color. Accordingly, rather than simple

causal relationships, the broadest possible correlations among proteins is displayed, therefore

U
this depiction indicates a full-scale potential PPI network bundled from the perspective of
N
AMPK-mediated metabolic control of MDR. For example, as shown in Fig 1A, there are five
A
groups of highlighted protein with intensive interactions in glycolysis, namely the first group

(HK1, SLC2A2, PC, LDHA, PFKM, PFKL, PGM1, PGAM1, GMPS, MDH2), the second
M

group (TALDO1, HK3, ENO4, ACACB, FH, ENO3, PFKFB4, PKM, PGK2, PGK1,
D

ACACA), the third group (TKT, LDHAL6B, MDH1, PFKP, LDHAL6A, GPI, SLC2A4), the
TE

fourth group (PFKFB3, HK2, FBP2, PDHA2, FBP1, PDHA1, ENO2, ENO1, ALDOC,

LDHB) and the fifth group (LDHC, ALDOA, ALDOB, TPI1, ME2, ME3, ME1, PKLR,
EP

PFKFB2), without priorities in sorting. Similarly, in Fig 1B, six groups are found with

intensive interactions in mitochondrial-related, including the first group (PPARGC1A, ME1,

CC

ME3, ME2, SUCLG2, PDHX, CS, IDH3A, DPYD), the second group (GMPS, ATP5C1,

PCK1, DLAT, IDH3G, ACO1, IDH3B, NDUFS2, NDUFS3), the third group (SDHAF2,
A

SDHD, TP53, FH, GOT2, ACLY, PDHB, PC, PDHA2, PDHA1, DHTKD1, BCKDHA,

ACACB, BCKDHB, ACACA), the fourth group (IDH1, GOT1, IDH2, MDH1, SUCLA2,

DLD, PDK2, PDK1, MTOR), the fifth group (SDHC, OGDH, SDHA, UQCRFS1, SUCLG1,

OGDHL, SDHB, MDH2, ATP5B, FXN), the sixth group (HSPD1, ACO2, VDAC1, CYCS,

RPTOR). There are several groups of highlighted protein with intensive interactions in fatty

27
acid synthesis related processes (Fig1C) and in glutamine-related processes (Fig1D).

Although most of proteins in these potential PPI networks have been confirmed by practical

experiments, only a few of them (such as PKM [83], PDK [18], etc.) are focused on MDR

improvement by metabolic control. Therefore, more proof and in-depth studies on this

specific topic are needed.

In addition to the associations between the proteins comprising each metabolic process, we

T
analyzed the core proteins directly interacted with AMPK (including PRKAA1, PRKAA2,

IP
PRKAB1, PRKAB2, PRKAG1, PRKAG2, and PRKAG3). Heat maps representing the

R
computed text-mining scores are shown in Figure 2, similarly broken down into the four

SC
identified metabolic pathways. More proteins across each pathway were found to preferably

interact with the catalytic subunits PRKAA1 and PRKAA2. Only a few proteins prefer to

U
interact with PRKAG1 and PRKAG2, the two regulatory subunits. For the catalytic subunits
N
of AMPK, four proteins were found to participate in all the metabolic process presented in
A
our study: ACACA (Acetyl-CoA carboxylase alpha, ACACA), ACACB (Acetyl-CoA

carboxylase beta, ACACB), RPTOR (Regulatory associated protein of MTOR complex 1,

M

RPTOR) and TSC2 (Tuberous sclerosis complex 2, TSC2). Collectively, as depicted in

D

Figure 3A, these directly linked proteins comprise a heterogeneous group with 22 members
TE

(ACACA, ACACB, ACLY, ASNS, CAB39, CAD, GOT2, HK2, HMGCR, LEP, MTOR,

PFKFB2, PFKFB4, PPARGC1A, RHEB, RPTOR, SLC2A4, STK11, TP53, TSC1, TSC2,
EP

ULK1). This grouping helps to visualize the central role of AMPK in the wider energy

metabolism network. The key proteins with the closest interactions with AMPK in each
CC

metabolic process (text-mining score greater than 0.500) were collected for further analysis,

revealing a large AMPK-based network with 165 proteins (Supplement 2) that could be
A

broken down into four groups: glycolysis (76 proteins) (Supplement 3), mitochondrial-

related (59 proteins) (Supplement 4), fatty acid metabolism (54 proteins) (Supplement 5),

glutamine-related (55 proteins) (Supplement 6). Figure 3B is a Venn diagram visualization

of the complex relationships between these four groups, with the proportion of shared

proteins amongst the groups represented as percentage values. The metabolic processes of

28
mitochondrial-related and FASN share the maximum amount of communal proteins, with 14

proteins accounting for 8.5% (ACO1, ACO2, CS, DHTKD1, DLAT, DPYD, OGDH,

OGDHL, PDHX, SDHA, SDHB, SDHC, SUCLA2, and SUCLG2). 9 proteins are shared

amongst all four groups, accounting for 5.5% (ACACA, ACACB, ACLY, GMPS, MDH1,

MDH2, MTOR, RPTOR, and TSC2). Other notable associations include 5 proteins (3%)

shared between glycolysis and others (GPI, HIF1A, PGAM1, PHGDH, and PSAT1), 5

T
proteins (3%) shared between mitochondrial-related and others (CAB39, GOT1, GOT2,

IP
TP53, and ULK1), 4 proteins (2.4%) shared among glycolysis, FASN, and others

(LDHAL6A, LDHAL6B, LDHB, and LDHC), 3 proteins (1.8%) shared among glycolysis,

R
mitochondrial-related, and FASN (ME1, ME3, and PC), 2 proteins (1.2%) shared among

SC
glycolysis, mitochondrial-related, and others (PPARGC1A and STK11), and 2 proteins

(1.2%) shared among mitochondrial-related, FASN, and others (IDH1 and IDH2).

U
N
We further analyzed and identified the most fundamental intersection between AMPK-
A
related energy metabolism network and metabolic adaptation in resistant cancer cells in order

to decipher the metabolic role of AMPK in MDR. In Figure 4, the key proteins (165 proteins)
M

derived from AMPK-based network through data mining and the altered proteins (137
D

proteins) in metabolic adaptation are compared. 28 proteins were found at this intersection,
TE

accounting for 10.2% of the total: ACACA, ACLY, ALDOA, ATP5B, ENO1, FASN, G6PD,

GAPDH, GLUL, GOT1, HK1, HK2, IDH1, LDHA, LDHB, ME1, PDHA1, PDK1, PDK2,
EP

PFKFB2, PFKFB3, PFKP, PGAM1, PGM1, PPARGC1A, SDHA, SDHB, and TKT. These

can be divided into several groups, fatty acid synthesis related group (Acetyl-CoA alpha,
CC

ACACA; ATP-citrate lyase, ACLY; Fatty acid synthase, FASN; Glucose-6-phosphate

dehydrogenase, G6PD), glycolysis group (Aldolase A, ALDOA; Enolase 1, ENO1;

A

Glyceraldehyde-3-phosphate, GAPDH; Hexokinase 1, HK1; Hexokinase 2, HK2; Lactate

dehydrogenase A, LDHA; Lactate dehydrogenase B, LDHB; Pyruvate dehydrogenase alpha

1, PDHA1; Pyruvate dehydrogenase kinase isozyme 1, PDK1; Pyruvate dehydrogenase

kinase isozyme 2, PDK2; 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 2, PFKFB2;

6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3, PFKFB3; Phosphofructokinase,

29
platelet, PFKP; Phosphoglycerate mutase 1, PGAM1; Phosphoglucomutase 1, PGM1;

Transketolase, TKT), mitochondrial-related group (ATP synthase, ATP5B; Isocitrate

dehydrogenase 1, IDH1; Malic enzyme 1, ME1; Peroxisome proliferator-activated receptor

gamma coactivator 1, PPARGC1A; Succinate dehydrogenase complex, subunit A, SDHA;

Succinate dehydrogenase complex, subunit B, SDHB), and glutamine-related group

(Glutamate-ammonia ligase, GLUL; Glutamic-oxaloacetic transaminase 1, GOT1).

T
These identified core proteins with dual roles in AMPK-mediated metabolic regulation in

IP
MDR may help take AMPK-mediated metabolic regulation from lab bench to clinical

R
bedside. Actually, there have been extensive studies on some of the pivotal proteins analyzed

SC
above, and the various proportions of contribution to the final metabolic phenotype or MDR

development were also investigated continuously in recent years. Finally, prior to verifying

U
these extracted proteins from literature analysis and data mining, the achievements of
N
experimental researches on AMPK-mediated network in overcoming MDR should also be
A
reviewed carefully.
M

4. The Perspective of Metabolic Control from the Clinic and in Drug Discovery

Metabolism reprogramming is one of the hallmarks of conceptual progress in cancer

D

research in the past few decades. Such approaches focus on intra-tumoral heterogeneity and
TE

the tumor microenvironment, in the context of cellular metabolic control [84]. Many studies

have applied this paradigm to drug discovery. In this section, we apply various gene networks
EP

to analyze 28 genes responsible for encoding the core proteins extracted from the AMPK-

mediated metabolic pathway in MDR. Mutation analysis of these genes in clinical data was
CC

conducted with the cBio portal method, an open access resource for interactive exploration of

cancer data sets [85, 86]. Greater than 30% of altered genes in these core 28 genes is criteria,
A

hence 57 studies were found according to the cancer genome atlas (TCGA) provisional data,

as shown in Figure 5A. With quite remarkable significance across 166 various studies, a high

alteration frequency (>80.0%) ranked three cancer types: glioma (100%) [87], breast cancer

(96.6%) [88] and melanoma (80.6%) [89]. Glioma data represents sequence analysis of 23

30
initial low-grade gliomas patients, and the recurrent tumors resected from these patients.

Breast cancer data are from a genetically-defined clonal population dynamics study using

patient-derived xenografts (PDXs). Melanoma data are from a genomic analysis of pre-

treatment melanoma biopsies.

For each caner type, genetic alterations for each gene involved in AMPK-mediated

metabolic pathways of MDR, which were found in at least three studies, are visualized

T
according to their percent alteration frequency in Figure 5B. In glioma, the gene alteration

IP
frequencies indicate the critical role of IDH1 mutation in resistance progression. Of the 28

R
genes across subtypes of glioma, IDH1 has 100%, 77%, and 73% of altered frequencies in

SC
low-grade glioma, brain lower grade glioma, and anaplastic oligodendroglioma or anaplastic

oligoastrocytomas tumors, respectively. The alteration frequency of IDH1 is much greater

U
than that of other core genes, which may distinguish it as the most important gene encoding
N
protein in metabolic control for glioma resistance treatment. Actually, IDH1 is an important

metabolic enzyme catalyzes oxidative decarboxylation of isocitrate to produce α-ketoglutarate

A

(α-KG) and carbon dioxide. Hot-spot mutations in IDH1 are extensively found in malignant
M

gliomas [90-92], and elevated levels of D-2-hydroxyglutarate (D2HG) caused by mutant

D

IDH1 can be used as a non-invasive imaging approach with high sensitivity and specificity
TE

before individualizing surgical strategies. Consistent with our findings regarding IDH1, the

high frequency of IDH1 mutations in malignant glioma are being studied in clinical trials, and
EP

mutant IDH inhibitors have been developed into a treatment [93]. Similarly, for breast cancer,

as shown in Figure 5C, the highest alteration frequency of genes in three independent studies
CC

are: for PDXs – PFKP (52%), PFKFB3 (38%), and SDHA (34%); for adenoid cystic

carcinoma – ATP5B (42%), PDK2 (25%), and FASN/ACLY/ACACA (17%); and for
A

invasive breast cancer – PFKFB2 (25%), GLUL (22%), and PDK2 (7%). Some of these have

been investigated with good potential, such as PFKP [94], PFKFB3 [34, 95], SDHA [96],

ATP5B [97], PDK2 [98], FASN [99-101], ACLY [102, 103], ACACA [104, 105], and GLUL

[106, 107]. In the context of AMPK-mediated metabolic control in MDR, the roles or

underlying mechanisms of the proteins identified above, including PFKP, PFKFB3, SDHA,

31
ATP5B, PDK2, FASN, ACLY, ACACA, PFKFB2, and GLUL, need to be further

investigated. Undoubtedly, the superposition of AMPK-mediated network and MDR

background makes metabolic modulation much more sophisticated than it was. And we have

to define the precise mechanisms with more thorough and cautious considerations before

more studies concentrated on this unique perspective. Additionally, as shown in Figure 5D of

melanoma, the most remarkable genes are FASN (19%, 10% and 5% for melanoma, skin

T
cutaneous melanoma, and desmoplastic melanoma respectively), ACACA (14% and 8% for

IP
melanoma and skin cutaneous melanoma), GLUL (16% and 5% for melanoma and

desmoplastic melanoma), ME1 (25% and 9% for desmoplastic melanoma and skin cutaneous

R
melanoma), PPARGC1A (25%, 14% and 7% for desmoplastic melanoma, melanoma, and

SC
skin cutaneous melanoma respectively), and PFKFB2 (14% and 7% for melanoma and skin

cutaneous melanoma), which might encode the most potential proteins in AMPK-mediated

U
metabolic pathway of melanoma MDR and be worthy of further study in the future [108-112].
N
For example, elevated expression of FASN is found to be associated with melanoma
A
progression [109], and a FASN inhibitor derived from Herba Epimedii exerts anti-melanoma
M

activities [110]. Besides the perspective of these extracted genes in data analysis of clinical

researches, there are some metabolic modifiers being studied on these proteins in drug
D

discovery, such as 2-Deoxy-D-glucose (2-DG) (targeting on HK), dichloroacetic acid (DCA)

TE

(targeting on PDK2), lonidamine (targeting on HK1), C75 (targeting FASN) and 6-NBDG

(targeting on HK1) [113, 114]. For example, DCA is an acid analogue of acetic acid
EP

inhibiting PDK2 with some ongoing clinical trials for safety and effectiveness

(NCT01029925; NCT01111097; NCT01163487; NCT00566410; NCT00540176;

CC

NCT00703859; NCT01386632). Given the further study on AMPK-mediated metabolic

control in MDR, more and more metabolic modifiers are expected to be discovered, and their
A

corresponding metabolic approaches will come to the fore in future drug discovery.

Conclusion

AMPK-mediated metabolic control in MDR is a promising prospective avenue in cancer

therapy. Based on our critical review of studies on metabolic modifiers that confer attenuation

32
in cancer resistance, and our subsequent presentation of the deep, central metabolic role of

AMPK, we posit that methods which interfere with AMPK metabolism would be compelling.

Through a stringent text mining approach, our present study provides a fully described

AMPK-mediated metabolic network in MDR represented by a vivid PPI figure, along with

analysis of the key proteins with the closest interactions with AMPK in each metabolic

process. A complex set of relationships among each AMPK-mediated metabolic process

T
illustrates the central role of AMPK in MDR, and an intersection analysis between AMPK-

IP
related energy metabolism network and metabolic adaption in resistant cancer cells identifies

the associated group of core genes. Undoubtedly, there are still underlying mechanisms

R
worthy to interpret and many details that need to be worked out, but the core genes identified

SC
here may serve as a guide for future studies, especially for deciphering the metabolic role of

AMPK in MDR. Meanwhile, we hold the opinion that future efforts should be focused on

U
alterations of specific metabolic proteins or pathways in corresponding cancer types, which
N
would provide much more valuable information for personalized medication from the view of
A
omics.
M

Author Contributions
D

WT wrote and revised this manuscript. ZFZ contributed significantly to manuscript

TE

preparation with constructive discussion and careful revision. RPC revised the entire

manuscript and edited the language for scientific presentation. YFM and JNL helped to revise
EP

the manuscript and provided valuable feedback to this conception. YTW and TRP conceived

and organized this study.

CC

Funding
A

This study was supported by the China Scholarship Council (CSC) and the Fundamental

Research Funds for the Central Universities (lzujbky-2018-135, lzujbky-2016-149).

Conflict of Interest

The authors declare no conflict of interest, financial or otherwise.

33
Acknowledgements

We want to thank all the colleagues and friends for their sincere and generous help.

References

T
IP
[1] L.A. Garraway, P.A. Janne, Circumventing cancer drug resistance in the era of

R
personalized medicine, Cancer Discov 2(3) (2012) 214-26.

SC
[2] M. Saraswathy, S. Gong, Different strategies to overcome multidrug resistance in

cancer, Biotechnol Adv 31(8) (2013) 1397-407.

U
[3] C. Holohan, S. Van Schaeybroeck, D.B. Longley, P.G. Johnston, Cancer drug
N
resistance: an evolving paradigm, Nat Rev Cancer 13(10) (2013) 714-26.
A
[4] K.O. Alfarouk, Tumor metabolism, cancer cell transporters, and
M

microenvironmental resistance, J Enzyme Inhib Med Chem 31(6) (2016) 859-66.

[5] R.J. DeBerardinis, N.S. Chandel, Fundamentals of cancer metabolism, Sci Adv 2(5)
D

(2016) e1600200.
TE

[6] R.S. Vidal, J. Quarti, F.D. Rumjanek, V.M. Rumjanek, Metabolic Reprogramming

During Multidrug Resistance in Leukemias, Front Oncol 8 (2018) 90.

EP

[7] R.V. Pusapati, A. Daemen, C. Wilson, W. Sandoval, M. Gao, B. Haley, A.R. Baudy,

G. Hatzivassiliou, M. Evangelista, J. Settleman, mTORC1-Dependent Metabolic

CC

Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells, Cancer

Cell 29(4) (2016) 548-562.

A

[8] G.J. Yoshida, Metabolic reprogramming: the emerging concept and associated

therapeutic strategies, J Exp Clin Cancer Res 34 (2015) 111.

[9] V. Lopes-Rodrigues, A. Di Luca, J. Mleczko, P. Meleady, M. Henry, M. Pesic, D.

Cabrera, S. van Liempd, R.T. Lima, R. O'Connor, J.M. Falcon-Perez, M.H.

34
Vasconcelos, Identification of the metabolic alterations associated with the multidrug

resistant phenotype in cancer and their intercellular transfer mediated by extracellular

vesicles, Sci Rep 7 (2017) 44541.

[10] G.P. Soriano, L. Besse, N. Li, M. Kraus, A. Besse, N. Meeuwenoord, J. Bader, B.

Everts, H. den Dulk, H.S. Overkleeft, B.I. Florea, C. Driessen, Proteasome inhibitor-

adapted myeloma cells are largely independent from proteasome activity and show

T
complex proteomic changes, in particular in redox and energy metabolism, Leukemia

IP
30(11) (2016) 2198-2207.

[11] L. Jerby, E. Ruppin, Predicting drug targets and biomarkers of cancer via genome-

R
scale metabolic modeling, Clin Cancer Res 18(20) (2012) 5572-84.

SC
[12] G. Corona, F. Rizzolio, A. Giordano, G. Toffoli, Pharmaco-metabolomics: an

emerging "omics" tool for the personalization of anticancer treatments and

U
identification of new valuable therapeutic targets, J Cell Physiol 227(7) (2012) 2827-
N
31.
A
[13] K. Yizhak, B. Chaneton, E. Gottlieb, E. Ruppin, Modeling cancer metabolism on
M

a genome scale, Mol Syst Biol 11(6) (2015) 817.

[14] G. Gentric, V. Mieulet, F. Mechta-Grigoriou, Heterogeneity in Cancer Metabolism:

D

New Concepts in an Old Field, Antioxid Redox Signal 26(9) (2017) 462-485.
TE

[15] A. Roesch, A. Vultur, I. Bogeski, H. Wang, K.M. Zimmermann, D. Speicher, C.

Korbel, M.W. Laschke, P.A. Gimotty, S.E. Philipp, E. Krause, S. Patzold, J. Villanueva,
EP

C. Krepler, M. Fukunaga-Kalabis, M. Hoth, B.C. Bastian, T. Vogt, M. Herlyn,

Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial

CC

respiratory chain of slow-cycling JARID1B(high) cells, Cancer Cell 23(6) (2013) 811-

25.
A

[16] V. Koshkin, L.E. Ailles, G. Liu, S.N. Krylov, Metabolic Suppression of a Drug-

Resistant Subpopulation in Cancer Spheroid Cells, J Cell Biochem 117(1) (2016) 59-

65.

[17] D. Sridaran, G. Ramamoorthi, R. MahaboobKhan, P. Kumpati, Oxystressed tumor

35
microenvironment potentiates epithelial to mesenchymal transition and alters cellular

bioenergetics towards cancer progression, Tumour Biol 37(10) (2016) 13307-13322.

[18] A. Kumar, S. Kant, S.M. Singh, Antitumor and chemosensitizing action of

dichloroacetate implicates modulation of tumor microenvironment: a role of

reorganized glucose metabolism, cell survival regulation and macrophage

differentiation, Toxicol Appl Pharmacol 273(1) (2013) 196-208.

T
[19] D. Tavares-Valente, F. Baltazar, R. Moreira, O. Queiros, Cancer cell bioenergetics

IP
and pH regulation influence breast cancer cell resistance to paclitaxel and doxorubicin,

J Bioenerg Biomembr 45(5) (2013) 467-75.

R
[20] M.A. Pierotti, F. Berrino, M. Gariboldi, C. Melani, A. Mogavero, T. Negri, P.

SC
Pasanisi, S. Pilotti, Targeting metabolism for cancer treatment and prevention:

metformin, an old drug with multi-faceted effects, Oncogene 32(12) (2013) 1475-87.
U
[21] C. Xue, C. Wang, Y. Sun, Q. Meng, Z. Liu, X. Huo, P. Sun, H. Sun, X. Ma, X. Ma,
N
J. Peng, K. Liu, Targeting P-glycoprotein function, p53 and energy metabolism:
A
Combination of metformin and 2-deoxyglucose reverses the multidrug resistance of
M

MCF-7/Dox cells to doxorubicin, Oncotarget 8(5) (2017) 8622-8632.

[22] C. Xue, C. Wang, Q. Liu, Q. Meng, H. Sun, X. Huo, X. Ma, Z. Liu, X. Ma, J. Peng,
D

K. Liu, Targeting P-glycoprotein expression and cancer cell energy metabolism:

TE

combination of metformin and 2-deoxyglucose reverses the multidrug resistance of

K562/Dox cells to doxorubicin, Tumour Biol 37(7) (2016) 8587-97.

EP

[23] J.G. Kim, S.J. Lee, Y.S. Chae, B.W. Kang, Y.J. Lee, S.Y. Oh, M.C. Kim, K.H. Kim,

S.J. Kim, Association between phosphorylated AMP-activated protein kinase and

CC

MAPK3/1 expression and prognosis for patients with gastric cancer, Oncology 85(2)

(2013) 78-85.
A

[24] Z. Wang, P. Liu, Q. Chen, S. Deng, X. Liu, H. Situ, S. Zhong, S. Hann, Y. Lin,

Targeting AMPK Signaling Pathway to Overcome Drug Resistance for Cancer Therapy,

Curr Drug Targets 17(8) (2016) 853-64.

[25] D.G. Hardie, AMPK--sensing energy while talking to other signaling pathways,

36
Cell Metab 20(6) (2014) 939-52.

[26] D.G. Hardie, B.E. Schaffer, A. Brunet, AMPK: An Energy-Sensing Pathway with

Multiple Inputs and Outputs, Trends Cell Biol 26(3) (2016) 190-201.

[27] S.P. Mathupala, Y.H. Ko, P.L. Pedersen, Hexokinase-2 bound to mitochondria:

cancer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy,

Semin Cancer Biol 19(1) (2009) 17-24.

T
[28] S.P. Mathupala, A. Rempel, P.L. Pedersen, Aberrant glycolytic metabolism of

IP
cancer cells: a remarkable coordination of genetic, transcriptional, post-translational,

and mutational events that lead to a critical role for type II hexokinase, J Bioenerg

R
Biomembr 29(4) (1997) 339-43.

SC
[29] R.G. Coelho, I.C. Calaca, D.M. Celestrini, A.H. Correia-Carneiro, M.M. Costa, P.

Zancan, M. Sola-Penna, Hexokinase and phosphofructokinase activity and intracellular

U
distribution correlate with aggressiveness and invasiveness of human breast carcinoma,
N
Oncotarget 6(30) (2015) 29375-87.
A
[30] A. Wolf, S. Agnihotri, J. Micallef, J. Mukherjee, N. Sabha, R. Cairns, C. Hawkins,
M

A. Guha, Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor

growth in human glioblastoma multiforme, J Exp Med 208(2) (2011) 313-26.

D

[31] W. Yi, P.M. Clark, D.E. Mason, M.C. Keenan, C. Hill, W.A. Goddard, 3rd, E.C.
TE

Peters, E.M. Driggers, L.C. Hsieh-Wilson, Phosphofructokinase 1 glycosylation

regulates cell growth and metabolism, Science 337(6097) (2012) 975-80.

EP

[32] N.H. Kim, Y.H. Cha, J. Lee, S.H. Lee, J.H. Yang, J.S. Yun, E.S. Cho, X. Zhang,

M. Nam, N. Kim, Y.S. Yuk, S.Y. Cha, Y. Lee, J.K. Ryu, S. Park, J.H. Cheong, S.W.
CC

Kang, S.Y. Kim, G.S. Hwang, J.I. Yook, H.S. Kim, Snail reprograms glucose

metabolism by repressing phosphofructokinase PFKP allowing cancer cell survival

A

under metabolic stress, Nat Commun 8 (2017) 14374.

[33] B.A. Webb, F. Forouhar, F.E. Szu, J. Seetharaman, L. Tong, D.L. Barber, Structures

of human phosphofructokinase-1 and atomic basis of cancer-associated mutations,

Nature 523(7558) (2015) 111-4.

37
[34] E. Domenech, C. Maestre, L. Esteban-Martinez, D. Partida, R. Pascual, G.

Fernandez-Miranda, E. Seco, R. Campos-Olivas, M. Perez, D. Megias, K. Allen, M.

Lopez, A.K. Saha, G. Velasco, E. Rial, R. Mendez, P. Boya, M. Salazar-Roa, M.

Malumbres, AMPK and PFKFB3 mediate glycolysis and survival in response to

mitophagy during mitotic arrest, Nat Cell Biol 17(10) (2015) 1304-16.

[35] S. Ros, C.R. Santos, S. Moco, F. Baenke, G. Kelly, M. Howell, N. Zamboni, A.

T
Schulze, Functional metabolic screen identifies 6-phosphofructo-2-kinase/fructose-

IP
2,6-biphosphatase 4 as an important regulator of prostate cancer cell survival, Cancer

Discov 2(4) (2012) 328-43.

R
[36] A. Silvestri, F. Palumbo, I. Rasi, D. Posca, T. Pavlidou, S. Paoluzi, L. Castagnoli,

SC
G. Cesareni, Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in

Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions, PLoS One 10(8)

(2015) e0136250. U
N
[37] D. Dixit, F. Ahmad, R. Ghildiyal, S.D. Joshi, E. Sen, CK2 inhibition induced
A
PDK4-AMPK axis regulates metabolic adaptation and survival responses in glioma,
M

Exp Cell Res 344(1) (2016) 132-42.

[38] C.A. Wu, Y. Chao, S.G. Shiah, W.W. Lin, Nutrient deprivation induces the Warburg
D

effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase,

TE

Biochim Biophys Acta 1833(5) (2013) 1147-56.

[39] Z. Liu, X. Chen, Y. Wang, H. Peng, Y. Wang, Y. Jing, H. Zhang, PDK4 protein
EP

promotes tumorigenesis through activation of cAMP-response element-binding protein

(CREB)-Ras homolog enriched in brain (RHEB)-mTORC1 signaling cascade, J Biol

CC

Chem 289(43) (2014) 29739-49.

[40] X. Huang, X. Li, X. Xie, F. Ye, B. Chen, C. Song, H. Tang, X. Xie, High
A

expressions of LDHA and AMPK as prognostic biomarkers for breast cancer, Breast 30

(2016) 39-46.

[41] K. Nam, S. Oh, I. Shin, Ablation of CD44 induces glycolysis-to-oxidative

phosphorylation transition via modulation of the c-Src-Akt-LKB1-AMPKalpha

38
pathway, Biochem J 473(19) (2016) 3013-30.

[42] A.B. Kohan, I. Talukdar, C.M. Walsh, L.M. Salati, A role for AMPK in the

inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids,

Biochem Biophys Res Commun 388(1) (2009) 117-21.

[43] R. Lin, S. Elf, C. Shan, H.B. Kang, Q. Ji, L. Zhou, T. Hitosugi, L. Zhang, S. Zhang,

J.H. Seo, J. Xie, M. Tucker, T.L. Gu, J. Sudderth, L. Jiang, M. Mitsche, R.J.

T
DeBerardinis, S. Wu, Y. Li, H. Mao, P.R. Chen, D. Wang, G.Z. Chen, S.J. Hurwitz, S.

IP
Lonial, M.L. Arellano, H.J. Khoury, F.R. Khuri, B.H. Lee, Q. Lei, D.J. Brat, K. Ye, T.J.

Boggon, C. He, S. Kang, J. Fan, J. Chen, 6-Phosphogluconate dehydrogenase links

R
oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling,

SC
Nat Cell Biol 17(11) (2015) 1484-96.

[44] S.M. Jeon, N.S. Chandel, N. Hay, AMPK regulates NADPH homeostasis to
U
promote tumour cell survival during energy stress, Nature 485(7400) (2012) 661-5.
N
[45] Y.Z. Lee, C.W. Yang, H.Y. Chang, H.Y. Hsu, I.S. Chen, H.S. Chang, C.H. Lee, J.C.
A
Lee, C.R. Kumar, Y.Q. Qiu, Y.S. Chao, S.J. Lee, Discovery of selective inhibitors of
M

Glutaminase-2, which inhibit mTORC1, activate autophagy and inhibit proliferation in

cancer cells, Oncotarget 5(15) (2014) 6087-101.

D

[46] L. Chen, T. Liu, S. Zhang, J. Zhou, Y. Wang, W. Di, Succinate dehydrogenase

TE

subunit B inhibits the AMPK-HIF-1alpha pathway in human ovarian cancer in vitro, J

Ovarian Res 7 (2014) 115.

EP

[47] C. Bardella, M. Olivero, A. Lorenzato, M. Geuna, J. Adam, L. O'Flaherty, P.

Rustin, I. Tomlinson, P.J. Pollard, M.F. Di Renzo, Cells lacking the fumarase tumor
CC

suppressor are protected from apoptosis through a hypoxia-inducible factor-

independent, AMPK-dependent mechanism, Mol Cell Biol 32(15) (2012) 3081-94.

A

[48] R. Naik, M. Won, H.S. Ban, D. Bhattarai, X. Xu, Y. Eo, Y.S. Hong, S. Singh, Y.

Choi, H.C. Ahn, K. Lee, Synthesis and structure-activity relationship study of chemical

probes as hypoxia induced factor-1alpha/malate dehydrogenase 2 inhibitors, J Med

Chem 57(22) (2014) 9522-38.

39
[49] H.S. Ban, X. Xu, K. Jang, I. Kim, B.K. Kim, K. Lee, M. Won, A Novel Malate

Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating

Mitochondrial Respiration, PLoS One 11(9) (2016) e0162568.

[50] S. Shah, W.J. Carriveau, J. Li, S.L. Campbell, P.K. Kopinski, H.W. Lim, N. Daurio,

S. Trefely, K.J. Won, D.C. Wallace, C. Koumenis, A. Mancuso, K.E. Wellen, Targeting

ACLY sensitizes castration-resistant prostate cancer cells to AR antagonism by

T
impinging on an ACLY-AMPK-AR feedback mechanism, Oncotarget 7(28) (2016)

IP
43713-43730.

[51] T. Migita, S. Okabe, K. Ikeda, S. Igarashi, S. Sugawara, A. Tomida, R. Taguchi, T.

R
Soga, H. Seimiya, Inhibition of ATP citrate lyase induces an anticancer effect via

SC
reactive oxygen species: AMPK as a predictive biomarker for therapeutic impact, Am

J Pathol 182(5) (2013) 1800-10.

U
[52] J.H. Lee, H. Jang, S.M. Lee, J.E. Lee, J. Choi, T.W. Kim, E.J. Cho, H.D. Youn,
N
ATP-citrate lyase regulates cellular senescence via an AMPK- and p53-dependent
A
pathway, FEBS J 282(2) (2015) 361-71.
M

[53] N. Scaglia, J.W. Chisholm, R.A. Igal, Inhibition of stearoylCoA desaturase-1

inactivates acetyl-CoA carboxylase and impairs proliferation in cancer cells: role of

D

AMPK, PLoS One 4(8) (2009) e6812.

TE

[54] C.J. Halbrook, C.A. Lyssiotis, Employing Metabolism to Improve the Diagnosis

and Treatment of Pancreatic Cancer, Cancer Cell 31(1) (2017) 5-19.

EP

[55] T.T. Hien, H.G. Kim, E.H. Han, K.W. Kang, H.G. Jeong, Molecular mechanism of

suppression of MDR1 by puerarin from Pueraria lobata via NF-kappaB pathway and
CC

cAMP-responsive element transcriptional activity-dependent up-regulation of AMP-

activated protein kinase in breast cancer MCF-7/adr cells, Mol Nutr Food Res 54(7)
A

(2010) 918-28.

[56] Z.F. Zhong, W. Tan, W.W. Qiang, V.L. Scofield, K. Tian, C.M. Wang, W.A. Qiang,

Y.T. Wang, Furanodiene alters mitochondrial function in doxorubicin-resistant MCF-7

human breast cancer cells in an AMPK-dependent manner, Mol Biosyst 12(5) (2016)

40
1626-37.

[57] L. Wang, J. Wang, H. Xiong, F. Wu, T. Lan, Y. Zhang, X. Guo, H. Wang, M.

Saleem, C. Jiang, J. Lu, Y. Deng, Co-targeting hexokinase 2-mediated Warburg effect

and ULK1-dependent autophagy suppresses tumor growth of PTEN- and TP53-

deficiency-driven castration-resistant prostate cancer, EBioMedicine 7 (2016) 50-61.

[58] N. Ishijima, K. Kanki, H. Shimizu, G. Shiota, Activation of AMP-activated protein

T
kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced

IP
by sorafenib, Cancer Sci 106(5) (2015) 567-75.

[59] C.H. Chang, C.Y. Lee, C.C. Lu, F.J. Tsai, Y.M. Hsu, J.W. Tsao, Y.N. Juan, H.Y.

R
Chiu, J.S. Yang, C.C. Wang, Resveratrol-induced autophagy and apoptosis in cisplatin-

SC
resistant human oral cancer CAR cells: A key role of AMPK and Akt/mTOR signaling,

Int J Oncol 50(3) (2017) 873-882.

U
[60] Z. Wang, L. Zhang, Z. Ni, J. Sun, H. Gao, Z. Cheng, J. Xu, P. Yin, Resveratrol
N
induces AMPK-dependent MDR1 inhibition in colorectal cancer HCT116/L-OHP cells
A
by preventing activation of NF-kappaB signaling and suppressing cAMP-responsive
M

element transcriptional activity, Tumour Biol 36(12) (2015) 9499-510.

[61] Y. Wu, Y. Qi, H. Liu, X. Wang, H. Zhu, Z. Wang, AMPK activator AICAR
D

promotes 5-FU-induced apoptosis in gastric cancer cells, Mol Cell Biochem 411(1-2)
TE

(2016) 299-305.

[62] S. Ling, Y. Tian, H. Zhang, K. Jia, T. Feng, D. Sun, Z. Gao, F. Xu, Z. Hou, Y. Li,
EP

L. Wang, Metformin reverses multidrug resistance in human hepatocellular carcinoma

Bel7402/5fluorouracil cells, Mol Med Rep 10(6) (2014) 2891-7.

CC

[63] C. Qu, W. Zhang, G. Zheng, Z. Zhang, J. Yin, Z. He, Metformin reverses multidrug

resistance and epithelial-mesenchymal transition (EMT) via activating AMP-activated

A

protein kinase (AMPK) in human breast cancer cells, Mol Cell Biochem 386(1-2)

(2014) 63-71.

[64] H.G. Kim, T.T. Hien, E.H. Han, Y.P. Hwang, J.H. Choi, K.W. Kang, K.I. Kwon,

B.H. Kim, S.K. Kim, G.Y. Song, T.C. Jeong, H.G. Jeong, Metformin inhibits P-

41
glycoprotein expression via the NF-kappaB pathway and CRE transcriptional activity

through AMPK activation, Br J Pharmacol 162(5) (2011) 1096-108.

[65] C. Oliveras-Ferraros, A. Vazquez-Martin, E. Cuyas, B. Corominas-Faja, E.

Rodriguez-Gallego, S. Fernandez-Arroyo, B. Martin-Castillo, J. Joven, J.A. Menendez,

Acquired resistance to metformin in breast cancer cells triggers transcriptome

reprogramming toward a degradome-related metastatic stem-like profile, Cell Cycle

T
13(7) (2014) 1132-44.

IP
[66] M.B. Aldonza, J.Y. Hong, S.Y. Bae, J. Song, W.K. Kim, J. Oh, Y. Shin, S.H. Lee,

S.K. Lee, Suppression of MAPK Signaling and Reversal of mTOR-Dependent MDR1-

R
Associated Multidrug Resistance by 21alpha-Methylmelianodiol in Lung Cancer Cells,

SC
PLoS One 10(6) (2015) e0127841.

[67] C. Ji, B. Yang, Y.L. Yang, S.H. He, D.S. Miao, L. He, Z.G. Bi, Exogenous cell-
U
permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced
N
apoptosis by promoting AMPK activation and mTORC1 inhibition, Oncogene 29(50)
A
(2010) 6557-68.
M

[68] S. Jaganathan, E. Malek, S. Vallabhapurapu, S. Vallabhapurapu, J.J. Driscoll,

Bortezomib induces AMPK-dependent autophagosome formation uncoupled from

D

apoptosis in drug resistant cells, Oncotarget 5(23) (2014) 12358-70.

TE

[69] D. Basu, R. Lettan, K. Damodaran, S. Strellec, M. Reyes-Mugica, A. Rebbaa,

Identification, mechanism of action, and antitumor activity of a small molecule

EP

inhibitor of hippo, TGF-beta, and Wnt signaling pathways, Mol Cancer Ther 13(6)

(2014) 1457-67.
CC

[70] W.D. Wu, Z.M. Hu, M.J. Shang, D.J. Zhao, C.W. Zhang, D.F. Hong, D.S. Huang,

Cordycepin down-regulates multiple drug resistant (MDR)/HIF-1alpha through

A

regulating AMPK/mTORC1 signaling in GBC-SD gallbladder cancer cells, Int J Mol

Sci 15(7) (2014) 12778-90.

[71] T.P. Tran, H.G. Kim, J.H. Choi, M.K. Na, H.G. Jeong, Reversal of P-glycoprotein-

mediated multidrug resistance is induced by mollugin in MCF-7/adriamycin cells,

42
Phytomedicine 20(7) (2013) 622-31.

[72] K.H. Eum, S.K. Ahn, H. Kang, M. Lee, Differential inhibitory effects of two Raf-

targeting drugs, sorafenib and PLX4720, on the growth of multidrug-resistant cells,

Mol Cell Biochem 372(1-2) (2013) 65-74.

[73] J.H. Ahn, M. Lee, Suppression of autophagy sensitizes multidrug resistant cells

towards Src tyrosine kinase specific inhibitor PP2, Cancer Lett 310(2) (2011) 188-97.

T
[74] Q. Zhang, Y. Feng, D. Kennedy, Multidrug-resistant cancer cells and cancer stem

IP
cells hijack cellular systems to circumvent systemic therapies, can natural products

reverse this?, Cell Mol Life Sci 74(5) (2017) 777-801.

R
[75] D. Uribe, A. Torres, J.D. Rocha, I. Niechi, C. Oyarzun, L. Sobrevia, R. San Martin,

SC
C. Quezada, Multidrug resistance in glioblastoma stem-like cells: Role of the hypoxic

microenvironment and adenosine signaling, Mol Aspects Med 55 (2017) 140-151.

U
[76] P. Mallini, T. Lennard, J. Kirby, A. Meeson, Epithelial-to-mesenchymal transition:
N
what is the impact on breast cancer stem cells and drug resistance, Cancer Treat Rev
A
40(3) (2014) 341-8.
M

[77] A. Schulze, A.L. Harris, How cancer metabolism is tuned for proliferation and

vulnerable to disruption, Nature 491(7424) (2012) 364-73.

D

[78] L.K. Boroughs, R.J. DeBerardinis, Metabolic pathways promoting cancer cell
TE

survival and growth, Nat Cell Biol 17(4) (2015) 351-9.

[79] D. Szklarczyk, A. Franceschini, S. Wyder, K. Forslund, D. Heller, J. Huerta-Cepas,

EP

M. Simonovic, A. Roth, A. Santos, K.P. Tsafou, M. Kuhn, P. Bork, L.J. Jensen, C. von

Mering, STRING v10: protein-protein interaction networks, integrated over the tree of
CC

life, Nucleic Acids Res 43(Database issue) (2015) D447-52.

[80] D. Szklarczyk, J.H. Morris, H. Cook, M. Kuhn, S. Wyder, M. Simonovic, A.

A

Santos, N.T. Doncheva, A. Roth, P. Bork, L.J. Jensen, C. von Mering, The STRING

database in 2017: quality-controlled protein-protein association networks, made

broadly accessible, Nucleic Acids Res 45(D1) (2017) D362-D368.

[81] A.J. Enright, S. Van Dongen, C.A. Ouzounis, An efficient algorithm for large-scale

43
detection of protein families, Nucleic Acids Res 30(7) (2002) 1575-84.

[82] P. Shannon, A. Markiel, O. Ozier, N.S. Baliga, J.T. Wang, D. Ramage, N. Amin,

B. Schwikowski, T. Ideker, Cytoscape: a software environment for integrated models

of biomolecular interaction networks, Genome Res 13(11) (2003) 2498-504.

[83] M. Talekar, Q. Ouyang, M.S. Goldberg, M.M. Amiji, Cosilencing of PKM-2 and

MDR-1 Sensitizes Multidrug-Resistant Ovarian Cancer Cells to Paclitaxel in a Murine

T
Model of Ovarian Cancer, Mol Cancer Ther 14(7) (2015) 1521-31.

IP
[84] J.R. Mayers, M.G. Vander Heiden, Famine versus feast: understanding the

metabolism of tumors in vivo, Trends Biochem Sci 40(3) (2015) 130-40.

R
[85] E. Cerami, J. Gao, U. Dogrusoz, B.E. Gross, S.O. Sumer, B.A. Aksoy, A. Jacobsen,

SC
C.J. Byrne, M.L. Heuer, E. Larsson, Y. Antipin, B. Reva, A.P. Goldberg, C. Sander, N.

Schultz, The cBio cancer genomics portal: an open platform for exploring
U
multidimensional cancer genomics data, Cancer Discov 2(5) (2012) 401-4.
N
[86] J. Gao, B.A. Aksoy, U. Dogrusoz, G. Dresdner, B. Gross, S.O. Sumer, Y. Sun, A.
A
Jacobsen, R. Sinha, E. Larsson, E. Cerami, C. Sander, N. Schultz, Integrative analysis
M

of complex cancer genomics and clinical profiles using the cBioPortal, Sci Signal

6(269) (2013) pl1.

D

[87] B.E. Johnson, T. Mazor, C. Hong, M. Barnes, K. Aihara, C.Y. McLean, S.D. Fouse,
TE

S. Yamamoto, H. Ueda, K. Tatsuno, S. Asthana, L.E. Jalbert, S.J. Nelson, A.W. Bollen,

W.C. Gustafson, E. Charron, W.A. Weiss, I.V. Smirnov, J.S. Song, A.B. Olshen, S. Cha,
EP

Y. Zhao, R.A. Moore, A.J. Mungall, S.J.M. Jones, M. Hirst, M.A. Marra, N. Saito, H.

Aburatani, A. Mukasa, M.S. Berger, S.M. Chang, B.S. Taylor, J.F. Costello, Mutational
CC

analysis reveals the origin and therapy-driven evolution of recurrent glioma, Science

343(6167) (2014) 189-193.

A

[88] P. Eirew, A. Steif, J. Khattra, G. Ha, D. Yap, H. Farahani, K. Gelmon, S. Chia, C.

Mar, A. Wan, E. Laks, J. Biele, K. Shumansky, J. Rosner, A. McPherson, C. Nielsen,

A.J. Roth, C. Lefebvre, A. Bashashati, C. de Souza, C. Siu, R. Aniba, J. Brimhall, A.

Oloumi, T. Osako, A. Bruna, J.L. Sandoval, T. Algara, W. Greenwood, K. Leung, H.

44
Cheng, H. Xue, Y. Wang, D. Lin, A.J. Mungall, R. Moore, Y. Zhao, J. Lorette, L.

Nguyen, D. Huntsman, C.J. Eaves, C. Hansen, M.A. Marra, C. Caldas, S.P. Shah, S.

Aparicio, Dynamics of genomic clones in breast cancer patient xenografts at single-cell

resolution, Nature 518(7539) (2015) 422-6.

[89] W. Hugo, J.M. Zaretsky, L. Sun, C. Song, B.H. Moreno, S. Hu-Lieskovan, B.

Berent-Maoz, J. Pang, B. Chmielowski, G. Cherry, E. Seja, S. Lomeli, X. Kong, M.C.

T
Kelley, J.A. Sosman, D.B. Johnson, A. Ribas, R.S. Lo, Genomic and Transcriptomic

IP
Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma, Cell 165(1)

(2016) 35-44.

R
[90] A.L. Cohen, S.L. Holmen, H. Colman, IDH1 and IDH2 mutations in gliomas, Curr

SC
Neurol Neurosci Rep 13(5) (2013) 345.

[91] L.E. Jalbert, A. Elkhaled, J.J. Phillips, E. Neill, A. Williams, J.C. Crane, M.P.
U
Olson, A.M. Molinaro, M.S. Berger, J. Kurhanewicz, S.M. Ronen, S.M. Chang, S.J.
N
Nelson, Metabolic Profiling of IDH Mutation and Malignant Progression in Infiltrating
A
Glioma, Sci Rep 7 (2017) 44792.
M

[92] H. Yan, D.W. Parsons, G. Jin, R. McLendon, B.A. Rasheed, W. Yuan, I. Kos, I.

Batinic-Haberle, S. Jones, G.J. Riggins, H. Friedman, A. Friedman, D. Reardon, J.

D

Herndon, K.W. Kinzler, V.E. Velculescu, B. Vogelstein, D.D. Bigner, IDH1 and IDH2
TE

mutations in gliomas, N Engl J Med 360(8) (2009) 765-73.

[93] M.S. Waitkus, B.H. Diplas, H. Yan, Biological Role and Therapeutic Potential of
EP

IDH Mutations in Cancer, Cancer Cell (2018).

[94] B.M. Lindqvist, S. Wingren, P.B. Motlagh, T.K. Nilsson, Whole genome DNA
CC

methylation signature of HER2-positive breast cancer, Epigenetics 9(8) (2014) 1149-

62.
A

[95] X. Ge, Z. Cao, Y. Gu, F. Wang, J. Li, M. Han, W. Xia, Z. Yu, P. Lyu, PFKFB3

potentially contributes to paclitaxel resistance in breast cancer cells through TLR4

activation by stimulating lactate production, Cell Mol Biol (Noisy-le-grand) 62(6)

(2016) 119-25.

45
[96] M.J. Kim, D.H. Kim, W.H. Jung, J.S. Koo, Expression of metabolism-related

proteins in triple-negative breast cancer, Int J Clin Exp Pathol 7(1) (2014) 301-12.

[97] A.T. Qattan, M. Radulovic, M. Crawford, J. Godovac-Zimmermann, Spatial

distribution of cellular function: the partitioning of proteins between mitochondria and

the nucleus in MCF7 breast cancer cells, J Proteome Res 11(12) (2012) 6080-101.

[98] T. Contractor, C.R. Harris, p53 negatively regulates transcription of the pyruvate

T
dehydrogenase kinase Pdk2, Cancer Res 72(2) (2012) 560-7.

IP
[99] E. Papaevangelou, G.S. Almeida, C. Box, N.M. deSouza, Y.L. Chung, The effect

of FASN inhibition on the growth and metabolism of a cisplatin-resistant ovarian

R
carcinoma model, Int J Cancer 143(4) (2018) 992-1002.

SC
[100] S. Al-Bahlani, H. Al-Lawati, M. Al-Adawi, N. Al-Abri, B. Al-Dhahli, K. Al-

Adawi, Fatty acid synthase regulates the chemosensitivity of breast cancer cells to
U
cisplatin-induced apoptosis, Apoptosis 22(6) (2017) 865-876.
N
[101] J.A. Menendez, R. Lupu, Fatty acid synthase (FASN) as a therapeutic target in
A
breast cancer, Expert Opin Ther Targets 21(11) (2017) 1001-1016.
M

[102] H. Liu, X. Huang, T. Ye, MiR-22 down-regulates the proto-oncogene ATP citrate

lyase to inhibit the growth and metastasis of breast cancer, Am J Transl Res 10(3)
D

(2018) 659-669.
TE

[103] K.S. Lucenay, I. Doostan, C. Karakas, T. Bui, Z. Ding, G.B. Mills, K.K. Hunt, K.

Keyomarsi, Cyclin E Associates with the Lipogenic Enzyme ATP-Citrate Lyase to

EP

Enable Malignant Growth of Breast Cancer Cells, Cancer Res 76(8) (2016) 2406-18.

[104] B. Corominas-Faja, E. Cuyas, J. Gumuzio, J. Bosch-Barrera, O. Leis, A.G.

CC

Martin, J.A. Menendez, Chemical inhibition of acetyl-CoA carboxylase suppresses

self-renewal growth of cancer stem cells, Oncotarget 5(18) (2014) 8306-16.

A

[105] M. Hilvo, C. Denkert, L. Lehtinen, B. Muller, S. Brockmoller, T. Seppanen-

Laakso, J. Budczies, E. Bucher, L. Yetukuri, S. Castillo, E. Berg, H. Nygren, M. Sysi-

Aho, J.L. Griffin, O. Fiehn, S. Loibl, C. Richter-Ehrenstein, C. Radke, T. Hyotylainen,

O. Kallioniemi, K. Iljin, M. Oresic, Novel theranostic opportunities offered by

46
characterization of altered membrane lipid metabolism in breast cancer progression,

Cancer Res 71(9) (2011) 3236-45.

[106] M. van Geldermalsen, Q. Wang, R. Nagarajah, A.D. Marshall, A. Thoeng, D.

Gao, W. Ritchie, Y. Feng, C.G. Bailey, N. Deng, K. Harvey, J.M. Beith, C.I. Selinger,

S.A. O'Toole, J.E. Rasko, J. Holst, ASCT2/SLC1A5 controls glutamine uptake and

tumour growth in triple-negative basal-like breast cancer, Oncogene 35(24) (2016)

T
3201-8.

IP
[107] Y. Wang, S. Fan, J. Lu, Z. Zhang, D. Wu, Z. Wu, Y. Zheng, GLUL Promotes Cell

Proliferation in Breast Cancer, J Cell Biochem 118(8) (2017) 2018-2025.

R
[108] P. Malvi, B. Chaube, V. Pandey, M.V. Vijayakumar, P.R. Boreddy, N. Mohammad,

SC
S.V. Singh, M.K. Bhat, Obesity induced rapid melanoma progression is reversed by

orlistat treatment and dietary intervention: role of adipokines, Mol Oncol 9(3) (2015)

689-703. U
N
[109] V. Pandey, M.V. Vijayakumar, A.K. Ajay, P. Malvi, M.K. Bhat, Diet-induced
A
obesity increases melanoma progression: involvement of Cav-1 and FASN, Int J Cancer
M

130(3) (2012) 497-508.

[110] J. Wu, J. Du, X. Fu, B. Liu, H. Cao, T. Li, T. Su, J. Xu, A.K. Tse, Z.L. Yu, Iciartin,
D

a novel FASN inhibitor, exerts anti-melanoma activities through IGF-1R/STAT3

TE

signaling, Oncotarget 7(32) (2016) 51251-51269.

[111] F. Vazquez, J.H. Lim, H. Chim, K. Bhalla, G. Girnun, K. Pierce, C.B. Clish, S.R.
EP

Granter, H.R. Widlund, B.M. Spiegelman, P. Puigserver, PGC1alpha expression defines

a subset of human melanoma tumors with increased mitochondrial capacity and

CC

resistance to oxidative stress, Cancer Cell 23(3) (2013) 287-301.

[112] V.N. Sumantran, P. Mishra, N. Sudhakar, Microarray analysis of differentially

A

expressed genes regulating lipid metabolism during melanoma progression, Indian J

Biochem Biophys 52(2) (2015) 125-31.

[113] Y. Zhao, E.B. Butler, M. Tan, Targeting cellular metabolism to improve cancer

therapeutics, Cell Death Dis 4 (2013) e532.

47
[114] W. Tan, Z. Zhong, S. Wang, H. Liu, H. Yu, R. Tan, X. Hu, T. Pan, Y. Wang, The

Typical Metabolic Modifiers Conferring Improvement in Cancer Resistance, Curr Med

Chem 24(34) (2017) 3698-3710.

T
R IP
SC
U
N
A
M
D
TE
EP
CC
A

48
Figure Captions

Figure 1 The specific interactions of AMPK in each identified metabolic process

The specific interactions of AMPK (represented as a grouped circle comprised of seven key

proteins) in each identified metabolic process, including glycolysis (A), OXPHOS related

T
process (B), fatty acid synthesis related process (C) and glutamine related process (D). Nodes

IP
represent proteins and edges (the lines between two nodes) represent protein-proteins

R
associations. The size of the nodes indicates the level of functional associations between

SC
proteins according to number of database annotations. Node color was derived from co-

expression data. Edge color represents the variance of interactions parsed across primary

databases during automated text mining data.

U
N
Figure 2 The core proteins directly interacted with AMPK in each metabolic process
A
The core proteins directly interacted with AMPK (including PRKAA1, PRKAA2, PRKAB1,
M

PRKAB2, PRKAG1, PRKAG2, and PRKAG3) were grouped by heat maps with the

computed text-mining scores, elucidating these closest interactions between AMPK and
D

glycolysis related proteins (A), OXPHOS related proteins (B), fatty acid synthesis related
TE

proteins (C) and glutamine related proteins (D).

EP

Figure 3 The central role of AMPK and the complex relationships between these four

metabolic processes
CC

These directly linked proteins comprise a heterogeneous group with 22 members indicate a

central role of AMPK in energy metabolism network (A), and a Venn diagram visualization
A

of the complex relationships between these four groups, with the proportion of shared

proteins amongst the groups represented as percentage values (B).

Figure 4 The intersection between AMPK-related energy metabolism network and

metabolic adaption in resistant cancer cells

49
The key proteins (165 proteins) derived from AMPK-based network through data mining and

the altered proteins (137 proteins) in metabolic adaptation are compared. 28 proteins were

found at this intersection, accounting for 10.2% of the total. These identified core proteins

with dual roles in AMPK-mediated metabolic regulation in MDR.

Figure 5 Gene networks analysis of 28 genes responsible for encoding the core proteins

extracted from the AMPK-mediated metabolic pathway in MDR

T
(A) Greater than 30% of altered genes in these core 28 genes is criteria, hence 57

IP
studies were found according to the cancer genome atlas (TCGA) provisional

R
data. With quite remarkable significance across 166 various studies, a high

SC
alteration frequency (>80.0%) ranked three cancer types: glioma (100%), breast

cancer (96.6%) and melanoma (80.6%). For each caner type, genetic alterations

U
for each gene involved in AMPK-mediated metabolic pathways of MDR, which
N
were found in at least three studies, are visualized according to their percent
A
alteration frequency, including glioma (B), breast cancer (C) and melanoma (D).
M
D
TE
EP
CC
A

50
 A

Fig 1B
Fig 1 A

CC
EP
TE
D
M
A
N
U
SC
R

51
IP
T
 A Fig 1 C

Fig 1 D
CC
EP
TE
D
M
A
N
U
SC
R

52
IP
T
 A

Fig 2B
Fig 2 A

CC
EP
TE
D
M
A
N
U
SC
R

53
IP
T
 A Fig 2 C

Fig 1 D
CC
EP
TE
D
M
A
N
U
SC
R

54
IP
T
 A

Fig 3B
Fig 3 A

CC
EP
TE
D
M
A
N
U
SC
R

55
IP
T
 A
CC
EP
TE
D
M
A
N
U
SC
R

56
IP
T
 A Fig 4

CC
EP
TE
D
M
A
N
U
SC
R

57
IP
T
 A

Fig 5 B
Fig 5 A

CC
EP
TE
D
M
A
N
U
SC
R

58
IP
T
 A

Fig D
Fig 5C

CC
EP
TE
D
M
A
N
U
SC
R

59
IP
T