Fatty Acid Metabolites in Rapidly Proliferating Breast
Joseph T. O’Flaherty1, Rhonda E. Wooten2, Michael P. Samuel2, Michael J. Thomas2, Edward A. Levine3,
L. Douglas Case4, Steven A. Akman5, Iris J. Edwards6*
1Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 2Department of Biochemistry, Wake Forest
School of Medicine, Winston-Salem, North Carolina, United States of America, 3Department of Surgical Oncology, Wake Forest School of Medicine, Winston-Salem, North
Carolina, United States of America, 4Department of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America,
5Department of Hematology and Oncology and Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 6Department
of Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America
Purpose: Breast cancers that over-express a lipoxygenase or cyclooxygenase are associated with poor survival possibly
because they overproduce metabolites that alter the cancer’s malignant behaviors. However, these metabolites and
behaviors have not been identified. We here identify which metabolites among those that stimulate breast cancer cell
proliferation in vitro are associated with rapidly proliferating breast cancer.
Experimental Design: We used selective ion monitoring-mass spectrometry to quantify in the cancer and normal breast
tissue of 27 patients metabolites that stimulate (15-, 12-, 5-hydroxy-, and 5-oxo-eicosatetraenoate, 13-hydroxy-
octadecaenoate [HODE]) or inhibit (prostaglandin [PG]E2and D2) breast cancer cell proliferation. We then related their
levels to each cancer’s proliferation rate as defined by its Mib1 score.
Results: 13-HODE was the only metabolite strongly, significantly, and positively associated with Mib1 scores. It was similarly
associated with aggressive grade and a key component of grade, mitosis, and also trended to be associated with lymph
node metastasis. PGE2and PGD2trended to be negatively associated with these markers. No other metabolite in cancer and
no metabolite in normal tissue had this profile of associations.
Conclusions: Our data fit a model wherein the overproduction of 13-HODE by 15-lipoxygenase-1 shortens breast cancer
survival by stimulating its cells to proliferate and possibly metastasize; no other oxygenase-metabolite pathway, including
cyclooxygenase-PGE2/D2pathways, uses this specific mechanism to shorten survival.
Citation: O’Flaherty JT, Wooten RE, Samuel MP, Thomas MJ, Levine EA, et al. (2013) Fatty Acid Metabolites in Rapidly Proliferating Breast Cancer. PLoS ONE 8(5):
Editor: Anthony Peter Sampson, University of Southampton School of Medicine, United Kingdom
Received January 29, 2013; Accepted March 28, 2013; Published May 2, 2013
Copyright: ? 2013 O’Flaherty et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from Golfers Against Cancer and National Institutes of Health (grant numbers P01 CA106742, R01 CA 115958, U10
CA081851, P30 CA12197, U1O CA76001, R01 AI064609, and R01 CA135288); the Quattro II mass spectrometer was acquired using funds from the NSF (BIR-
9414018, MJT) and the 600 MHz Bruker NMR was purchased in part with funds from the NIH Shared Instrumentation Program (1S10RR13875, MJT). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The growth rate of cancer is commonly estimated by measuring
a cell proliferation-related protein, Ki-67, with Mib1 monoclonal
antibody. The Mib1 score provides a particularly strong prognos-
tic marker in human breast cancer [1,2]. However, its relation to
this cancer’s content of linoleic acid (LA), arachidonic acid (AA),
and the oxygenase-derived metabolites of these two fatty acids (FA)
is not clear. Application of LA or AA to breast cancer cell cultures
stimulates proliferation apparently because these cells over-express
5-lipoxygenase (LO), 12-LO, 15-LO-1, 15-LO-2, cyclooxygenase
(CO)-1 and/or CO-2, and thereby over-produce LA and/or AA
metabolites some of which feedback to up-regulate their parent
cells’ proliferation. Since dietary excesses of these FA may promote
human breast cancer, one or more of these autocoid loops may
contribute to this disease [3,4].
5-LO and 12-LO are over-expressed in the breast cancer of
patients who suffer poor survival [5,6,7]. Two 5-LO products, 5-
hydroxy-eicosatetraenoate (HETE) and 5-oxo-eicosatetraenoate
(5-oxo-ETE), and a 12-LO product, 12-HETE, stimulate breast
cancer cells in culture to proliferate and may thereby mediate the
effect of these enzymes on survival [8,9,10,11], although one study
did find 12-LO over expression to be associated with an improved
disease-free survival . Low 15-LO-1/15-LO-2 ratios are also
associated with shortened breast cancer survival [5,13]; this
suggests that a 15-LO-1 product slows or a 15-LO-2 product
speeds the cancer’s growth. Of the metabolites made by these
LOs, 15-HETE slows proliferation in several types of cancer cells
 but its effect on breast cancer cells is unclear, and 13-hydroxy-
octadecaenoate (HODE) mediates breast cancer cell proliferation
responses to growth factors but its direct effect on these cells also is
not clear [4,14].
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Over-expression of CO-2 in breast cancer has been reported to
reduce survival [5,7,15,16], not impact survival [12,15,16,17,18],
reduce survival when associated with high Ki-67 levels [19,20],
and improve survival when associated with low Ki-67 levels .
In spite of this confusion, CO-2 appears especially important since
its pharmacological inhibition reduces the prevalence and
progression of this cancer in many, although not all, studies
[21,22,23,24]. CO-1 over-expression is associated with increased
breast cancer invasiveness ; its activity may explain the
protective effect in breast cancer of aspirin, which blocks both CO-
1 and -2, compared to some disappointing results with drugs that
selectively inhibit CO-2 . Aspirin’s targeting of CO-1 may also
explain its preventative effects in breast cancers that under-express
CO-2 . In any case, both COs metabolize AA to prostaglandin
(PG)E2 and D2, which, perhaps paradoxically, inhibit the
proliferation of cultured breast cancer cells [26,27,28].
The varying results of the cited studies may reflect an imperfect
relation between levels of the oxygenases and their metabolites: an
oxygenase’s level does not indicate its activity status, its substrate
availability, or the action of other oxygenases which make the
same metabolite(s). To clarify the roles of the oxygenases in breast
cancer, levels of their metabolites need to be defined and, similar
to their parent enzymes, related to factors in the disease that alter
survival. Radioimmunoassay studies conducted .20 years ago
found that PG-like material in breast cancer was associated with
an increase, no change, or a decrease in survival  and that
PGE-like material was unrelated to tumor grade, estrogen
receptors, node metastasis, or survival [30,31,32]. Perhaps because
of these results and the need for fresh, specially processed tissues
for study, newer measurement methods that unambiguously
identify the metabolites of the COs and also the LOs in this
disease are lacking. We have assayed the growth-promoting
activity in human breast cancer cell lines of metabolites whose
effects are unknown, measured the metabolites in breast cancer by
mass spectrometry (MS)-based selective ion monitoring, and
related the findings of these studies to patient Mib1 scores and
grade (which includes mitotic index as one of its parameters) as
well as node metastasis and other markers of disease severity.
Materials and Methods
We measured the metabolites in the malignant and normal
breast tissue and the fatty acids (FA) in malignant and normal
breast tissue, plasma, and RBC of 27 women undergoing surgery
for breast cancer at Wake Forest University Medical Center. The
study was approved by the Institutional Review Board of Wake
Forest University School of Medicine and IRB-approved, written
informed consent was obtained from each subject before surgery.
Consenting patients were taken in sequence with no knowledge of
their disease status; patients with recurrent disease, inflammatory
breast cancer, a malignancy other than adenocarcinoma, in situ
disease, neo-adjuvant treatment, or failure to have a Mib1 score
were excluded retrospectively. All patients included in the study
were diagnosed as having invasive ductal carcinoma and were
categorized by the following assessments of their cancer: Mib1
score of #20 vs. .20 (13 vs. 14 patients, respectively); grade I and
II vs. III (Nottingham score [Bloom-Richardson grading system]
of 3–5 vs. 6–9; 10 vs. 17 patients) and grade’s component indices,
mitosis (2–3 vs. 1; 19 vs. 8 patients), nuclear pleomorphism (3 vs. 2
& 1 19 vs. 8 patients; only 1 patient had a score of 1 [omitting this
patient did not effect the statistical significance of results]), and
tubule formation (3 vs. 2, 24 vs. 3 patients)(no patient had a tubule
index of 1); Her2 negative vs. positive (histological score of 0 or I
vs. II or III; 21 vs. 6 patients); estrogen receptor negative vs.
positive (histological score of #10 vs. .10; 12 vs. 15 patients);
progesterone receptor negative vs. positive (histological score of
#10 vs. .10; 15 vs. 12 patients); triple negative vs. receptor
positive for Her2, estrogen, and/or progesterone receptors (12 vs.
15 patients); and tumor size I vs. II–IV (,2 cm vs. $2 cm or
spread to chest wall or skin; 13 vs. 14 patients). Patients were also
categorized based on self-reported race of Caucasian vs. African
American (21 vs. 6 patients; no Hispanic, Oriental, or Native
Americans occurred in the patient sequence studied); age .50 or
#50, (7 vs. 20 patients); lymph node metastasis absent (N0) vs.
present (N1–N3; 21 vs. 6 patients); and body mass index (BMI) of
#30 vs. .30 (20 vs. 7 patients).
Breast tissue was removed by lumpectomy or mastectomy and
identified visually as malignant (subject to histological confirma-
tion) or normal. Within 15 minutes of removal for lumpectomy or
mastectomy specimens, malignant and normal (taken from sites
.0.5–1 and .2 cm, respectfully, from malignant tissue) tissues
were sampled, placed on ice and dissected into 3 approximately
equal-sized specimens (two for metabolites, one for FA). Speci-
mens designated as malignant and normal from the same patient
were immediately weighed (wet weight of 3–110 mg for malig-
nant, 5–90 mg for normal) and within 20 min of dissection,
separately processed for content of metabolites and FA. For
metabolites, sections were put in 1 ml of tris-buffered saline (4uC,
pH 7.4) containing 100 mM of diethylenetriaminepentaacetic acid
(DTPA), 80 mM of butylated hydroxytoluene (BHT), and 5 ng of
the deuterated metabolites listed in Table 1; acidified to pH 3.5;
and extracted with hexane:ethyl acetate (1:1). Extracts were blown
dry under a stream of N2, taken up in ethanol containing 100 mM
of triphenylphosphine (PPh3) and 80 mM of BHT, and stored
under argon at 280uC. For FA, breast tissue sections were placed
in ethanol (4uC) containing 80 mM of BHT and 100 mM of PPh3.
Whole blood (4 ml) was drawn into ethylenediaminetetracetic
acid-containing tubes from patients just before surgery; placed on
ice; made 100 mM in DTPA and 80 mM in BHT; and centrifuged
(1000 g, 5 min, 4uC) to obtain cell-free plasma and erythrocytes
(RBC). RBC were washed in tris-buffered saline (pH 7.4) and
suspended in this buffer with 80 mM of BHT and 100 mM of PPh3.
Extracts and prepared RBC and plasma were stored at 280uC
Breast tissue extracts were quantified for metabolites by
measuring their precursor and parent ions and correcting for
processing losses based on the recovery of their deuterated internal
standards by liquid chromatography (LC)-tandem MS running in
the multiple reaction-monitoring mode. Table 1 lists the metab-
olites, internal standards, and precursor/product ion m/z values
monitored. The MS system was a Waters Quattro II MS with a Z-
spray interface automated by a Spark Holland LC and a Reliance
Autosampler and Conditioned Stacker maintained at 4uC. We
used a cone voltage of 35 V and a capillary voltage of 2.4 kV for
HETE, hydroxy-eicosapentaenoate (HEPE), and leukotriene (LT)
metabolites and 50 V and 3.5 kV for PG metabolites. The LC
system for HETE, HODE, leukotriene (LT), and HEPE metab-
olites was a Waters Corp YMC ODS-AQ 1 mm I.D.6100 mm
length column eluted at 0.05 ml/min with 2 mM ammonium
acetate in H2O, pH 8.0, as solvent A and methanol as solvent B in
the following gradients: 0 min, 70% B; 0–4 min to 90% B; 4–
5 min, 90% B; 5–6 min to 70% B; 6–30 min, 70% B. The LC
system for PGs was a Phenomenex Luna Phenylhexyl 1 mm
Fatty Acid Metabolites and Breast Cancer
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I.D.6150 mm length column eluted at 0.07 ml/min with H2O as
solvent A and CH3CN, 0.1% formic acid, as solvent B in the
following gradients: 0–6 min, 20% B; 6–6.1 min to 45% B; 6.1–
7.1 min, 45% B; 7.1–7.2 min to 65% B; 7.2–9.2 min, 65% B; 9.2–
9.3 min to 20% B; 9.3–15 min, 20% B. We validated this
method’s accuracy and precision and determined the linearity of
recoveries over 1 pg–100 ng of each metabolite in cultured
prostate cancer cells; several repetitions of the response curves
gave closely agreeing results and yielded metabolite levels that
varied by ,15%. The method reliably detected 1 pg of the PG’s
and 5 pg of the other metabolites; in general, it showed ,15%
variations in breast tissue extracts examined in duplicate.
Just before analysis, specimens were spiked with an internal FA
standard, extracted, ran on gas chromatography, and quantified as
described . FA distributions were consolidated into 7
categories: total v6 FA, AA, LA, total v3 FA, the ratio of total
v3 to total v-6 FA (v3/v6 ratio), total saturated FA (Sat), and
Metabolites and Other Reagents
Deuterated metabolites (Cayman Chemical) and MCF-7 and
MDA-MB-231 breast cancer cell lines (American Type Culture
Collection (Manassas, VA) were purchased. We prepared 13-
HODE and 15-HETE by incubating LA and AA, respectively,
with soybean type II LO and 5-oxo-ETE by chemically oxidizing
5-HETE as described [34,35]. Products of these reactions were
purified by reverse- followed by normal-phase high-performance
LC; the structure and purity of the products were defined by UV
spectroscopy, nuclear magnetic resonance spectroscopy, and MS.
The other non-deuterated metabolites were purchased (Cayman
Cell Growth Assays
Cell growth was assayed with Cell Titer96 Aqueous One
Solution Cell Proliferation Assays (Promega) as described .
Cells, cultured in DMEM with 10% fetal bovine serum, were
grown to 50–70% confluency and then challenged with culture
medium 6 metabolites for 48 hr.
Data Presentation and Statistical Analyses
Cell growth data are given in OD units at 490 nm for cultures
processed in the Promega assay. Metabolite levels are reported as
pg/mg of wet tissue weight; FA levels are in mg/mg of wet tissue
weight or percentage of total recovered FA. Differences between
outcomes observed in normal and malignant tissue were assessed
using paired t-tests while differences in outcomes between
independent groups (e.g., grade I/II vs. III) were assessed using
Student t-tests with the Welch correction used if the equality of
variances p-value was ,0.05. Correlations between parameters
are presented as Pearson coefficients. All probability tests were
two-sided with their significances being corrected, where indicat-
ed, for multiple observations by the false discovery rate method
. Corrected p-values of ,0.05 were considered significant.
Since the effects of 13-HODE and 15-HETE on growth of
breast cancer cells are unclear, we tested them for this. Both
metabolites increased cell number in MDA-MB-231 and MCF-7
cultures at $100 pM, achieving responses similar to the known
 effects of 12-HETE (Fig. 1). 5-HETE and 5-oxo-ETE also
stimulate these cell lines to proliferate [8,10] while PGD2and E2
exhibit anti-proliferative effects [26,27,28].
Metabolites in Breast Tissue
We measured the 10 metabolites and 8 deuterated internal
standards listed in Table 1 in malignant and non-malignant breast
tissue. PGE3and LTB4were undetectable in all tissues; 5-HEPE
was undetectable in 24 malignant and 27 normal tissues. These
metabolites were excluded from further analyses. 13-HODE, 15-
HETE, 12-HETE, 5-HETE, 5-oxo-ETE, PGD2, and PGE2were
Table 1. Molecular weights of the precursor and product
ions21of the metabolites and their deuterated internal
standards selectively monitored by LC-tandem MS.
PrecursorProduct Precursor Product
351271 15-HETE319 218.8
5-HETE 31911512-HETE 319179
5-HEPE317 11513-HODE 295194.7
5-oxo-ETE 317 203d4-13-HODE 299197.7
aInternal standard for PGE2and PGE3.
bInternal standard for 5-HETE and 5-HEPE.
Figure 1. Cell growth. MCF-7 (upper panels) and MDA-MB-231 (lower
panels) cell cultures were challenged with a metabolite for 48 hr and
assayed for cell density. One-way ANOVA gave the statistical
significances shown between comparisons of cells treated with 0
(culture media) or 100 pM–1 mM of the indicated metabolite. Data are
means 6SEM in ODU490of 3–6 cultures.
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detected in virtually all malignant and most normal tissues. Their
levels, corrected for processing losses by the recoveries of their
analogous deuterated internal standards, are given in Fig. 2A.
Each metabolite except 5-oxo-ETE was significantly more
abundant in malignant than normal tissue with 13-HODE being
the dominant metabolite in both tissues.
Metabolites and Mib1
Mib1 scores of .20 and #20 classify breast cancer into
respectively faster and slower proliferating diseases with corre-
sponding poorer or better survivals. In breast cancer tissue, 13-
HODE stood alone in being significantly related to this
classification: it was .3.3-fold higher in patients with .20 Mib1
scores (Fig. 2B). 15-HETE, 5-HETE, and 5-oxo-ETE were slightly
higher in tissue with high Mib1 scores; 12-HETE was almost
identical regardless of the tissue’s Mib1 score; and PGD2and E2
trended 60% and 40% lower, respectively, in tissue with the higher
Mib1 scores. In sharp contrast to the findings in cancer tissue, no
metabolite was significantly higher in the normal tissue of patients
with .20 compared to #20 Mib1scores (Fig. 2C). Particularly
relevant to this result, the level of 13-HODE in cancer tissue
(49.6614.0 pg/mg, mean 6SEM) was significantly (P,0.05,
paired t-test) higher than that of normal tissue (23.865.1) in
patients with .20 Mib1 scores yet was virtually identical in
patients with #20 Mib1 scores, i.e. 15.061.8 in cancer, 14.261.9
in normal tissue.
Metabolites and Grade
Our study population had 1 grade I, 9 grade II, and 17 grade III
tumors. Increasing grade predicts increasingly more aggressive
disease and poorer survival. We compared grade I and II to grade
III (omitting grade I did not alter the significance of this
comparison). In malignant tissue, 13-HODE levels were .3-fold
higher (p,0.05) and each PG trended lower by .43% in Grade
III disease. The other metabolites were at similar levels irrespective
of grade (Fig. 2D). 13-HODE’s level in the cancer tissue of patients
with grade III disease (44.7611.8 pg/mg, mean 6SEM; Fig. 2D)
was significantly (P,0.05, paired t-test) higher than that in normal
tissue (21.664.4; not shown); for patients with grade I & II disease,
these respective levels were 14.761.8 (Fig. 2D) and 15.062.1 (not
shown). The agreement of grade with Mib1 scores reflected their
common basis: grade scores are a combination of 3 proliferation-
related indices: mitosis, nuclear pleomorphism, and tubule
formation (higher indices for each suggest poorer prognoses). 13-
HODE was significantly higher in the cancer (Fig. 3A) but not
normal tissue (results not shown) of patients with higher indices for
each grade component. PGE2and D2trended lower in the cancer
of patients with higher indices for mitosis but not the other two
components (Fig. 3A).
Metabolites and Metastasis
The level of 13-HODE was 2-fold higher in the cancer tissue of
patients with $1 lymph node positive for disease compared to
those with node negative disease (Fig. 3B). This difference only
trended toward significance (P=0.15, Students unpaired t-test)
although we stress that there were only 6 node positive patients in
our study. 15-, 12-, and 5-HETE and 5-oxo-ETE showed far
smaller elevations, and PGD2and E2were respectively 35 and
20% lower, in node positive disease (Fig. 3B).
Figure 2. Metabolites and Mib1. Levels of the metabolites are compared by tissue type (panel A), Mib1 score in malignant (panel B) or normal
(panel C) tissue; and grade in malignant tissue (panel D). Probability values were defined by paired (panel A) or unpaired (panels B, C, and D) Student
t-tests and were corrected for the 7 comparisons made in each panel by the false discovery rate method.
Fatty Acid Metabolites and Breast Cancer
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Metabolites and Other Markers
We examined 8 other prognostic markers: African or Caucasian
American, Her2 receptor presence or absence; age .50 or
#50 yrs; BMI .30 or # 30; absence or presence of estrogen or
progesterone receptors; triple negativity for Her2, estrogen, and
progesterone receptors or presence of at least one of these
receptors; and tumor size of .2 or #2 cm. The first category in
each marker carries a poorer prognosis except age which at .50
years is associated with more frequent but not more severe disease.
13-HODE (Fig. 3C), PGE2(Fig. 3D), and PGD2, 15-HETE, 12-
HETE, 5-HETE, and 5-oxo-ETE (results not shown) levels did not
differ significantly between alternate categories of race, Her2, age,
BMI, estrogen receptor; triple negative for Her2, and tumor size
except PGE2 which was significantly (p,0.01) less in larger
tumors. In addition, no metabolite showed a significant difference
as a function of progesterone receptor (results not shown).
In normal breast tissue, 13-HODE levels did not correlate
significantly with those of 15-HETE, 12-HETE, PGE2, or PGD2
(Pearson correlation coefficients of 20.15, 0.31, 20.20, and
20.10, respectively). In cancer tissue, however, 13-HODE was
strongly and significantly correlated with 15-HETE (r=0.63;
P,0.01) but not with 12-HETE, PGD2, or PGE2(r=0.49, 20.01,
and 0.15, respectively; P values for these correlations are corrected
for the 4 observations made in each tissue). Similar result occurred
in tissues from patients with .20 Mib1 scores: 13-HODE and 15-
HETE levels were significantly correlated in malignant (r=0.72,
P,0.01) but not in normal (r=20.04) tissue. In sharp contrast to
this result, correlations between 13-HODE and 15-HETE in
malignant (r=20.25) and normal (r=0.18) tissues of patients with
#20 Mib1 scores were not statistically significant. 13-HODE also
failed to correlate significantly with 12-HETE, PGE2, or PGD2
levels in patients with #20 Mib1 scores.
FA and Mib1
No FA parameter, measured as mg/mg of tissue or percentage
of total recovered FA, in malignant (Figs. 4A, 4C) or normal
(Figs. 4B and 4D) breast tissue varied significantly as a function of
patient Mib1 scores; importantly, this included the precursor to
13-HODE, LA, and the precursor to PGs, AA. A similar lack of
relation to Mib1 scores occurred with RBC and plasma from these
patients (results not shown).
FA and Metabolites
There were no significant correlations between the levels of 13-
HODE, PGE2, or PGD2 in malignant tissue and their FA
precursors in cancer or normal breast tissue, RBC, or plasma
(Table 2). A similar lack of significant correlations occurred in
comparing LA and AA levels in these tissues to cancer tissue levels
Figure 3. Metabolites and other markers. Malignant tissue levels of the indicated metabolites were compared for poorer or better prognoses by
mitosis, nuclear pleomorphism, and tubule formation indices (panel A) or nodal metastasis (panel B). 13-HODE (panel C) and PGE2(panel D) levels
were compared by poorer vs. better prognoses for: race, African (closed bars) or Caucasian American (open bars); Her2 score, 2 & 3 (closed bars) or 0
& 1 (open bars); age .50 years (closed bars) or #50 years (open bars); body mass index (BMI) .30 (closed bars) or #30 (open bars); estrogen
receptors (ER) negative (closed bars) or positive (open bars); triple negative (tri (2)) for estrogen, progesterone, & Her2 receptors (closed bars) or not
(open bars); tumor size, .2 (closed bars) or # 2 cm (open bars). p Values are from Students t-test corrected for the 3 comparisons in each component
of growth (panel A), for the 7 metabolite comparisons (panel B), or for the 7 marker comparisons (panels C and D) by the false discovery rate method.
Analysis of these two metabolites for progesterone receptors or for 15-HETE, 12-HETE, 5-HETE, 5-oxo-ETE, and PGD2in all 8 marker categories found
no significant differences (data not shown).
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of the 3 metabolites in patients with Mib1.20 scores. Tissue levels
of v6 FA, v3 FA, total Sat FA, oleate, and v3/v6 ratios in the 4
tissues also failed to correlated significantly with cancer tissue levels
of the 3 metabolites in all patients or patients with .20 Mib1
scores (results not shown).
Based on the results in Fig. 1 and the literature (see
Introduction), 13-HODE, 15-HETE, 12-HETE, 5-HETE, and
5-oxo-ETE, if impacting proliferation in vivo, would be elevated
while PGE2and D2would be reduced in malignant breast tissue
with high Mib1 scores. There was evidence for this with 13-
HODE: it was the predominant metabolite in breast tissue and its
levels were higher in malignant than normal tissue (Fig. 2A) and
even higher in malignant tissue from patients with .20 vs. #20
Mib1 scores (Fig. 2B). Normal tissue did not show this difference
(Fig. 2C). 13-HODE levels were also significantly higher in
malignant than normal tissue of patients with .20 but not #20
Mib1 scores. No other metabolite presented this pattern. LA levels
were not appreciably elevated in malignant breast, normal breast,
RBC, or plasma as a function of Mib1 scores (Fig. 4) or
significantly correlated with the levels of 13-HODE in malignant
tissue (Table 2). Thus, elevated 13-HODE is strongly, positively
Figure 4. FA and Mib1. Levels of the indicated FA are presented as mass (upper panels) or percentage of total recovered FA (lower panels) in
malignant (left panels) and normal (right panels) breast tissue of patients with high or low Mib1 scores. Comparison of the 7 FA parameters on the
basis of high or low Mib1 score by Students t-test gave no significant differences even before correction for multiple comparisons; the same analysis
in RBC and plasma likewise revealed no significant differences as a function of Mib1 scores (results not shown).
Table 2. Correlations of LA and AA levels (as masses or
percentages of total FA) in malignant breast tissue, normal
breast tissue, RBC, and plasma with the levels of 13-HODE,
PGD2and PGE2in malignant breast tissue.
FA mass FA percentage
Tissue 13-HODE PGD2
Malignant breast LA 0.021
Malignant breast AA0.06
Normal breast LA
Normal breast AA
20.03 0.04 0.19
1Pearson correlation coefficients between the cited FAs and metabolites. None
of the correlations attained statistical significance. There were also no
significant correlations between the FA in patients with .20 Mib1 scores (data
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and, within the range of metabolites tested, uniquely associated
with breast cancer proliferation; this does not appear to result from
an abundance of its precursor FA. The same pattern of
significantly elevated levels of 13-HODE, but not the other
metabolites, also associated with the poor prognostic feature of
grade (Fig. 2D) and its mitosis, nuclear, and tubular components
(Fig. 3A). Neither 13-HODE nor the other metabolites was
significantly associated with metastasis (Fig. 3B), race, age, BMI,
Her2, estrogen receptor, progesterone receptor, and triple
receptor negativity markers (Fig. 3C). Based on these results, 13-
HODE appears to fuel proliferation, mitosis, and other compo-
nents of an aggressive morphology but is less related or unrelated
to the remaining markers of severe disease that we tested. There is
a proviso here. 13-HODE was far higher, although not
significantly, in the cancer of patients with node metastasis
(Fig. 3B). Since proliferation markers reflect the potential for,
rather than presence of, metastasis, 13-HODE may fuel time-
dependent metastases not captured by a single time point study:
absent intervention, patients with high Mib1 scores, grade scores,
and 13-HODE levels may develop metastasis sooner than those
with lower values for these indicators.
15-LO-1 catalyses the oxygenation of AA to 15-HETE and 12-
HETE in a 89:11 ratio, prefers LA over AA as substrate, and
makes 13-HODE in excess when both FA are available. This FA
preference along with the higher levels of LA compared to AA
(Fig. 4) may be responsible for the preferential incremental
accumulation of 13-HODE over 15-HETE in rapidly compared to
more slowly proliferating cancers (Fig. 2B, 2D, and 3A).
Nonetheless, CO-1 and -2 also make 13-HODE [37,38,39] and
thereby appear to contribute to, for example, the ability of LA to
stimulate the growth of MDA-MB-231 cell explants in mice by a
CO inhibitor-dependent mechanism . In any event, 15-LO-2
oxygenates AA to 15-HETE but does not make 12-HETE and,
like 12-LO and 5-LO, does not attack LA to make 13-HODE
[41,42,43]. Thus, the effect of CO-1, CO-2, and/or 15-LO-1 on
breast cancer survival may reflect their production of 13-HODE.
However, we found that: a) 13-HODE and 15-HETE levels were
highly and significantly correlated in the cancer but not the
normal breast tissue of all patients and patients with .20 Mib1
scores; b) patients with #20 Mib1 scores showed none of these
cancer tissue findings; and c) no significant correlations occurred
between 13-HODE and the other metabolites. This result and the
oxygenases’ metabolic profiles argue that the oxygenase capable of
making 15-HETE and 13-HODE, 15-LO-1, is the major
contributor to 13-HODE overproduction in rapidly proliferating
breast cancer. This does not exclude a lesser but still significant
role for CO-1/2 in adding to 13-HODE levels in this tissue.
Indeed, the beneficial actions of CO inhibitors in breast cancer
may reflect such a role. It should also be noted that although
human breast cancer cells express all of the relevant oxygenases
([5,13] and our own unpublished data), the study of breast tissues
as a whole does not inform on the cell type originating FA
metabolites. An influx of immune cells into a developing tumor
may dramatically increase the availability of 13-HODE. The
generation of 13-HODE by tissue macrophages is a major feature
of late atherosclerotic lesions , a mechanism that may
extrapolate to malignancies. Analysis of these tissues for the
oxygenases by immunohistochemistry may also fail to identify the
cells of origin since, as indicated in Introduction, the presence of
an oxygenase does not necessarily indicate its metabolite
PGE2and D2trended lower in the cancer tissue of patients with
Mib1.20 scores (Fig. 2B), grade II & III disease (Fig. 2D), high
mitosis rate (Fig. 3A), and node metastasis (Fig. 3B). While these
trends failed to attain statistical significance, they did not occur
with any other metabolite (Figs. 2B, 2D, and 3A) or marker (except
PGE2and tumor size, Fig. 3D). These results are compatible with
a notion that reduced levels of PGE2and D2favor breast cancer
In conclusion, the metabolites and pathophysiology behind the
contributions of FA oxygenases to poor survival in breast cancer
has been ill-defined. We find that among the metabolites of the
oxygenases known or found here to stimulate breast cancer cell
proliferation, 13-HODE stands alone in associating with rapidly
proliferating, rapidly dividing, aggressive grade, and perhaps
metastasizing breast cancer. Three oxygenases make 13-HODE
but correlation studies suggest that its major producer in rapidly
proliferating breast cancer is 15-LO-1. Since 15-LO-1 makes other
metabolites that are not characterized for proliferative activity in
breast cancer cells or measured here, 13-HODE’s contribution to
proliferation, division, and metastasis may be complemented or
even superseded by other products of 15-LO-1. This caveat also
applies to the trends of PGE2and D2to be negatively associated
with these parameters of aggressive disease. Nonetheless, our
results indicate that 13-HODE is a marker for breast cancer
severity and the 15-LO-1/13-HODE pathway is associated with a
rapidly proliferating, dividing, and possibly metastasizing pheno-
type. We propose that the over expression of this pathway speeds
breast cancer’s growth and spread. Over expression of the other
oxygenase-metabolite pathways, including the CO/PGE2/D2
pathways, do not use this specific mechanism to worsen the
The content of this manuscript is solely the responsibility of the authors and
does not necessarily represent the official views of the National Cancer
Institute or the National Institutes of Health.
Conceived and designed the experiments: JTO SAA IJE. Performed the
experiments: REW MPS. Analyzed the data: JTO LDC. Contributed
reagents/materials/analysis tools: MJT EAL. Wrote the paper: JTO SAA
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