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Published in final edited form as:

Gynecol Oncol. 2014 April ; 133(1): 9097. doi:10.1016/j.ygyno.2013.12.026.

Obesity increases tumor aggressiveness in a genetically

engineered mouse model of serous ovarian cancer
Liza Makowskia,b,1, Chunxiao Zhouc, Yan Zhongc, Pei Fen Kuanb,d, Cheng Fanb, Brante P.
Sampeye, Megan Difuriof, and Victoria L. Bae-Jumpb,c,*,2
aDepartment
bLineberger
cDivision

of Nutrition, University of North Carolina, Chapel Hill, NC, USA

Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA

of Gynecologic Oncology, University of North Carolina, Chapel Hill, NC, USA

dDepartment

of Biostatistics, University of North Carolina, Chapel Hill, NC, USA

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eMetabolon,

Inc., Durham, NC, USA

fDepartment

of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC,

USA

Abstract
ObjectivesObesity is associated with increased risk and worse outcomes for ovarian cancer.
Thus, we examined the effects of obesity on ovarian cancer progression in a genetically
engineered mouse model of serous ovarian cancer.

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MethodsWe utilized a unique serous ovarian cancer mouse model that specifically deletes the
tumor suppressor genes, Brca1 and p53, and inactivates the retinoblastoma (Rb) proteins in adult
ovarian surface epithelial cells, via injection of an adenoviral vector expressing Cre (AdCre) into
the ovarian bursa cavity of adult female mice (KpB mouse model). KpB mice were subjected to a
60% calories-derived from fat in a high fat diet (HFD) versus 10% calories from fat in a low fat
diet (LFD) to mimic diet-induced obesity. Tumors were isolated at 6 months after AdCre injection
and evaluated histologically. Untargeted metabolomic and gene expression profiling was
performed to assess differences in the ovarian tumors from obese versus non-obese KpB mice.
ResultsAt sacrifice, mice on the HFD (obese) were twice the weight of mice on the LFD (nonobese) (51 g versus 31 g, p = 0.0003). Ovarian tumors were significantly larger in the obese versus
non-obese mice (3.7 cm2 versus 1.2 cm2, p = 0.0065). Gene expression and metabolomic profiling
Presented as an oral presentation at the 2013 Annual Meeting of the Society of Gynecologic Oncology in Los Angeles, CA.
2013 Elsevier Inc. All rights reserved.
*
Corresponding author at: University of North Carolina (UNC), Division of Gynecologic Oncology, CB# 7572, Physicians Office
Building Rm# B105, Chapel Hill, NC 27599, USA. Fax: +1 919 843 5387. [email protected] (V.L. Bae-Jump).
1LM was supported by UNC University Cancer Research Fund, NIH AA017376; NIH ES019472; NIH P30DK056350 Nutrition
Obesity Research Consortium (NORC).
2VBJ was supported by Gynecologic Cancer Foundation/Florence & Marshall Schwid Ovarian Cancer Research Grant, The North
Carolina Translational and Clinical Sciences Institute/NC TraCS $50 K Pilot Grant Program, National Institutes of Health Grant
DK056350 to the UNC Nutrition Obesity Research Center, OC110163 Department of Defense/Ovarian Cancer Research Program
(DOD/OCRP) Translational Pilot Award.
Conflict of interest statement
The authors declare that there are no conflicts of interest.

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indicated statistically significant differences between the ovarian tumors from the obese versus
non-obese mice, including metabolically relevant pathways.

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Keywords
Obesity; Ovarian cancer; Mouse model; Metabolomics; Genomics; Biomarkers

Introduction
Obesity has been linked to increased risk of many cancers, including breast, colon,
endometrial, among others [1]. Currently, new cancer cases are in the order of 1.5 million
with half a million cancer deaths per year, and nearly one in five are due to obesity [1,2]. It
is postulated that hyperglycemia and hyperinsulinemia resulting from over-nutrition in obese
patients provide abundant nutrients and growth factors to cancer cells, resulting in the ideal
environment for tumor initiation and promotion [3]. Chronic inflammation and
immunosuppression are also thought to be a link between obesity and cancer [3].

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Epithelial ovarian cancer (OC) is one of the most deadly cancers with an overall 5-year
survival of only 3040%. Increasing evidence suggests that obesity is a significant risk
factor for OC and associated with worse outcomes for this disease [1,420]. Given the
overall poor prognosis of OC and the rising rate of obesity, it is imperative to investigate
obesity as a potential modifiable risk factor that may reverse risk and lead to the prevention
and improvement of outcomes for OC. We hypothesize that the metabolic consequences of
obesity may play a contributing role in the pathogenesis of OC and may lead to biologically
and phenotypically different cancers than those that arise in normal weight women, possibly
necessitating distinct treatment strategies. Herein, we assessed the impact of obesity on OC
development and progression in a genetically engineered mouse model of serous OC and
comprehensively interrogated the obesity-induced carcinogenesis signature through genomic
and metabolomic analysis.

Materials and methods

Obesity and the

;p53fl/fl;Brca1fl/fl mouse model

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The
;p53 fl/fl;Brca1fl/fl (KpB) mouse model (Terry Van Dyke, PhD, NIH) is a
unique serous OC mouse model, wherein the tumor suppressor genes, Brca1 and p53 are
specifically and somatically deleted and the retinoblastoma (Rb) proteins are inactivated in
the adult ovarian surface epithelium [21]. Inactivation of all 3 Rb proteins by T121 (a
fragment of the SV40 large T antigen) is driven by the keratin 18 (K18) promoter [21].
Expression of the T121 transgene and knockout of p53 and Brca1 are conditional and only
activated via injection of an adenoviral vector expressing Cre (AdCre) into the ovarian bursa
cavity of adult female mice. At approximately 6 months after AdCre injection, tumors
develop in the affected ovary, while the un-injected ovary remains normal.
All experimental animals were maintained in accordance with the Institutional Animal Care
and Use Committee (IACUC) and the NIH guide for the Care and Use of Laboratory
Animals. Recombinant adenovirus Ad5-CMV-Cre (AdCre) was purchased from the

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University of Iowa Transfer Vector Core at a titer of 10111012 infectious particles/ml. To

maximize weight gain, mice were provided a high-fat diet (HFD, obese group) (60% kcal
from fat, Research Diets, New Brunswick, NJ) and control mice (non-obese group) were
provided a low-fat diet (LFD) (10% kcal from fat, Research Diets, New Brunswick, NJ) ad
libitum, beginning at 6 weeks of age. AdCre injection occurred at 8 weeks to induce OC 6
months later (at 8 months of age) [21]. Thirty-six hours following superovulation, the mice
were anesthetized, and a single 1 cm incision was made on the dorsal surface of each mouse.
The AdCre was then injected via a needle introduced into the oviduct near the infundibulum
and into the ovarian bursa, and the incision was closed. All mice were sacrificed at 8 months
of age.
The primary outcome comparison between non-obese and obese mice was the response of
tumor growth to the obesity exposure. This was assessed via direct measurement of the
tumor at the time of sacrifice. At the time of sacrifice, the ovarian tumors were harvested,
wet tumor weights recorded, and tissue was snap frozen in liquid nitrogen for later harvest
of mRNA for microarray analysis and metabolites for metabolomic analysis.
Body weight & composition

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Prior to starting mice on diet and weekly until sacrifice, body weight was measured. Body
composition, including lean mass, fat mass, free water content and total water content, of
non-anesthetized mice was also measured at pre- and post-diet exposures using the
EchoMRI-100 quantitative magnetic resonance whole body composition analyzer (Echo
Medical Systems, Houston, TX).
Blood glucose
Random blood glucose was measured prior to start of diet and at sacrifice using a Bayer
Contour Blood Glucose Monitor (Bayer HealthCare LLC, Tarrytown, NY).
mRNA isolation

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Approximately 2550 mg of frozen OC tissue in small fragments was homogenized in RLT

lysis buffer. Total RNA was isolated using the RNeasy mini kit and QIAshredder kit
(Qiagen Inc., Mississauga, ON) following the manufacturers instructions. RNA quantity
and quality were analyzed by Nanodrop (Thermoscientific, Wilmington, DE).
Gene expression profiling
Microarrays were performed on ovarian tumors from non-obese and obese mice (N = 5/
group) using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. These samples were
processed in the Lineberger Comprehensive Cancer Center Genomics Core Facility. The
image files were analyzed with GenePix Pro 4.1 and pre-processed via the UNC-Chapel Hill
Microarray Database (https://genome.unc.edu) where a Lowess normalization procedure
was performed to adjust for Cy3 and Cy5 channel biases [22]. In addition, probes with
missing values in 3 or more samples in each of the obese and non-obese groups were
removed. Two-class SAM (Significance Analysis of Microarrays, http://wwwstat.stanford.edu/~tibs/SAM/) was performed to identify significantly differentially
expressed genes using FDR < 0.2. EASE (Expression Analysis Systematic Explorer, http://
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david.niaid.nih.gov/david/ease.htm) analysis was used to interpret and identify biological

themes (gene ontology categories) overrepresented in the gene list obtained from SAM
results. The EASE Score was used as statistical measure of overrepresentation of a
biological theme. Specifically, the EASE Score is a jackknifed one-tailed Fishers exact
probability which is calculated by removing one gene within the given category from the list
and penalizes the statistical significance of categories supported by fewer genes; thus is a
more robust measure than the Fishers exact probability [23].
Metabolomic profiling

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Gas chromatography time-of-flight mass spectrometry (GC-TOFMS, Leco Corporation, St

Joseph, MI) and liquid chromatography coupled with time-of-flight mass spectrometry (LCTOFMS, Agilent Corporation, Santa Clara, CA) were used to analyze tumors from nonobese and obese mice (N = 5/group). Metabolite extraction followed previous publication
with minor revision through the UNC/Nutrition Obesity Research Center (NORC) Core
facility [24]. Briefly, 50 mg samples were extracted with 0.5 ml of
methanol:chloroform:water = 3:1:1 (v:v:v) with homogenization for 3 min using 1-mm inner
diameter balls in a Bullet Blender (Next Advance, Averill Park, NY). Two aliquots of 150 l
of supernatant were used for GC-TOFMS and LC-TOFMS analysis, separately. After
removal of the extra supernatant, the remainder was extracted with 500 l of methanol. Two
aliquots of 150 l of supernatant were combined into the tube containing first step extraction
for GC and LC-TOFMS analysis, separately. Metabolite annotation was performed by
comparing the mass spectrum and retention time to an in-house library and NIST library
(GC-TOMFS) or HMDB (LC-TOFMS) [25,26].
Statistical methods

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Unpaired Students t-test was used to determine statistical difference between non-obese and
obese treatment groups using STATA software (College Station, TX). A p-value <0.05 was
considered significant. For metabolomics, after normalization to the internal standard and
sample weight, the data set was imported into SIMCA-p software (Ume, Sweden) for
multivariate analysis. Principle component analysis (PCA) was first performed to check the
outliers and the separation tendency (data not shown). A supervised orthogonal partial least
squares-discriminant analysis (OPLS-DA) analysis was then performed. Differentiating
metabolites were selected with the criteria of the variable importance in the projection (VIP)
value >1 and p value (Students t test) lower than 0.05.

Results
Obesity drove significant tumor progression in KpB mice
KpB mice were subjected to 60% calories-derived from fat in a high fat diet (HFD) versus
10% calories from fat in a low fat diet (LFD) to induce diet-induced obesity (N = 14/group)
starting at 6 weeks of age and until sacrifice. After 8 months of exposure to the HFD or
LFD, obese mice weighed significantly greater than non-obese mice (p = 0.003, Table 1).
There was no effect of HFD on non-fasted blood glucose levels in KpB mice over the course
of the diet (Table 1). Body composition was significantly altered in obese KpB mice
compared to non-obese controls. Percent body fat was six-fold greater in obese mice (Table

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1, p = 0.0001), while percent lean mass increased by 25% (p = 0.0006, Table 1). The ovarian
tumors were tripled in size in the obese mice as compared to non-obese mice (mean size of
3.7 cm2 versus 1.2 cm2, Fig. 1, p = 0.0065).
Obesity induces genomic differences between obese and non-obese ovarian tumors

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439 genes were found to be significantly up-regulated (417 genes) or down-regulated (22
genes) in the ovarian tumors from obese KpB mice versus non-obese mice (FDR < 0.2,
Supplemental Table 1). Fig. 2 is a heat map of 131 genes up- and down-regulated at a FDR
< 0.1. Metabolically relevant genes were significantly upregulated in the ovarian tumors
from the obese versus non-obese mice, such as lipocalin (2.7 fold), fatty acid amide
hydrolase (2.7 fold), fatty acid 2-hydroxylase (2.2 fold), glycerol-3-phosphate
acyltransferase (1.5 fold), protein phosphatase (1.2 fold), AMP deaminase 3 (1.6 fold), and
protein kinase C (1.7 fold) (Supplemental Table 1). Arginase 1 was the most upregulated
gene (7.3 fold) and plays a role in the urea cycle, tissue remodeling and inflammation. Other
upregulated genes identified in the ovarian tumors from the obese mice were related to cell
adhesion, including neurotrimin (2.2 fold) and desmoglein 1-alpha (2.0 fold). Increased
expression of histone 1 (2.3 fold), endothelin-1 (5.8 fold), ectonucleoside triphosphate
diphosphohydrolase (3 fold) and serotonin transporter solute carrier family 6 member 4
(Slc6a4) (5.4 fold) were also associated with obesity in the KpB mouse model. Significantly
downregulated genes with obesity included spermidine synthase and thrombospondin 4.
In the ovarian tumors from the obese versus non-obese mice, EASE over-representation
analysis revealed significant enrichment in phospholipid binding (EASE score of 0.008),
regulation of apoptosis (EASE score of 0.014), lipid binding (EASE score of 0.015),
endopeptidase activity (EASE score of 0.03) and cellcell signaling (EASE score of
0.44) for those identified genes.
Metabolic differences between ovarian tumors from obese and non-obese KpB mice

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Principle component analysis defined a clear separation between obese and non-obese
samples (Fig. 3, 3 components, R2X = 0.563, R2Ycum = 0.95, Q2cum = 0.411).
Differentiating metabolites were selected with the criteria of the variable importance in the
projection (VIP) value >1 and p value (Students t test) lower than 0.05. Twenty metabolites
were identified using this criterion, all of which were up-regulated in the ovarian tumors of
the non-obese versus obese KpB mice (Table 2).
Metabolites involved in inflammatory signaling and protein/collagen metabolism were
down-regulated in the ovarian tumors of obese mice as compared to non-obese mice,
including arginine (p = 0.0268), N-glycylproline (p = 0.0043) and 3-amino-2-piperidone (p
= 0.0099). Components and markers of oxidative stress were also downregulated in the
tumors from obese mice: glutathione (p = 0.0313), oxidized glutathione (p = 0.0047),
gluconolactone (p = 0.0311) and 8-hydroxy-deoxyguanosine (p = 0.0230). Lower levels of
nucleotides (i.e. cytidine (p = 0.0122 and p = 0.0424), cytosine (p = 0.0158), guanosine
diphosphate (GDP, p = 0.0404)) and adenosine monophosphate (AMP, p = 0.0257) were
detected with obesity. The serotonin metabolite, 5-hydroxyindoleacetic acid (5HIAA, p =
0.0498), and the catecholamine metabolites, vanillactic acid (p = 0.0079) and

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phenylethanolamine (p = 0.0446), were found to be lower in the ovarian tumors of obese

versus non-obese mice. Glutamate (p = .0318), N-acetylaspartic acid (p = 0.0059) and
succinic acid (p = 0.0465) are involved in energy metabolism, and were decreased in the
ovarian tumors of obese KpB mice. LysoPC(16:1(9Z)) (p = 0.0205), a lysophospholipid,
was also lower in the ovarian tumors from obese animals.

Discussion
Recent evidence suggests that obesity may be a significant risk factor and associated with
worse outcomes for OC [1,420]. Therefore, a metabolic approach to the diagnosis and
treatment of OC may provide a novel strategy to improve outcomes for this invariably lethal
disease. Hence, we induced obesity in the KpB mouse, a faithful murine model of serous
OC, to ask if obesity alters tumorigenesis. KpB mice fed a HFD had significant increases in
their body weight and fat mass compared to mice fed a LFD. Herein, we report that obesity
promoted tumor progression in the KpB mouse model of OC with a tripling of ovarian
tumor size. Obesity has been associated with more rapid tumor growth in animal models of
other cancer types, such as breast, colon and lung cancer [27,28], but this is the first study to
demonstrate this for OC.

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Genomic and metabolomic analyses were utilized to identify obesity-induced alterations in

tumors with the intention of identifying significant pathways or biomarkers to aid in
explaining why obese mice developed larger, more aggressive tumors. The metabolically
relevant genes, lipocalin, ectonucleoside triphosphate diphosphohydrolase and fatty acid
amide hydrolase, were upregulated in the ovarian tumors from the obese versus non-obese
mice. Lipocalin, particularly lipocalin 2, has been previously found to be upregulated in
number of different cancers, including OCs [29,30]. The primary function of lipocalin is the
transport of small ligands such as steroids, bilins, retinoids and lipids. In addition to its role
in lipid transport, lipocalin has also been implicated in the inflammatory response. Another
gene significantly upregulated was ectonucleoside triphosphate diphosphohydrolase, which
is involved in the extracellular hydrolysis of ATP to generate adenosine, which signals
through G-protein coupled receptors and regulates metabolic pathways and inflammation.
Chronic inflammation is well known to play a role in obesity-driven cancers which could
also explain the increased expression of both lipocalin and ectonucleoside triphosphate
diphosphohydrolase in the ovarian tumors of obese KpB mice.
Fatty acid amide hydrolase (FAAH) is a serine hydrolase that metabolizes Nacylethanolamines (i.e. N-arachidonoylethanolamine, N-oleoylethanolamine and Npalmitoylethanolamine), also known as endocannabinoids, to fatty acids plus ethanolamine.
The endocannabinoid system is thought to be important in the regulation of cancer cell
apoptosis, proliferation, migration, adhesion and invasion. Increased expression of the
cannabinoid receptors (CB1R and CB2R) and FAAH has been documented in prostate and
breast cancer and has been associated with worse outcomes [31]. FAAH inhibitors are under
development for the treatment of pain and inflammation [31], but may also be useful in
cancer. Our data suggests that FAAH inhibitors might be a potential targeted agent for
obesity-driven cancers.

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Other unique, metabolically relevant genes that were associated with obesity and OC
development in the KpB mouse model included fatty acid 2-hydroxylase, glycerol-3phosphate acyltransferase, protein phosphatase, protein kinase C and AMP deaminase. Fatty
acid 2-hydroxylase (FA2H) catalyzes the synthesis of 2-hydroxysphingolipids, a subset of
sphingolipids that contain 2-hydroxy fatty acids. FA2H is thought to be involved in the cell
differentiation of Schwann cells, keratinocytes and adipocytes. Glycerol-3-phosphate
acyltransferase is an enzyme that participates in glycerolipid metabolism and
glycerophospholipid metabolism. Protein phosphatases are essential to protein
phosphorylation, an important form of reversible protein posttranslational modification
involved in cell signaling cascades. The protein kinase C (PKC) family represents a number
of protein kinase enzymes that are involved in regulating the function of other proteins
through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues
on these proteins. The PKC family of enzymes has been implicated in the regulation of
signal transduction, cell proliferation, metabolism and differentiation through its effects on
regulation of the cell cycle. PKC inhibitors are already being evaluated in clinical trials for a
variety of different cancers, including OC [32]. AMP deaminase 3 is a highly regulated
enzyme that catalyzes the hydrolytic deamination of adenosine monophosphate to inosine
monophosphate, a branch point in the adenylate catabolic pathway. AMP deaminase 3 is
thought to be a potent regulator of energy metabolism in cells. Increased expression of AMP
deaminases has been documented in hepatocellular carcinomas [33] but has not been
explored in OC.

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Although many metabolically relevant genes were found to be associated with obesitydriven cancers in the KpB mouse model, other up-regulated genes and pathways were
identified. This included genes related to cell adhesion, including neurotrimin and
desmoglein 1-alpha. Expression of neurotrimin and desmoglein 1-alpha has not been
previously documented in OCs. Increased expression of histone 1 in the ovarian tumors was
also associated with obesity in the KpB mice. Histones are the chief protein component of
chromatin and are critical for gene regulation. Endothelin-1 (ET-1) is a highly potent vasoconstrictive peptide and was found to be upregulated 5.8 fold in the ovarian tumors from
obese mice. Overexpression of ET-1 has been implicated in the epithelialmesenchymal
transition, a mechanism by which transformed epithelial cells acquire the ability to
proliferate, invade, resist apoptosis and metastasize [34]. In chemoresistant ovarian cancer
cells, ET-1 has been found to be upregulated, leading to enhanced signaling through the
MAPK and mTOR/Akt pathway, increased cell proliferation and reduced sensitivity to
cisplatin and paclitaxel [35]. Endothelin receptor antagonists are being developed as
potential chemotherapeutic agents for cancer [34]. In the ovarian tumors from the obese
versus non-obese mice, DAVID functional annotation analysis revealed significant
enrichment in phospholipid binding, regulation of apoptosis, lipid binding,
endopeptidase activity and cellcell signaling. Thus, the increase in aggressiveness, as
manifested by a tripling of tumor size, in the obese KpB mice was accompanied by
upregulation of genes involved in metabolic, apoptotic and cell signaling pathways.
Metabolic analysis revealed that 20 metabolites were identified as significantly regulated. In
general, metabolomic analysis revealed that multiple metabolites contributed to separation
of non-obese and obese mice with each metabolite being down-regulated in tumors derived
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from obese mice. Arginase 1 was the most up-regulated gene in obese tumors, which
explains the lower detection of arginine concentrations. Catabolic disease states such as
sepsis, injury and cancer cause an increase in arginine utilization, which can exceed normal
body production, leading to arginine depletion. Arginase 1 converts L-arginine into Lornithine and urea. Nitric oxide (NO) synthase and arginase compete for the same substrate
(L-arginine); hence high arginase activity will blunt NO production, limiting potential proinflammatory responses necessary in tumoricidal immune responses. Indeed, arginase 1 is a
marker of the M2, alternatively activated, macrophage that is often associated with more
aggressive tumors [36]. Arginase also drives polyamine (such as spermidine) synthesis
necessary for proliferation. Spermidine synthase in spermidine synthesis was a downregulated gene in tumors from obese animals, perhaps in a negative feedback mechanism
due to elevated delivery of ornithine generated by arginase 1 (30% lower levels of
spermidine were detected in ovarian tumors of obese mice but this did not reach statistical
significance). Ornithine can also be converted to the delta-lactam 3-amino-2-piperidine, and
this was significantly blunted in tumors from obese mice. Finally, arginase generates
ornithine which is used to generate proline (necessary for collagen synthesis) and glutamate/
glutamine. Glutamate was found at lower levels suggesting that arginase was directing
ornithine production to modulate collagen synthesis in tumors derived from obese mice.
AMP and arginine both activate AMP kinase (AMPK) which stimulates substrate
metabolism, while arginine can also activate mTOR [37,38]. Decreased concentrations of
both AMP and arginine in the ovarian tumors from obese versus non-obese mice may be a
reflection of increased turnover of these metabolites in the rapidly growing tumors in the
obese mice, and potential regulation of substrate metabolism.
N-glycylproline, which had the highest VIP contributing to separation between non-obese
and obese tumors, was significantly lower in obese tumors relative to non-obese tumors in
KpB mice (Table 2, p = 0.0043). N-glycylproline is an end product of collagen metabolism,
but may be recycled into collagen synthesis, and this suggests a potential difference in tissue
remodeling between non-obese and obese mice. Overall, Fig. 4 depicts metabolites and
genes related to arginine/polyamine/collagen/glutamine metabolism that were decreased in
the ovarian tumors from obese mice, suggesting that diet-induced alterations in the stromal
components and extracellular matrix are associated with greater growth of the ovarian
tumors in obese animals.

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Although glutathione disulfide (GSSG) and glutathione (GSH) were significantly regulated
by diet, the ratio of the two (as an indicator of oxidative stress) was not significantly
different between lean (0.5/+0.048) and obese (0.45/+0.284) tumors, suggesting that there
was no active oxidative stress. However, a more stable marker of oxidative stress-induced
DNA modification, 8-hydroxy-deoxyguanosine, was detected at significantly lower
concentrations in obese versus non-obese tumors. Lower concentrations of gluconolactone,
an oxidized derivative of glucose, were also found in tumors from obese animals relative to
lean, providing further evidence of changes in reductionoxidation status between the
ovarian tumors in the non-obese versus obese group. In sum, in ovarian tumors in obese
KpB mice, there appears to be less DNA modification and markers of oxidized metabolites
due to oxidative stress, suggesting that oxidative stress is not a major driver of obesitydriven tumorigenesis in the KpB mice or that compensatory mechanisms exist.
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Alternatively, it could be that the greater growth of ovarian tumors in the obese animals was
driven by inflammatory cytokines produced in adipose tissue and distributed to the tumor
through the circulation.
Lower concentrations of nucleotides (i.e. cytidine, cytosine, guanosine diphosphate (GDP),
adenosine monophosphate (AMP)) may be reflective of increased cell turnover and
alterations in utilization and production of these building blocks in the ovarian tumors from
obese versus non-obese mice. We postulate that the observed heightened proliferation in the
ovarian tumors from the obese versus non-obese mice, as evidenced by a tripling of tumor
size, may result in the increased consumption of nucleotides. In the genomic analysis, we
also found a 3-fold increase in ectonucleoside triphosphate diphosphohydrolase. This
enzyme catalyzes the breakdown of multi-phosphated nucleotides (i.e. ATP, ADP, etc.) and
removes free nucleotides and upstream compounds like AMP and GDP, all of which were
significantly decreased in the ovarian tumors from the obese mice. In addition, low AMP
detected in the ovarian tumors from obese mice suggests possible elevations in anabolic,
ATP-burning processes such as lipid synthesis as well as protein, RNA and DNA synthesis.

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5-Hydroxyindoleacetic acid (5HIAA) was significantly lower in obese versus non-obese

tumors. 5HIAA is a breakdown product of serotonin. Interestingly, the serotonin transporter
solute carrier family 6 member 4 (Slc6a4) was upregulated 5.4 fold by obesity. In addition to
its function as a neurotransmitter in the central nervous system, increasing evidence suggests
that peripheral serotonin may have pro-proliferative and anti-apoptotic effects and act as a
mitogen in cancer cells [39,40]; hence, the obesity-mediated regulation of serotonin is of
interest.
The catecholamine metabolites, vanillactic acid and phenylethanolamine, were lower in
ovarian tumors derived from obese animals. Catecholamines, including epinephrine and
norephinephrine, are known to regulate lipolysis [41]. Several studies report that
catecholamine responses are blunted in obese versus non-obese individuals at rest and in
response to physical activity, suggestive of decreased sympathetic nervous system activity
[41]. A decrease in the catecholamine response in the obese mice could lead to reductions in
lipolysis and an increase in fat stores that could be advantageous for cancer cell growth.

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Succinic acid and glutamate were also significantly decreased by obesity in tumors (Table 2,
p = 0.0465 and p = 0.0318). Succinate is a metabolite of the tricarboxylic acid cycle (TCA)
cycle and an electron donor to complex II (Succinate-Q oxidoreductase) in oxidative
metabolism. Glutamate is also the metabolic intermediate of glutaminolysis, which would
feed into the TCA cycle upstream of succinic acid at alpha-ketoglutarate. Interestingly,
fructose-6-phosphate did not reach statistical significance (non-obese vs. obese ratio 1.62, p
= 0.0684) but contributed to principle component analysis variance (VIP was 1.67). Fructose
6-phosphate is an important intermediate in glycolysis. Taken together, low AMP, succinate,
glutamate, and fructose 6-phosphate suggest that KpB tumors in obese mice have a
substantially altered metabolic phenotype compared to tumors that have arisen in non-obese
controls. We are currently investigating the role of glycolysis and oxidative metabolism,
along with AMPK and mTOR signaling, in ovarian cancers from obese and non-obese
patients.

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Finally, cytidine is a precursor of cytodinetriphosphate, which is needed to create

phosphatidylcholine (PC) and phosphatidylethanolamine. Interestingly, lysoPC(16:1(9Z))
was also downregulated in the ovarian tumors from obese versus non-obese mice.
Lysophospholipids (LPLs) can play a role in signaling through G-protein coupled receptors,
and are a readily accessible fat source for cancer cells [42]. LPLs are generated via
inflammatory-responsive phospholipase A (PLA) activity, suggesting that there may be
altered inflammatory signaling between non-obese and obese tumors, which is currently
being explored. In our genomic analysis, significant enrichment was found in phospholipid
binding in the ovarian tumors from the obese mice, potentially corresponding to increased
utilization of lysophospholipids in the setting of obesity and depletion of cytidine and
lysoPC.

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In conclusion, we demonstrate that the obese state can promote tumor progression in the
KpB mouse model of serous OC, resulting in genomic and metabolic differences between
tumors arising in the obese versus non-obese state. Our work suggests that the metabolic
consequences of obesity may be crucial in the pathogenesis of OC, resulting in biologically
distinct cancers than those that arise in normal weight women. This may have important
implications for the treatment of this disease, such that obesity status may be a critical factor
in the individualization of management strategies. Further work will be focused on the
investigation of the identified obesity-dependent metabolic bio-markers as well as potential
novel targets of treatment that may be specific to obesity-driven OCs.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ygyno.
2013.12.026.

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HIGHLIGHTS

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Obesity promotes tumor progression in the KpB mouse model of serous ovarian
cancer.

Gene expression and metabolomic profiling indicated significant differences

between ovarian tumors from obese versus non-obese mice, including
metabolically relevant pathways.

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Fig. 1.

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Obesity increases tumor size in KpB mice. KpB mice were fed low fat or high fat diets to
induce obesity for 6 months during tumorigenesis. (A) Comparison of tumor size from nonobese and obese mice (N = 14). These mice were sacrificed 6 months after ovarian tumor
induction via injection of AdCre into the ovarian bursa cavity. For the calculation of tumor
size, the greatest longitudinal diameter (length) and the greatest transverse diameter (width)
were determined and multiplied (m2). *p = 0.0065. (B) MRI images of tumors (arrow) from
non-obese (top image) and obese (bottom image) mice demonstrate representative tumors.

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Fig. 2.

Genomic differences between ovarian tumors from obese versus non-obese KpB mice reveal
alterations in metabolically relevant genes. Heat map representation of 131 genes found to
be significantly up- or down-regulated in the ovarian tumors from the obese versus nonobese KpB mice (FDR < 0.1). Many metabolically relevant genes, such as lipocalin, fatty
acid amide hydrolase, ectonucleoside triphosphate diphosphohydrolase, fatty acid 2hydroxylase, glycerol-3-phosphate acyltransferase, protein phosphatase, protein kinase C
and AMP deaminase 3, were upregulated in obese tumors.

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Fig. 3.

Several metabolites define a clear separation using principal component analysis between
the ovarian tumors in the non-obese group and obese group. PLS-DA scores plot of the
ovarian tumors in the non-obese group (low fat diet) and obese (high fat diet) group.

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Fig. 4.

Obesity-induced alterations in arginine/polyamine/collagen/glutamine metabolism.

Metabolomic profiling of ovarian tumors from obese and non-obese KpB mice revealed
significant decreases in a number of metabolites related to arginine/polyamine/collagen/
glutamine metabolism, suggesting that diet-induced alterations in the stromal components
and extracellular matrix are associated with greater growth of the ovarian tumors in obese
animals.

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Table 1

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Diet-induced metabolic characteristics in non-obese and obese KpB mice.

Non-obese
Weight (g)
Glucose (mg/dl)

Obese

p-Value

31.14 5.26

50.71 16.73

p = 0.0003

186.81 26.99

214.38 58.11

p = 0.053

% fat

3.28 1.51

19.58 7.88

p = 0.00001

% lean

22.89 2.11

28.66 5.24

p = 0.0006

N = 14 mice per group. Mean SD. % fat or % lean = each mass / total body mass as measured by MRI.

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0.0498

1.52

1.85

1.90

1.69

3.05

4.97

2.39

LC-ES+

GCTOF

GCTOF

LC-ES+

GCTOF

LC-ES

GCTOF

LC-ES+

LC-ES+

LC-ES+

LC-ES-

LC-ES+

LC-ES+

LC-ES+

LC-ES+

GCTOF

LC-ES+

LC-ES

LC-ES+

LC-ES+

Analysis method

HMDB

Std

NIST

HMDB

NIST

HMDB

Std

Std

Std

Std

HMDB

HMDB

HMDB

Std

Std

NIST

HMDB

HMDB

Std

Std

The metabolites were identified by in-house library (Std), NIST library (NIST) or HMBD database (HMDB).

Fold change with a value larger than 1 indicates a relatively higher concentration in tumors from non-obese (low fat diet-fed) KpB mice, while a value less than 1 means a relatively lower concentration as
compared to tumors from obese (high fat diet-fed) KpB mice.

1.76

5-Hydroxyindoleacetic acid

0.0446
0.0465

p value was calculated from Students t test.

1.80
1.78

Phenylethanolamine

Succinic acid

0.0439

0.0318

3.10

2.97

1.93

1.61

2.45

1.83

4.11

4.52

1.75

2.23

2.31

3.45

1.95

Fold change (non-obese/obese)c

Variable importance in the projection (VIP) was obtained from OPLS-DA with a threshold of 1.0.

0.0424

1.81
1.80

Cytidine

Guanosine diphosphate

Inodxyl glucuronide

0.0404

1.89
1.82

Glutamate

0.0311
0.0313

1.89
1.89

Gluconolactone

0.0268

0.0257

0.0230

0.0205

0.0158

0.0122

0.0099

0.0079

0.0059

0.0047

0.0043

pb

Glutathione

1.94
1.93

Adenosine monophosphate

Arginine

1.99
1.97

8-Hydroxy-deoxyguanosine

2.05

LysoPC(16:1(9Z))

2.10

2.17

Vanillactic acid

Cytosine

2.22

N-acetylaspartic acid

Cytidine

2.25

Oxidized glutathione

2.14

2.27

N-glycylproline

3-Amino-2-piperidone

VIPa

Compound name

Identification methodd

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Metabolic alterations in tumors from non-obese and obese KpB mice.

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Table 2
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