Oncotarget, 2019, Vol. 10, (No. 4), pp: 511-526
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Research Paper
Serum
amyloid
A
predisposes
inflammatory
microenvironment in triple negative breast cancer
tumor
Rosa Mistica C. Ignacio1, Carla R. Gibbs1, Soohyun Kim2, Eun-Sook Lee3, Samuel E.
Adunyah1 and Deok-Soo Son1
1
Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN, USA
2
Department of Veterinary Sciences, College of Veterinary Medicine, Kon-Kuk University, Seoul, Republic of Korea
3
Department of Pharmaceutical Sciences, College of Pharmacy, Florida A&M University, Tallahassee, FL, USA
Correspondence to: Deok-Soo Son, email: dson@mmc.edu
Keywords: serum amyloid A; proinflammatory; tumor microenvironment; triple negative breast cancer; interleukin-1β
Received: November 12, 2018
Accepted: December 29, 2018
Published: January 11, 2019
Copyright: Ignacio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY
3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
Acute-phase proteins (APPs) are associated with a variety of disorders such
as infection, inflammatory diseases, and cancers. The signature profile of APPs in
breast cancer (BC) is poorly understood. Here, we identified serum amyloid A (SAA)
for proinflammatory predisposition in BC through the signature profiles of APPs,
interleukin (IL) and tumor necrosis factor (TNF) superfamily using publicly available
datasets of tumor samples and cell lines. Triple-negative breast cancer (TNBC)
subtype highly expressed SAA1/2 compared to HER2, luminal A (LA) and luminal B
(LB) subtypes. IL1A, IL1B, IL8/CXCL8, IL32 and IL27RA in IL superfamily and CD70,
TNFSF9 and TNFRSF21 in TNF superfamily were highly expressed in TNBC compared
to other subtypes. SAA is restrictedly regulated by nuclear factor (NF)-κB and IL1β, an NF-κB activator highly expressed in TNBC, increased the promoter activity of
SAA1 in human TNBC MDA-MB231 cells. Interestingly, two κB-sites contained in SAA1
promoter were involved, and the proximal region (-96/-87) was more critical than the
distal site (-288/-279) in regulating IL-1β-induced SAA1. Among the SAA receptors,
TLR1 and TLR2 were highly expressed in TNBC. Cu-CPT22, TLR1/2 antagonist,
abrogated IL-1β-induced SAA1 promoter activity. In addition, SAA1 induced IL8/
CXCL8 promoter activity, which was partially reduced by Cu-CPT22. Notably, SAA1/2,
TLR2 and IL8/CXCL8 were associated with a poor overall survival in mesenchymallike TNBC. Taken together, IL-1-induced SAA via NF-κB-mediated signaling could
potentiate an inflammatory burden, leading to cancer progression and high mortality
in TNBC patients.
as a non-coding RNA [7], while SAA4 is constitutively
expressed as a non-inducible protein [8, 9]. On the other
hand, the synthesis of SAA1 and SAA2 is inducible under
inflammatory conditions [9], such as inflammation, trauma
and infection, increasing to several hundredfold [10, 11].
SAA is a family of apolipoproteins associated
with high-density lipoprotein, playing a role in AA-type
amyloidosis and cholesterol metabolism and transport
[12, 13]. Emerging studies of SAA have been implicated
in promoting cellular migration [14, 15], augmenting
cytokines and chemokines expression [16–18], stimulating
angiogenesis [19, 20], inducing the transcription factor
INTRODUCTION
Serum am4yloid A (SAA), an acute phase
protein (APP), is mainly produced in the liver, while
its extrahepatic synthesis has been reported in skin [1],
in atherosclerotic lesions [2], in synovial tissues [3], in
adipocytes and smooth muscle cells [4, 5]. The human
SAA acute phase protein family contains four different
isoforms namely, SAA1, SAA2, SAA3 and SAA4.
Both SAA1 and SAA2 show high degree of homology
in the mRNA and the protein sequences which are hard
to be identified [6]. SAA3 is a pseudogene categorized
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nuclear factor (NF)-κB, and activating signaling pathways,
such as ERK1/2, p38 and JNK [17, 20–23]. Cumulative
studies have shown that SAA is upregulated in a wide
range of malignancies, such as lung [24–26], ovarian
[23, 27], pancreatic [28, 29], prostate [30], uterine serous
papillary [31] and renal cell [32] carcinomas. We have
reported that SAA is preferentially localized to ovarian
epithelial cells and the thecal-interstitial layers compared
to granulosal cell layers in a mouse ovary [33], and
ovarian carcinoma show tumor necrosis factor α (TNF)induced SAA production [23]. However, expression of
SAA in breast cancer (BC) is poorly understood.
BC is the most frequently diagnosed cancer and the
second most common cause of cancer deaths in women
in the US [34]. One probable reason for this is that we
lack a complete picture of the biologic heterogeneity of
this disease. BC is divided into 4 intrinsic subtypes with
a molecular basis as follows: luminal A (LA), luminal B
(LB), human epidermal growth factor receptor 2 (HER2)enriched, and basal-like (BL) BC [35–38]. Triple negative
breast cancer (TNBC), lacking estrogen receptor (ER),
progesterone receptor (PR) and HER2, is known to be the
most heterogeneous and comprises largely of the basallike subtype [39]. LA-BC is characterized by ER or PR
positive but HER2 negative, while LB-BC presents with
ER or PR and HER2 positive [40]. The HER2-enriched
subtype is characterized by HER2 positive, both ER and
PR negative, and high expression of proliferation-related
genes [40]. The BL subtype includes plenty of TNBC and
high expression of EGFR and proliferation-related genes
[36, 41]. Furthermore, TNBC, the most heterogeneous BC,
were defined into several subtypes namely, basal-like 1
and 2 (BL1, BL2), mesenchymal-like (ML) and luminal
androgen (LAR) [42]. BL-TNBC subtype is characterized
by highly activated cell cycle and DNA [43]. ML-TNBC is
associated with poor prognosis due to enhanced epithelialto-mesenchymal transition (EMT) [43] and elevated
expression of genes involved in growth factor pathways
[42, 44]. LAR-TNBC expresses differentially the estrogen/
androgen metabolism pathways and is driven by androgen
signaling [43, 45]. BL-BC representing TNBC is of
particular interest because of aggressiveness, early pattern
of metastasis, greater size of tumor, and lack of welldefined therapeutic target sites due to ER-/PR-/HER2negative status [39, 46]. Most patients with TNBC have
experienced higher rate of distant recurrence compared to
patients with other BC subtypes, requiring identification
of molecular drivers for TNBC.
To date, the signature profile of APPs in BC is not
well defined. Here, we analyzed the signature profiles of
APPs, interleukin (IL) family and TNF superfamily using
publicly available datasets of breast tumor samples and cell
lines. Based on the results analyzed, we found SAA as a
main factor for proinflammatory predisposition in TNBC and
proposed IL-1-induced SAA via NF-κB-mediated signaling
as a molecular driver for the aggressiveness of TNBC.
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RESULTS
SAA1 and SAA2 APPs are predominantly
expressed in human BL-BC subtype and TNBC
cells
We investigated the APP signature in human breast
tumor tissues and cell lines. We used The Human Cancer
Genome Atlas (TCGA)-based dataset for human BC tissues
to define the APP signature in breast tumor heterogeneity.
Analysis of TCGA-based dataset by Gitools 2.3.1 revealed
the following dominant APP signature: BL-BC subtype
representing TNBC highly expressed SAA1, SAA2, SAA4
and TF. Both BL and HER2 subtypes highly expressed
ORM1 and CP (Figure 1A and 1B), while LA subtype
highly expressed SERPINA1 and SERPINA2 (Figure 1A
and 1B). In addition, the analysis of the National Center
for Biotechnology Information (NCBI) Gene Expression
Omnibus (GEO) dataset on 51 human BC cell lines
revealed the following signature of APP: SAA1/2 was
highly expressed in both BL- and ML-TNBC; C3 and FN1
were predominantly expressed in BL-and ML-TNBC cells,
respectively (Figure 1C and Supplementary Figure 1).
Based on the intersection of APP signature between human
BC tissues and cell lines (Figure 1D) to exclude the tumor
heterogeneity, SAA1 and SAA2 were highly expressed both
TNBC tissues and cell lines (Figure 1D).
IL1A, IL1B, IL8/CXCL8 and IL32 are highly
expressed in human BL-BC subtype and TNBC
cells
We analyzed the IL superfamily signature in human
breast tumor tissues and cell lines. We also used TCGAbased dataset for human BC tissues and NCBI-GEO
dataset for 51 human BC cell lines. Particularly, BLBC subtype representing TNBC highly expressed IL1A,
IL1B, IL23A, IL32 and IL34 (Figure 2A and 2B). Both
BL- and HER2-BC subtypes predominantly expressed
IL7 and IL8, while LA-BC subtype highly expressed
IL33 (Figure 2A and 2B). In addition, both BL- and MLTNBC human cell lines highly expressed IL1A, IL1B,
IL6, IL8/CXCL8 and IL32, BL-TNBC cell lines highly
expressed IL18, and LA cell lines dominantly expressed
IL20 (Figure 2C and Supplementary Figure 2). Based
on the intersection of interleukin superfamily signature
between human BC tissues and cell lines (Figure 2D) to
exclude the tumor heterogeneity, both TNBC tissues and
cell lines dominantly expressed IL1A, IL1B, IL8, and IL32
(Figure 2D).
IL27RA is predominantly expressed in human
BL-BC subtype and TNBC cells
We also assessed the IL receptor superfamily
signature in human breast tumor tissues and cell lines.
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to the analysis on TCGA-based dataset for human BC
tissues, CD70 and TNF are highly expressed in BL-BC
representing TNBC, while TNFSF12 is highly expressed
in LA-BC subtype (Figure 4A and 4B). Both BL- and
HER2-BC subtypes highly expressed FASLG, LTA and
LTB (Figure 4A and 4B). Moreover, BL- and ML-TNBC
human cell lines highly expressed CD70 and TNFSF9
(Figure 4C and Supplementary Figure 3). Based on the
intersection of TNF superfamily signature between
human BC tissues and cell lines (Figure 4D) to exclude
the tumor heterogeneity, both TNBC tissues and cell lines
dominantly expressed CD70 and TNFSF9 (Figure 4D).
Analysis based on TCGA-based dataset for human BC
tissues revealed that BL-BC subtype representing TNBC
highly expressed the following receptors: IL1R2, IL1RAP,
IL1RL2, IL12RB2, IL15RA, IL17RD, IL18R1, IL20RB,
IL22RA1, IL22RA2, and IL27RA (Figure 3A and 3B).
HER2-BC subtype highly expressed IL13RA1 and LABC subtype highly expressed IL6ST (Figure 3A and 3B).
Both BL- and HER2-BC subtypes highly expressed the
following receptors: IL2RA, IL2RB, IL2R6, IL12RB1,
IL18RAP and IL21R (Figure 3A and 3B). However, only
IL7R (BL- and ML-TNBC) and IL27RA (BL-TNBC)
are highly expressed on human cell lines (Figure 3C and
Supplementary Figure 2). Based on the intersection of
interleukin receptor superfamily signature between human
BC tissues and cell lines (Figure 3D) to exclude the tumor
heterogeneity, IL27RA was predominantly expressed in
both TNBC tissues and cell lines (Figure 3D).
TNFRSF21 is mainly expressed in human BLBC subtype and TNBC cells
We further assessed the TNF receptor superfamily
signature in human breast tumor tissues and cells lines.
Analysis on TCGA-based dataset for human BC tissues
showed that BL-BC subtype representing TNBC highly
expressed FAS, LTBR, TNFRSF10B, TNFRSF10D,
TNFRSF11A, TNFRSF13B, TNFRSF21, and TNFRSF25
(Figure 5A and 5B). LA-BC subtype highly expressed
CD70 and TNFSF9 are predominantly expressed
in human BL-BC subtype and TNBC cells
We checked the signature of TNF superfamily in
human breast tumor tissues and cells lines. According
Figure 1: Acute-phase protein signatures in BC tissues and cell lines. (A) Heatmap for APP expression profiles in human
BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of APP expression intensity in human BC tissues. (C)
Heatmap for RNA expression levels of APPs based on analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using
Gitools 2.3.1. (D) Intersection of APP signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high
expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and
HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
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SAA has multiple receptors, including the FPR2, the TLRs
TLR2 and TLR4, the scavenger receptor SR-BI, and the
ATP receptor P2X7 [47]. Analysis on TCGA-based dataset
for human BC tissues revealed that the BL-BC subtype
representing TNBC highly expressed SCARB1, TLR1,
TLR2, and TLR6, while LA-BC subtype dominantly
expressed TLR3 (Figure 6A and 6B). Both BL and HER2subtypes highly expressed TLR8 and TLR9 (Figure 6A and
6B). In addition, the analysis of NCBI GEO dataset on
human BC cell lines revealed that TLR1 and TLR3 were
highly expressed in BL-TNBC, while TLR2 was highly
expressed in BL- and ML-TNBC and HER2 subtype
(Figure 6C and Supplementary Figure 3). Based on the
intersection of SAA receptors and TLR superfamily
signature between human BC tissues and cell lines (Figure
6D) to exclude the tumor heterogeneity, TLR1 and TLR2
were highly expressed in both TNBC tissues and cell lines
(Figure 6D).
NGFR, TNFRSF10C, TNFRSF14 and TNFRSF19
(Figure 5A and 5B). Both BL- and HER2-BC subtypes
highly expressed TNFRSF4, TNFRSF8, TNFRSF9,
TNFRSF10A, TNFRSF12A and TNFRSF17 (Figure 5A
and 5B). However, our analysis of NCBI GEO dataset on
51 human BC cell lines showed that only TNFRSF21 is
highly expressed in BL- and ML-TNBC cells (Figure 5C
and Supplementary Figure 3). Based on the intersection
of TNF receptor superfamily signature between human
BC tissues and cell lines (Figure 5D) to exclude the
tumor heterogeneity, both TNBC tissues and cell lines
dominantly expressed TNFRSF21 (Figure 5D).
TLR1 and TLR2 are predominantly expressed in
human BL-BC subtype and TNBC cells
We investigated the SAA receptor and TLR family
signature in human breast tumor tissues and cell lines.
Figure 2: Interleukin superfamily signatures in BC tissues and cell lines. (A) Heatmap for IL superfamily expression profiles
in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of IL superfamily expression intensity in human
BC tissues. (C) Heatmap for RNA expression levels of IL superfamily based on analysis of GEO dataset (Accession: GSE12777) with
51 human BC cell lines using Gitools 2.3.1. (D) Intersection of IL superfamily signature between human BC tissues and cell lines. Red,
yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify
high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triplenegative breast cancer.
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IL-1β augments SAA1 promoter activity via both
NF-κB like and consensus sites
SAA1-P319 containing κB-like and consensus sites have
a similar induction level, while SAA1-P139 containing
only the κB-consensus site has a lower induction level
(Figure 7B). This result suggests that both κB-like and
consensus sites is critical in regulating IL-1β-induced
SAA1 promoter activity in TNBC cells. Based on SAA1P319LUC (−319/+43) promoter generated previously
[23], we further mutated each κB-site in the promoter
to investigate which NF-κB sites could be critical in
regulating IL-1β-induced SAA1 promoter activity. IL1β fully induced SAA1-P319 promoter activity, while
all of mutants abrogated IL-1β-induced effects (Figure
7C). The mutation of κB-like site at -279/-288 region
abrogated IL-1β-induced SAA1 promoter activity
without decreasing the basal activity, while the mutation
of κB-consensus site at -97/-87 region abrogated both
basal and IL-1β-induced activity (Figure 7C). The κBconsensus site at the proximal region -97/-87 appeared
to be more critical site in regulating SAA1 even by
eliminating IL-1β-induced SAA1 promoter activity,
compared to the distal κB-like site (Figure 7C).
We utilized the Align Sequences Nucleotide and
Protein BLAST (https://blast.ncbi.nlm.nih.gov) to
check the identities of promoters, mRNAs and proteins
of SAA1 and SAA2. The identity of SAA1 and SAA2
is 88% with the same NF-κB like (-287/-278) and
consensus (-95/-86) sites (Figure 7A). The identities
of mRNAs and proteins for SAA1 and SAA2α are
97% and 95%, SAA1 and SAA2β are 97% and 95%,
while the identities of SAA2α and SAA2β are 99%
and 99%, respectively (Figure 7A). SAA is restrictedly
regulated by NF-κB signaling [21–23] and TNBC
cells highly express IL1A and IL1B (Figure 2) which
lead to production of IL-1α and IL-1β. We confirmed
IL-1β-induced SAA1 promoter activity in TNBC cells
to validate a high level of SAA1/2 in TNBC cells.
SAA1-P401, SAA1-P319 and SAA1-P139 deletions
were induced by IL-1β, whereas SAA1-P85 without κB
sites was not induced (Figure 7B). The SAA1-P401 and
Figure 3: Interleukin receptor superfamily signatures in BC tissues and cell lines. (A) Heatmap for IL receptor superfamily
expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of IL receptor superfamily
expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of IL receptor superfamily based on analysis of GEO
dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of IL receptor superfamily signature
between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and
LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like,
LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
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TLR1/2-mediated signaling is involved in
regulating IL-1β-induced SAA and SAA1induced CXCL8 promoter activity in TNBC
cells
coefficient of determination (R2) between SAA1/2 and
TNBC-dominant IL and TNF subfamilies in 51 human
BC cell lines as follows: R2 = 0.86 with IL1A, R2 = 0.88
with IL1B, R2 = 0.59 with CXCL8, R2 = 0.62 with IL32, R2
= 0.08 with IL27RA, R2 = 0.017 with CD70, R2 = 0.11 with
TNFSF9, R2 = 0.027 with TNFRSF21, R2 = 0.002 with
TLR1, and R2 = 0.002 with TLR2 (Supplementary Figure
4). Furthermore, we evaluated Kaplan-Meier overall
survival (OS) for SAA1/2, TLR2 and IL8/CXCL8 which
were highly expressed in ML-TNBC. The high expression
levels of SAA1/2 (HR: 2.29, 95% CI: 1.04–5.04), TLR2
(HR: 2.78, 95% CI: 1.25–6.18) and IL8/CXCL8 (HR: 2.88,
95% CI: 1.31–6.36) were associated with poor OS in MLTNBC patients (Figure 8C).
Because both TLR1 and TLR2 among the SAA
receptors are highly expressed in TNBC cells (Figure 6),
we utilized Cu-CPT22, antagonist of both TLR1 and
TLR2, to confirm the involvement of TLR1/2 in IL-1βinduced SAA1 promoter activity. Cu-CPT22 significantly
reduced IL-1β-induced SAA1 promoter activity in MDAMB231 cells (Figure 8A). IL-8/CXCL8, a well-known
proinflammatory chemokine [48], were highly expressed
in human BL-BC tissues and TNBC cells (Figure 2).
Using the previously generated CXCL8 promoter [48],
we investigated if SAA1 could induce CXCL8 promoter
activity. SAA1 fully induced the CXCL8 promoter
activity in MDA-MB231 cells (Figure 8B). Furthermore,
Cu-CPT22 partially reduced SAA1-induced CXCL8
promoter activity (Figure 8B). In addition, we analyzed
DISCUSSION
A main finding in this study is that interaction
of SAA and proinflammatory cytokines is potentiated
in TNBC compared to other BC subtypes, probably
Figure 4: TNF superfamily signatures in BC tissues and cell lines. (A) Heatmap for TNF superfamily expression profiles in
human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of TNF superfamily expression intensity in human
BC tissues. (C) Heatmap for RNA expression levels of TNF superfamily based on analysis of GEO dataset (Accession: GSE12777) with
51 human BC cell lines using Gitools 2.3.1. (D) Intersection of TNF superfamily signature between human BC tissues and cell lines. Red,
yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify
high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like, LAR; luminal androgen receptor and TNBC; triplenegative breast cancer.
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leading to inflammatory tumor environment followed
by cancer progression and a high mortality. Among
expression profiles of APPs, SAA1/2 is emerging as
a novel biomarker in TNBC. SAA is an acute-phase
protein known to mediate proinflammatory response
and generated primarily in the liver. Serum of healthy
donors expresses SAA at relatively low levels [49]. SAA
protein levels were higher in patients with ER-negative
breast tumors compared to those with ER-positive [50],
supporting our findings of higher levels of SAA1/2 in
TNBC (Figure 1 and Supplementary Figure 1). High
levels of SAA were related to survival time of patients
less than one year with breast invasive ductal carcinoma
as a useful marker in BC recurrence [51]. Elevated
SAA was associated with reduced OS in BC [52]. On
the other hand, SAA was linked to poor recurrence-free
survival in BC but not OS [53]. Our results indicate that
SAA is associated with poor OS in ML-TNBC subtype
(Figure 8C) but not all of BC. Interestingly, elevated SAA
in tumor-associated macrophage and breast tumor cells
was associated with both lymphovascular invasion and
lymph node metastasis [53]. In a BC mouse model, ectopic
expression of SAA1 or SAA3 in tumor cells potently
promoted widespread metastasis [54]. SAA in stages II,
III and IV BC patients had a higher value compared to
those of the healthy, benign and stage I groups. Also, BC
patients with lymph node metastasis or distant metastasis
were found to have significantly higher SAA levels [55].
These findings indicate a metastatic effect of SAA in BC,
probably contributing to aggressiveness of TNBC which
highly expresses SAA (Figure 1 and Supplementary
Figure 1). The expression of SAA is restrictedly regulated
by proinflammatory cytokines, such as IL-1, IL-6 and
TNF [21–23, 56]. Because IL1A, IL1B, IL8/CXCL8,
and IL32 are highly expressed in TNBC (Figure 2 and
Supplementary Figure 2), these cytokines can be directly
Figure 5: TNF receptor superfamily signatures in BC tissues and cell lines. (A) Heatmap for TNF receptor superfamily
expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of TNF receptor superfamily
expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of TNF receptor superfamily based on analysis of GEO
dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of TNF receptor superfamily signature
between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-, HER2 (H2)-, LA- and
LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML; mesenchymal-like,
LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
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involved in upregulating SAA in TNBC. TNBC tumor
irradiation in a mouse model significantly increased the
plasma level of IL-1β, which was associated with lung
metastases [57]. The basal levels of IL-1β were higher in
TNBC cells compared with non-TNBC cells and IL-1β
increased invasiveness of TNBC cells [58]. IL-1β also
significantly increases IL-8/CXCL8 in TNBC cells [59].
IL-1β was found to induce SAA1 promoter activity in an
NF-κB-dependent manner in TNBC cells (Figure 7) and
SAA was able to induce IL8/CXCL8 promoter activity
(Figure 8B), indicating that interaction of SAA and
proinflammatory cytokines could enhance inflammatory
burden in TNBC. IL-8/CXCL8 is a blood biomarker for
TNBC, significantly increased in TNBC cells compared
to non-TNBC, and increase the invasiveness and growth
of TNBC cells [60–62]. IL-8/CXCL8 was associated
with short disease-free survival and OS in TNBC [60,
63], supporting poor OS to high levels of IL8 in MLTNBC subtype (Figure 8C). IL-32 increased migration
and invasion capacities of TNBC cells [64] and promoted
TNBC cell growth [65]. In addition, IL-32 is a potential
immunotherapy target antigen in HLA-A2-positive
TNBC [66]. Based on these results, a high level of IL32
in TNBC (Figure 2) may be involved in aggressiveness of
TNBC despite unclear direct relationship between SAA
and IL32. Although IL27RA is found to be predominantly
expressed in BL-TNBC (Figure 3 and Supplementary
Figure 2), a functional role of IL27RA in BC is poorly
understood at this point. CD70 expression levels were
significantly higher in BL-BC compared to LA [67],
supporting our findings of high levels of CD70 in TNBC
(Figure 4 and Supplementary Figure 3). A functional role
of CD70 in BC is poorly understood at this point. TNFSF9
(4-1BB/CD137) antibody favored the propagation of
CD8+ tumor-infiltrating lymphocytes (TILs) from TNBC
tumors, being capable of cytotoxic functions [68]. This
indicates that high levels of TNFSF9 in TNBC (Figure 4
and Supplementary Figure 3) might inhibit CD8+TILs to
reduce cytotoxicity, leading to enhanced aggressiveness of
TNBC. TNFRSF21 (DR6) level is known to be increased
in a grade-dependent manner in BC [69], although the
functional role of TNFRSF21 in TNBC is still unclear.
Based on the correlations of APPs with both
dominant IL and TNF superfamily in TNBC found by
the present studies and other studies, the interrelationship
between SAA, IL-1 and IL8/CXCL8 appears to be a main
driver in aggressiveness of TNBC. IL-1β-induced SAA1
promoter activity is critically involved in NF-κB-mediated
Figure 6: SAA receptor and TLR family signatures in BC tissues and cell lines. (A) Heatmap for SAA receptor and TLR
family expression profiles in human BC tissues from TCGA-based dataset using Gitools 2.3.1. (B) Statistical analysis of SAA receptor and
TLR family expression intensity in human BC tissues. (C) Heatmap for RNA expression levels of SAA receptor and TLR family based on
analysis of GEO dataset (Accession: GSE12777) with 51 human BC cell lines using Gitools 2.3.1. (D) Intersection of SAA receptor and
TLR family signature between human BC tissues and cell lines. Red, yellow, blue and green dots specify high expression levels in BL-,
HER2 (H2)-, LA- and LB-BC subtypes, respectively. Pink letters specify high expression levels in both BL- and HER2-BC subtypes. ML;
mesenchymal-like, LAR; luminal androgen receptor and TNBC; triple-negative breast cancer.
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pathway (Figure 7A and 7B) which upregulates IL8/
CXCL8 [48]. IL-8/CXCL8 secretion by SAA correlates
with NF-κB activation through FPRL1/LXA4R, a G
protein-coupled receptor, in neutrophils [70]. SAA induced
IL-8 in monocytes and dendritic cells [71] and peripheral
blood mononuclear cells (PBMCs) [72]. SAA1 induced
IL-8/CXCL8 via TLR4-mediated NF-κB signaling in
human bone marrow-derived mesenchymal stem cells
(hMSCs) [73]. In addition, SAA released TNF, IL-1β and
IL-8 in neutrophils [16]. Although TLR2 among multiple
receptors of SAA cells are highly expressed in TNBC
(Figure 6), the interrelation between TLR2 and TNBC is
poorly understood. We demonstrate that IL-1β-induced
SAA1 and SAA1-induced CXCL8 promoter activities are
partially mediated by TLR2 signaling in MDA-MB231
cells (Figure 8A and 8B). Other studies have also shown
that SAA could activate NF-κB via TLR2-mediated
pathway in HeLa cells and mouse macrophages [72,
73]. TLR2 is expressed in normal mammary epithelia
and inhibition of TLR2 reduces growth of human
BC cells [74]. Expression of TLR2 was increased in
circulating tumor cell-positive patients [75]. TLR2 was
highly expressed by two-fold in mammary cancer stem
cells and inhibition of TLR2 signaling impaired in vitro
mammosphere generation in MDA-MB-231 cells [76].
TLR2 mediates invasion by activating NF-κB in MDA-
Figure 7: IL-1β increases human SAA1 promoter activity via NF-κB signaling. (A) DNA sequence and homology of
the human SAA1 and SAA2 promoters. (B) Effects of IL-1β on luciferase activity in deletion constructs of the SAA1 promoter. After
transfection with deletion constructs of SAA1 (SAA1-P401, SAA1-P319, SAA1-P139 and SAA1-P85) luciferase vectors in MDA-MB231
TNBC cells overnight, a luciferase promoter activity assay was performed at post-treatment of IL-1β (10 ng/ml) for 6 h. (C) Effects of
IL-1β on luciferase activity in NF-κB mutated constructs of the SAA1 promoter. Site-directed mutants were generated from the SAA1P319LUC using primers with mutant κB-like sites (-287/-278) and κB-consensus site (-95/-86). After transfection with SAA1-P319LUC
and its mutant κB-site luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay was performed at
post-treatment of IL-1β (10 ng/ml) for 6 h. Results were normalized to the protein level and expressed as a fold increase compared to
non-treated control. Gray circles indicate κB site mutants. *, ** indicate significant (p < 0.05) increase compared to each control, when a
Student’s-t test was analyzed. Also, significant (p < 0.05) change exists between * and ** groups. Representative results are shown from
triplicated experiments.
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MB-231 cells [77]. Based on these findings, high levels
of TLR2 in TNBC (Figure 6) might be closely involved
in aggressiveness of TNBC, presenting poor OS to TLR2
in ML-TNBC (Figure 8C). Furthermore, SAA is known to
activate the NF-κB signaling pathway in TLR2-dependent
manner in other model systems [78–85]. Coefficient of
determination indicates that SAA1/2 are associated with
proinflammatory cytokines such as IL1 and IL8/CXCL8 in
human BC cell lines (Supplementary Figure 4).
In conclusion, IL-1-induced SAA could predispose
proinflammatory tumor microenvironment in TNBC,
leading to aggressiveness of TNBC followed by a higher
mortality.
MATERIALS AND METHODS
Reagents
Recombinant human (rh) proteins and inhibitors
were purchased as follows: IL-1β from Life Technologies
(Carlsbad, CA, USA) and Apo-SAA1 from Peprotech
Inc. (Rocky Hill, NJ, USA) and Cu-CPT22 (TLR1 and
TLR2 inhibitor) from MilliporeSigma (St. Louis, MO,
USA). Antisense and sense oligonucleotides were obtained
from Eurofins MWG Operon (Huntsville, AL, USA).
Lipofectamine 2000 and all liquid culture media were
acquired from Invitrogen (Grand Island, NY, USA). The
Figure 8: Abrogated effects of Cu-CPT22, a TLR1/2 inhibitor, on IL-1β-induced SAA1 and SAA1-induced CXCL8
promoter activities and overall survival (OS) of SAA1/2, TLR2 and CXCL8 expression levels. (A) Effect of Cu-CPT22, a
TLR1/2 inhibitor, on IL-1β-induced SAA1 promoter activity. After transfection with SAA1-P319 luciferase vectors in MDA-MB231 TNBC
cells overnight, a luciferase promoter activity assay was performed at post-treatment of IL-1β (10 ng/ml) for 6 h with a pre-treatment of CuCPT22 (1 μM) for 0.5 h. (B) Effect of Cu-CPT22, a TLR1/2 inhibitor, on SAA1-induced CXCL8/IL8 promoter activity. After transfection
with human CXCL8 promoter (-322/+10) luciferase vectors in MDA-MB231 TNBC cells overnight, a luciferase promoter activity assay
was performed at post-treatment of recombinant human SAA1 (500 ng/mL) for 6 h with a pre-treatment of Cu-CPT22 (1 μM) for 0.5 h.
Results were normalized to the protein level and expressed as a fold increase compared to non-treated control. *, # indicate significant (p ≤
0.05) increase and decrease, respectively, when ANOVA test was analyzed. Representative results are shown from triplicated experiments.
(C) Kaplan-Meier OS for SAA1/2, TLR2 and CXCL8 in ML-TNBC patients (n = 73). The black and red lines indicate low and high
expression levels, respectively.
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instructions (Stratagene, La Jolla, CA). The mutant
constructs of the SAA1 promoter were confirmed by DNA
sequencing analysis. Human CXCL8 promoter (-322/+10)
was generated as described previously [48].
Luciferase Reporter Assay System was obtained from
Promega (Madison, WI, USA).
Data analysis from GEO and the TCGA datasets
Data analysis was performed using publicly
available microarray data sets deposited in NCBI-GEO
(http://www.ncbi.nlm.nih.gov/geo/) database under
accession number GSE12777. Raw microarray data
for APPs, IL family and TNF superfamily were RNA
expression levels prepared from 51 human breast cancer
cell lines. The basal acute-phase proteins, cytokines
and their receptors expression levels were determined
by global gene expression profiling of BC cell lines,
while molecular subtyping was determined using gene
expression and HER2 status by fluorescent in situ
hybridization. We utilized Gitools 2.3.1 (http://www.
gitools.org) based on Oracle Java 7, an open-source tool
to perform Genomic Data Analysis and Visualization as
interactive heat-maps [86]. Breast invasive carcinoma
dataset for TCGA individual projects was used for BC
subtypes as follows: n = 140 for BL-BC, n = 67 for HER2BC, n = 419 for LA-BC and n = 192 for LB-BC patients
(http://www.gitools.org/datasets/tcga).
Transient transfection and luciferase assay
TNBC MDA-MB231 cells at approximately 50%
confluency in 24-well plates were washed once with fresh
media without additives and then transiently transfected
with SAA1 constructs and κB-site mutants for 24 h at
37°C using Lipofectamine solution. Transfected cells were
treated as outlined in Results and incubated for 6 h. After
rinsing cells with cold 1X PBS and adding lysis buffer
(Promega, Madison, WI), cell lysates mixed with BrightGloTM Assay Reagent (Promega, Madison, WI) were used
for determination of luciferase activity using a microplate
luminometer. Luciferase activity, expressed as relative
light units, was normalized to measured protein levels.
Overall survival analysis
Kaplan-Meier plotter database was utilized to
assess overall survival (OS) using proportional hazards
regression to estimate Hazard Ratio (HR) and 95%
Confidence Interval (CI) with auto computed cutoff value
based on the gene expression levels of SAA1/2, TLR2 and
IL8/CXCL8 from 73 ML-TNBC patients downloaded
from GEO (Affymetrix HGU133A and HGU133+2
microarrays) [87].
Construction of the SAA1 promoter, its deletion
constructs and the SAA1 κB-site mutants
Human SAA1 (−490/+43) promoter was generated
as described previously [23]. The following primers were
designed: 5′-GGG ATT ATA GGA GTG AGC CAC-3′ for
sense and 5′-CTC CTC ACC TGA TCT GTG CTG-3′ for
antisense. The PCR was performed for 35 cycles at 94°C
for 1 min, 58°C for 1 min and 74°C for 1 min, followed
by a final extension at 74°C for 10 min. The amplified
SAA1 DNA fragment was then subcloned into pGEM-T
easy vector (Promega, Madison, WI). Deletion constructs
of pGL4.12 Luciferase Reporter Vector were produced
from the SAA1 DNA fragment inserted in pGEM-T Easy
Vector under the same PCR conditions using the following
primers containing the XhoI and Hind III sites: : 5′-TAA
CTC GAG ATC TGC CAT GTG GCC CAG CAG-3′ for
SAA1-P401, 5′-TAA CTC GAG ACA CCT TCC AGC
AGC CCA GGT-3′ for SAA1-P319, 5′-GCA CTC GAG
CCA GGA ACT TGT CTT AGA CCG-3′ for SAA1-P139,
and 5′-TAC CTC GAG CCA GGG ACC ACA TCC AGC
TTT-3′ for SAA1-P85.
We found two κB sites in SAA1 promoter and
generated mutant constructs by mutating each κB site.
Primers for mutation of κB-consensus site (lowercase)
were designed as follows: 5′-TGA CCT GCA aGG ACT
TTC tCC AGG GAC CAC-3′ for -104/-74 and κBlike site; 5′-TGC CGC CAT CAC aGG GCT CCt ACT
CTC AAC-3′ for -299/-269. The mutation of κB sites
was performed by PCR-based mutagenesis using a sitedirected mutagenesis kit according to manufacturer's
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Statistics
Data were analyzed by coefficient of determination
(R2), the paired Student’s t-test and one-way analysis
of variance (ANOVA) as appropriate. If statistical
significance (p ≤ 0.05) was determined by ANOVA,
the data were further analyzed by Tukey’s pairwise
comparisons to detect specific differences between
treatments.
Abbreviations
ANOVA, one-way analysis of variance; APPs,
acute-phase proteins; BC: breast cancer; BL, basal-like;
CI, Confidence Interval; EMT, epithelial-to-mesenchymal
transition; ER, estrogen receptor; GEO: gene expression
omnibus; HER2, epidermal growth factor receptor 2;
hMSCs, human bone marrow-derived mesenchymal
stem cells; HR, Hazard Ratio; IL, interleukin; LA,
luminal A; LAR, luminal androgen; LB, luminal B;
ML, mesenchymal-like; NCBI: National Center for
Biotechnology Information; NF, nuclear factor; OS,
overall survival; PBMCs, peripheral blood mononuclear
cells; PR, progesterone receptor; SAA, serum amyloid
A; TCGA: The Cancer Genome Atlas; TILs, tumor521
Oncotarget
tumors: implication for a role in ovarian tumorigenesis.
J Histochem Cytochem. 2010; 58:1015–23. https://doi.
org/10.1369/jhc.2010.956821.
infiltrating lymphocytes; TNBC, triple negative breast
cancer; TNF, tumor necrosis factor.
Author contributions
6. Yamamoto K, Migita S. Complete primary structures of
two major murine serum amyloid A proteins deduced
from cDNA sequences. Proc Natl Acad Sci U S A. 1985;
82:2915–9.
SK, EL, SA, DS: Conceived and designed the
experiments; RI, DS: Performed the experiments; RI, SK,
EL, DS: Analyzed the data; RI, CG, SK, EL, SA, DS:
Wrote the paper.
7. Kluve-Beckerman B, Drumm ML, Benson MD.
Nonexpression of the human serum amyloid A three
(SAA3) gene. DNA Cell Biol. 1991; 10:651–61. https://doi.
org/10.1089/dna.1991.10.651.
CONFLICTS OF INTEREST
8. Steel DM, Sellar GC, Uhlar CM, Simon S, DeBeer FC,
Whitehead AS. A constitutively expressed serum amyloid
A protein gene (SAA4) is closely linked to, and shares
structural similarities with, an acute-phase serum amyloid
A protein gene (SAA2). Genomics. 1993; 16:447–54.
https://doi.org/10.1006/geno.1993.1209.
The authors declare no conflicts of interest in this
work.
GRANT SUPPORT
9. Uhlar CM, Whitehead AS. Serum amyloid A, the major
vertebrate acute-phase reactant. Eur J Biochem. 1999;
265:501–23.
This research was supported by National
Institutes of Health through the following grants:
R01ES024756 (E.L.), NIMHD U54MD007593 (S.E.A),
U54CA16306 (S.E.A), NIAID SC1AI089073 (D.S.)
and NCI SC1CA200519 (D.S.). Its contents are solely
the responsibility of the authors and do not necessarily
represent the official views of NIH. S.K was supported
by the National Research Foundation of Korea (NRF2015R1A2A2A01003472, NRF-2014M3A6A4075058,
NRF-2015R1A2A1A15051472) as BK21 plus project
fund.
10. Urieli-Shoval S, Linke RP, Matzner Y. Expression and
function of serum amyloid A, a major acute-phase protein,
in normal and disease states. Curr Opin Hematol. 2000;
7:64–9.
11. Chiba T, Han CY, Vaisar T, Shimokado K, Kargi A, Chen
MH, Wang S, McDonald TO, O’Brien KD, Heinecke
JW, Chait A. Serum amyloid A3 does not contribute to
circulating SAA levels. J Lipid Res. 2009; 50:1353–62.
https://doi.org/10.1194/jlr.M900089-JLR200.
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