Wyatt et al. Breast Cancer Research(2019) 21:131
https://doi.org/10.1186/s13058-019-1224-y

RESEARCH ARTICLE

Open Access

Cross-talk between SIM2s and NFκB
regulates cyclooxygenase 2 expression inbreast cancer
Garhett L. Wyatt1, Lyndsey S. Crump2,3, Chloe M. Young2,3, Veronica M. Wessells2,3, Cole M. McQueen1,
Steven W. Wall1, Tanya L. Gustafson1, Yang-Yi Fan4, Robert S. Chapkin4, Weston W. Porter1* and Traci R. Lyons2,3*

Abstract
Background: Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is anincreasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges
in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway
contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of thispathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast
carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here,
we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated ininhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2.
Methods: For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter
activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin
immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments,
MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent
tumors harvested for immunostaining and analysis.
Results: Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter
assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction invarious NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling
proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of
SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that
NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 isnegated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB
sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s
decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in reexpression of SIM2.
Conclusion: Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.
Keywords: SIM2, COX-2, NFκB, Inflammation, Breast cancer, Prostaglandin E2* Correspondence: wporter@cvm.tamu.edu; traci.lyons@ucdenver.edu
1
Department of Integrative Biosciences, College of Veterinary Medicine, Texas
A&M University, College Station, TX, USA
2
Department of Medicine, Division of Medical Oncology, University ofColorado Anschutz Medical Campus, Aurora, USA
Full list of author information is available at the end of the article© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Wyatt et al. Breast Cancer Research(2019) 21:131

Background
Ductal carcinoma in situ (DCIS) is a heterogeneous disease characterized by tumor cells that are confined tothe ductal system of the breast [1]. DCIS progresses toinvasive ductal carcinoma (IDC) through events such asepithelial mesenchymal transition (EMT), basement
membrane degradation, inflammatory signaling, andother pathways associated with a wound-healing milieu[2–4]. It is estimated that ~ 20% of mammographydetected breast cancers are DCIS [5] and ~ 65,000 casesof DCIS are diagnosed per year [6]. Provided that DCIS
is removed surgically, as is the standard of care, awoman diagnosed with DCIS without recurrence is more
likely to die of other causes than of breast cancer [7].
However, it is estimated that ~ 15–20% of DCIS patientsdevelop invasive disease within a decade [8, 9]. Recently
identified risk factors for DCIS recurrence include age <40 at diagnosis, African American ethnicity, hormone
receptor negativity, and HER2 positivity [7]. However,these high-risk groups only account for 20% of the DCIS
patient population [9]. Therefore, identifying additionalrisk factors for, or markers that will predict, DCIS
aggressiveness is an extremely important goal for preventing invasive cancer in DCIS patients.
There is increasing evidence that inflammation plays a
key role in breast cancer progression [10]. One such specific inflammatory pathway is nuclear factor kappa b(NFκB). The NFκB signaling pathway includes five members: NFκB1 (p105/p50), NFκB2 (p100/p52), RelA (p65),
RelB, and c-Rel. Dimers of the aforementioned proteins
are held in the cytoplasm by inhibitor kappaB kinase(IκB) proteins, primarily IκBα. The mechanism of activation of NFκB requires phosphorylation of IκBα by inhibitor of kappaB kinase (most commonly IKKα and IKKβ),
which leads to degradation of IκBα. Upon degradation of
the IκBα, NFκB heterodimers, specifically the canonicalheterodimer p50/p65, translocate to the nucleus andbind to promoters of target genes, leading to activationof their transcription [11, 12]. Known transcriptional targets of NFκB include mediators of inflammation, such as
cytokines/chemokines and immunoreceptors, as well asproteins involved in antigen presentation, cell adhesion,stress response, apoptosis, growth factor receptor signaling, and transcription [13]. Two NFκB consensus sites
are located in the promoter region of the human PTGS2
gene, which encodes for pro-inflammatory enzyme cyclooxygenase 2 (COX-2) [14]. These NFκB consensus
sites contribute not only to cancer progression by preventing apoptosis but also to the activation of COX-2mediated signaling [15]. COX-2 is the inducible form of
cyclooxygenase, which is the key enzyme involved in thebiosynthesis of the pro-inflammatory prostaglandins[16–21]. Importantly, COX-2 has been implicated in
DCIS progression through promotion of proliferation,

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migration, invasion, and metastatic spread in pre-clinicalmodels [22–24]. Additionally, expression of COX-2 isfrequently observed in patients with invasive disease andis associated with DCIS recurrence. Furthermore, thetherapeutic benefit of inhibiting COX-2 has been observed in the colon, esophagus, lung, bladder, breast,and prostate cancers [18, 19, 25–35]. Thus, it is logicalto expect that inhibition of COX-2 signaling in breast
cancer patients could enhance overall prognosis.
Previously, we have shown that Singleminded-2s(SIM2s; expressed from SIM2), a member of the bHLH/
PAS family of transcription factors, is a tumor suppressor that is expressed in breast epithelial cells and downregulated in the transition from DCIS to IDC [36–39].
Specifically, using the MCF10-DCIS.COM progression
model, we demonstrated that re-expression of SIM2s inhibits growth, invasive phenotypes, and progression tometastasis. Furthermore, overexpression of SIM2s inbreast cancer cells promotes a more luminal-like phenotype, whereas downregulation of SIM2s leads to increased invasive potential [39]. Consistent with the rolefor SIM2s in cancer progression, we have also shownthat the NFκB signaling pathway is negatively regulated
by SIM2s in normal mammary tissues during postpartum mammary involution [40], which has been identified
as a driver of tumor progression and metastasis. In thisstudy, we demonstrate a relationship between SIM2s,
the NFκB signaling pathway, and COX-2 in breast cancer cells. We suggest that re-expression of SIM2s could
be mediated by inhibition of COX-2 signaling, which
may serve to reduce breast cancer progression.

Methods
Cell culture

MCF7 and SUM159 cells were purchased from American Type Culture Collection (ATCC) and were maintained in accordance to their guidelines. MCF10ADCIS.COM (DCIS.COM) cells were generously donatedby Dr. Dan Medina (Baylor College of Medicine, Houston, TX, USA). Cells were plated into 6-well plates for
RNA isolation experiments according the guidelines
from ThermoFisher Scientific. Celecoxib experiments
were performed as follows: cells were first dosed with
10 μM celecoxib for 24 h, then media was changed andtreatment was performed at 20 μM celecoxib for 24 h,and then cells were harvested for analysis. DHA andPGE2 experiments on cell lines were performed as follows: cells were dosed with 50 μM DHA or 100 μM for
24 h and then harvested for analysis.
Generation of cell lines

Point mutations in the SIM2 gene were generated via
long cDNA synthesis (Invitrogen). Plasmids were amplified using Subcloning Efficiency DH5a competent cells

Wyatt et al. Breast Cancer Research(2019) 21:131

Page 3 of 12(Life Technologies). Plasmid DNA was isolated using the
HiPure Plasmid Maxiprep Kit (Life Technologies) or the
ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Viral transduction was performed as previously
described [38]. Puromycin selection (2 μg/mL) wasstarted the following day and maintained for a week.

Chromatin immunoprecipitation

Animal models

Oncomine analysis

Two hundred thousand DCIS.COM cells stably expressing
anti-COX-2 shRNAs (a generous gift from Kornelia
Polyak and Andriy Marusyk) were orthotopically injected
and tumors harvested as previously described [22, 23].

Analysis of SIM2 in primary breast cancer versus metastasis was performed using the Oncomine software(oncomine.org). The TCGA dataset was analyzed forSIM2 with the threshold p value set at 0.05 and thethreshold fold change set at 2. Additionally, using Oncomine gene expression signatures, we evaluated the breast
cancer metastasis concept, setting the odds ratio threshold at 2 and the p value at 0.01.

RNA isolation and real-time PCR

RNA isolation and real-time PCR were performed aspreviously described [39]. Primers can be found in Additional file 1: Table S2.

For chromatin immunoprecipitation (ChIP) assays, 2 μgof plasmid containing repressor and 2 μg of plasmid containing the SIM2 promoter construct were transfected
into 293T cells in a 10-cm plate. Chromatin was harvested 2 days after transfection.

Statistical analysis
Immunoblotting

Cells were washed with cold PBS and lysed in high-salt
lysis buffer (50 mM HEPES, 500 mM NaCl, 1.5 mM
MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA),
10% glycerol, 1% Triton X-100, pH 7.5) supplemented
with 1 mM Na3VO4 (Sigma) and 1 mM complete
ULTRA tablets mini EDTA-free Easy pack (Roche). Protein concentration was determined using the DC Protein
Assay (Bio-Rad) with bovine serum albumin as a standard. Immunoblotting and zymography were then conducted as previously described [38]. Antibodies can befound in Additional file 1: Table S1. Blots were imaged
on a ChemiDoc MP (Bio-Rad) after incubating in ProSignal Pico ECL Spray (Genesee Scientific) for 3 min.
Quantification was performed using ImageJ.
Immunohistochemistry

Immunohistochemistry (IHC) for COX-2 was performed
as previously described [22]. Analysis for positive nuclei
was performed as previously described [24]. Antibodies
can be found in Additional file 1: Table S1.
Transient transfection

MCF7 or 293T cells were used for transfections for luciferase activity. One hundred nanograms (0.1 μg) of
plasmid containing transcription factor was mixed withup to 1 μg (amount varies per construct) of plasmid containing promoter construct. Three microliters of Genejuice (Novagen) was used per microgram of DNA. DNA
and Genejuice were mixed in Opti-MEM media (Invitrogen). Protein was harvested 2 days after transfection,using Reporter Lysis Buffer (Promega). Luciferase activity and total protein were measured as described previously [37]. Luciferase activities were normalized to total
protein values and are represented as the means ± SE forthree wells per condition.

To address scientific rigor, all experiments in cell lines
and xenografts were conducted at a minimum in biological triplicates and technical duplicates and were repeated three times. Normal distribution was confirmed
before conducting unpaired t test. ANOVA analysis was
performed using JMP Pro 14 to asses that means are infact different, and then the post hoc Student’s t test was
performed. Correlation analysis was performed using
Prism7; Pearson’s r and a two-tailed test were performed
to examine significance. Significance was considered at
p < 0.05 unless otherwise noted.

Results
SIM2s downregulates NFκB signaling

To test the hypothesis that SIM2 negatively regulates
NFκB/p65-mediated transcription in breast cancer cells,
we co-transfected a reporter plasmid encoding a NFκB
binding site upstream of the luciferase gene (5x NFκBluc) with the p65 subunit along with SIM2s in MCF7
cells and measured relative light units as a readout for
NFκB activity. As expected, we observed that p65
strongly activated the reporter construct, but this response was blocked by co-transfection of SIM2s (Fig. 1a).
Furthermore, we repeated the 5x NFκB-luc transfectionin MCF7 cells with stable transduction of a SIM2 shRNA or control plasmid (Additional file 1: Figure S1). In
the shSIM2 cells, the 5x NFκB-luc was significantly
increased over the controls (Fig. 1b). To determine
whether inhibition of the 5x NFκB-luc reporter by
SIM2s was dependent on the transcriptional repressor
activity of SIM2s, the transfection was repeated with aSIM2s expression construct missing the Pro/Ala transcriptional repression domain (SIM2sΔR). Interestingly,
this construct also significantly attenuated the activationof the 5x NFκB-luc construct by NFκB/p65, demonstrating that the repression domain of SIM2s is not requiredWyatt et al. Breast Cancer Research(2019) 21:131

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Fig. 1 a Luciferase activity in MCF7 cells co-transfected with 5x kB binding sites upstream of the luciferase gene (5x NFκB-luc) and NFkB p65 and/or SIM2s. (Diagram of promoter construct is shown above for reference.) b Luciferase activity in MCF7 control or MCF7 SIM2-shRNA cells with 5x
NFκB-luc. c Luciferase activity in MCF7 cells co-transfected with 5x NFκB-luc and NFkB p65 and/or SIM2s with its repression domain deleted(SIM2sΔR). d SUM159 plpcx emp (control), SUM159 plpcx SIM2s-FLAG (overexpression), MCF7 psil SCR (control), and MCF7 psil SIM2-shRNA(knockdown) were analyzed by western blot for levels of IKKα, IKKβ, phospho-p65, p65, and beta actin as a loading control. e SUM159 plpcx emp(control), SUM159 plpcx SIM2s-FLAG (overexpression), MCF7 psil SCR (control), and MCF7 psil SIM2-shRNA (knockdown) were analyzed by western
blot for levels of phospho-AKTs473, pan AKT, and GAPDH as loading control. ANOVA and Student’s t test were performed to test significance. a,b, c All significant at p < 0.05, *p < 0.05. Analysis was performed via ImageJ for comparison of protein expression

for inhibition of NFκB signaling (Fig. 1c; Additional file
1: Figure S2A,B). As an alternative, we performed immunoblot analysis of key players in the NFκB signaling
pathway to determine whether SIM2 modulates expression levels of key mediators of NFκB/p65 signaling inour breast cancer cell lines that could downregulate signaling in a posttranscriptional/posttranslational manner.
We found that IKKα, IKKβ, phosphorylated-p65, andp65 protein expression, all of which mediate NFκB activation, are decreased in SIM2s overexpressing SUM159

cells (Fig. 1d). Similarly, we found that these same NFκB
pathway protein levels are increased in SIM2s knockdown MCF7 cells (Fig. 1d). These results suggest that
SIM2s may affect NFκB-mediated transcription via
modulation of protein expression and/or phosphorylationof key mediators of NFκB signaling. Akt is a known activator of NFκB signaling through its ability to phosphorylate
and activate IKKα/IKKβ, which leads to nuclear translocation [41, 42]. Thus, we analyzed whether activation/phosphorylation of Akt was modulated by SIM2 expression.

Wyatt et al. Breast Cancer Research(2019) 21:131

Indeed, we observed that overexpression of SIM2s results
in a modest decrease in pAkt, while SIM2s knockdown
strongly restored pAkt. Together, our results suggest aSIM2s-mediated negative regulation of NFκB/p65 involvesde-activation of Akt signaling.
NFκB signaling downregulates SIM2s expression

Unexpectedly, we also observed that stable overexpression of inhibitor kappa kinase beta (IKKβ), which normally functions to phosphorylate IκBα in the cytoplasm,
allowing for activation of NFκB-mediated signaling, significantly decreases SIM2s gene expression in the DCIS.COM cells suggesting a reciprocal relationship between
NFκB and SIM2s (Fig. 2a). Conversely, when we suppressed NFκB activation via stable transduction of theinhibitor kappaB alpha super repressor (IκB-SR), which
maintains the NFκB heterodimer (p50/p65) in the cytosol, SIM2s expression was increased (Fig. 2b). To confirm that activation of NFκB downregulates SIM2
expression in breast cancer cells, we cloned a 2-kb portion of the SIM2 promoter upstream of the luciferase
gene and co-transfected with increasing amounts of p65
in MCF7 cells. We observed dose-dependent repressionof SIM2s promoter activity (Fig. 2c; Additional file 1:
Figure S2C). Importantly, co-transfection with IκB-SR,
as well as IκB-SR with NFκB p65, successfully reversed
the downregulation of SIM2 promoter activity (Fig. 2d;
Additional file 1: Figure S2D), suggesting that this
response was not a dominant negative effect. Analysis of
the SIM2 promoter identified two consensus NFκB binding sites near the transcriptional start site for SIM2. Utilizing ChIP analysis, we showed that p65 directly binds
to the SIM2 promoter around the transcriptional startsite (Fig. 2e). To determine whether binding of p65 to
the NFκB binding sites is necessary for downregulationof SIM2s expression, we mutated the two NFκB sites in
the SIM2 promoter reporter construct and performed
additional co-transfection experiments with p65. The
NFκB double mutant site promoter failed to inhibit
SIM2 promoter activity when compared to the wild-type
promoter (Fig. 2f; Additional file 1: Figure S2E), implicating a direct interaction of NFκB/p65 on the SIM2
promoter to repress SIM2s transcription. These results
suggest that NFκB-mediated transcriptional repressionof SIM2s may serve to reverse SIM2-mediated downregulation of NFκB signaling, allowing for its activation andpromotion of transcription of known pro-inflammatory
target genes such as PTGS2.
SIM2s expression downregulates COX2

To explore the relationship between SIM2s and PTGS2
expression in breast cancer, we analyzed three different
breast cancer cell lines including MCF7, DCIS.COM,
and SUM159 cells. The non-invasive MCF7 cell line andPage 5 of 12

the highly invasive triple-negative SUM159 cell line were
utilized to examine the differential expression of SIM2s,
and subsequent regulation of PTGS2, as it relates to invasion. DCIS.COM cells (also triple negative) were used
for their unique ability to mimic basal-like DCIS in vivo
and their ability to progress to invasive disease upon acquisition of COX-2 protein expression [22, 43]. We havepreviously shown that the invasive competent DCIS.COM cells have more SIM2s expression when comparedwith the non-invasive MCF7 [37, 38]. Confirming andextending this observation, qPCR analysis reveals thelowest PTGS2 expression in MCF7 cells, which was increased 130-fold in DCIS.COM cells and the highest in
the SUM159 cells (Additional file 1: Figure S3). To determine whether reduction of SIM2s in the non-invasive
cells could increase expression of PTGS2, we analyzed
control and shRNA-SIM2s DCIS.COM and MCF7 cells
by qPCR. Our results revealed that downregulation of
SIM2s led to a significant increase in PTGS2 gene expression in both cell lines (Fig. 3a, b). Moreover, wefound that overexpression of SIM2s in highly invasive
SUM159 cells significantly inhibited PTGS2 expression(Fig. 3c). In previous studies, we showed that overexpression of SIM2s in DCIS.COM cells blocked invasionin vivo, whereas loss of SIM2s or overexpression of theprotein product of PTGS2, COX-2, resulted in increased
invasion and metastasis [22, 39]. To determine the relationship between SIM2s and COX-2 protein expressionin vivo, we performed IHC analysis for COX-2 in tumors
derived from control and SIM2s DCIS.COM xenografts
to reveal that COX-2 levels were decreased with overexpression of SIM2s (Fig. 3d). Taken together, our results
suggest that SIM2s may repress invasion in the DCIS.COM model by promoting downregulation of COX-2.
COX-2 downregulation restores SIM2s

Since the invasive potential in DCIS.COM positively correlates with, and depends upon, expression and activityof COX-2 [22, 44], we tested the hypothesis that the lossof invasive phenotype observed with blocking of COX-2
expression was due, in part, to re-expression of SIM2s.
Thus, we measured SIM2 protein levels by IHC analysisof tumors generated from control and shPTGS2 DCIS.COM cells, which are less invasive [22]. Our results reveal an increase in positive nuclear staining for SIM2
with PTGS2 knockdown (Fig. 4a, b; Additional file 1:
Figure S4A). We also observed a significant negative correlation between expression of SIM2 and COX-2 andconfirmed increased SIM2 in shPTGS2 DCIS.COM andcontrol cells via immunoblot (Fig. 4c, d; Additional file
1: Figure S4B). Additionally, in this study, 87.5% of tumors in the control group had progressed to fully invasive disease compared with 25% in the shPTGS2 group(Fig. 4e). To determine whether COX-2 activity drives

Wyatt et al. Breast Cancer Research(2019) 21:131

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Fig. 2 a SIM2s expression in DCIS.COM control cells and cells overexpressing IKKβ by qPCR as fold change. b SIM2s expression in DCIS.COM
control cells and cells overexpressing IκB-SR by qPCR as fold change. c SIM2s promoter activity in MCF7 cells co-transfected with SIM2 promoter
upstream of the luciferase gene and increasing amounts of NFκB p65 (50 ng, 100 ng, and 150 ng). d SIM2s promoter activity in MCF7 cells afterco-transfection of promoter with control vector (pcDNA3), NFκB p65, and/or IκB-SR. e ChIP assay for NFκB p65 binding after transient transfectionof SIM2 promoter with NFκB p65 in HEK293T cells. f SIM2s promoter activity in MCF7 cells co-transfected with SIM2s promoter upstream of theluciferase gene and 150 ng NFκB p65 compared with the SIM2s promoter activity in MCF7 cells co-transfected with NFκB double mutant SIM2s
promoter upstream of the luciferase gene. ANOVA and Student’s t test were performed to test significance. a, b All significant at p < 0.05,
unpaired t test: *p < 0.05

the inverse relationship between SIM2 and COX-2 andcell invasion, we treated the highly invasive SUM159
cells with a dose of the selective COX-2 inhibitor celecoxib that had previously been shown to decrease invasion of COX-2-expressing cells [22]. We observed asignificant increase in SIM2s expression (Fig. 4f). Additionally, celecoxib also inhibited activation of the 5x
NFκB-luc reporter MCF7 cells, suggesting that COX-2
activity can also feedback to promote NFκB-mediatedsuppression of SIM2s (Fig. 4g). Indeed, treatment of
MCF7 cells with PGE2, the product of COX-2 activity,
inhibited SIM2s gene expression (Fig. 4g). Furthermore,
we extend our findings to show that docosahexaenoic(DHA), a n-3 polyunsaturated fatty acid (PUFA) that canalso result in a shift to a more anti-inflammatory gene
expression profile [45] and can reduce COX-2 expression [46–49], significantly increases SIM2s expression(Fig. 4i). Thus, our driving hypothesis is that reductionof inflammatory pathways via inhibition of activity and/or decreased COX-2 expression results in re-expressionof SIM2s and may be one mechanism for preventing
progression of DCIS to invasive or metastatic breast
cancer [23]. Consistent with this hypothesis, Oncomine
analysis reveals that SIM2s is in the top 5–10% of
under-expressed genes in a breast cancer metastasis concept signature and in the top 10% of copy number lossWyatt et al. Breast Cancer Research(2019) 21:131

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Fig. 3 a PTGS2 expression in MCF7 control cells and cells overexpressing SIM2s by real-time qPCR as fold change. b PTGS2 expression in
DCIS.COM control cells and cells with SIM2-shRNA by real-time qPCR as fold change. c PTGS2 expression in SUM159 control cells and cells
overexpressing SIM2s by real-time qPCR as fold change. d Immunohistochemistry for COX-2 in DCIS.COM cells stable transduced with control
vector or SIM2s-FLAG (overexpression). Unpaired t test: *p < 0.05

genes (Table 1). Further, overall expression is significantly lower in a small number of metastasis in thisdataset when compared to the expression in the primaries (Additional file 1: Figure S5), supporting our previous
studies showing loss of SIM2s with DCIS progression to
IDC [39, 50]. Thus, our results may be relevant for preventing metastasis in women with breast cancer.

Discussion
Through transgenic mouse models and in vitro studies,SIM2s has been identified as a novel player in several
key aspects of mammary gland development. Specifically, genetic ablation of SIM2s in mammary epithelial
cells revealed that SIM2s is required for ductal morphogenesis and differentiation of luminal cells for milk
production during lactation. Furthermore, mammaryspecific overexpression of SIM2s resulted in a delay inpost-lactational mammary gland involution throughsuppression of Stat3 and NFκB signaling, as well as
maintenance of markers of epithelial cell differentiation
normally observed only during lactation. These results
suggest that SIM2s has tumor-suppressive activities in
the mammary gland through maintenance of epithelial

cell differentiation. Consistent with this, loss of SIM2s
expression in the mammary epithelium results in EMT
events, such as loss of E-cadherin and increases inmatrix metalloprotease activity, and similar results arealso observed in breast cancer cell lines. SIM2s is also
downregulated in breast cancer patient samples, further
validating its potential role in tumor suppression [39].
In this study, we demonstrate a novel role for SIM2s as
a negative regulator of tumorigenesis via downregulation of the NFκB pathway, which normally results intranscriptional activation and expression of the proinflammatory/pro-tumorigenic enzyme COX-2, which
in turn promotes DCIS invasion. Interestingly, we also
identify a novel link between SIM2s and preventing
signaling of the pro-tumor/pro-survival kinase Akt,
which has been shown to promote tumorigenesis inpart through NFκB-mediated COX-2 expression [51].
Additionally, we also show that SIM2s is directly targeted for suppression by NFκB signaling, suggesting a
regulatory pro-tumorigenic feedback loop. Consistent
with a role for SIM2s preventing this pro-tumorigenic
cycle, loss of SIM2s also drastically increases COX-2
expression, while loss of COX-2 activity and expression

Wyatt et al. Breast Cancer Research(2019) 21:131

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Fig. 4 a IHC analysis for COX-2 positive nuclei in tumors generated from control (Ctl) and shPTGS2 (KD2) DCIS.COM cells. Prism7 was utilized forstatistical significance. Unpaired t test, *p value < 0.02. b IHC analysis for SIM2s positive nuclei in tumors generated from control (Ctl) and shPTGS2(KD2) DCIS.com cells. Prism7 was utilized for statistical significance. Unpaired t test, *p value < 0.0001. c Correlation data for SIM2s and COX-2
positive nuclei in tumors generated from control and shPTGS2 DCIS.com cells. Prism7 was utilized for statistical significance. Unpaired t test, **pvalue < 0.01. d Images of IHC analysis for SIM2s in tumors generated from control and shPTGS2 DCIS.COM cells (left); DCIS.COM control, shPTGS2(KD1), and shPTGS2 (KD2) were analyzed by western blot for SIM2s and GAPDH as loading control (right). e Pie Chart to show percent tumor
progression DCIS+IDC or IDC only in the control group (n = 8) and shPTGS2 (n = 4). f SIM2s expression in SUM159 control cells and cells dosedwith 20 μM celecoxib by qPCR as fold change. g SIM2s expression in DCIS.COM control cells and cells dosed with 50 μM DHA by qPCR as foldchange. h SIM2s expression in MCF7 cells dosed with vehicle or 100 μM PGE2 for 24 h by qPCR, unpaired t test: p < 0.08. i SIM2s expression in
DCIS.COM cells treated with vehicle (control) or 50 μM DHA by qPCR. Unpaired t test: *p < 0.05

results in re-expression of SIM2s and downregulationof tumor cell invasion. Thus, we have identified a reciprocal relationship between a molecule with known
tumor-suppressive activities, SIM2s, and a wellestablished tumor promotional pathway that involves

pro-survival and pro-invasive signaling mediated by
Akt, NFκB, and COX-2 (Fig. 5).
Based on our previous results reporting a role for
COX-2 in promotion of DCIS invasion [22], and results
showing that SIM2 is lost in IDC compared with DCIS

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Table 1 Oncomine analysis
Concept type

Concept name

Size

DatasetExpression

Top %Oncomine gene expression
signatures

Breast cancer - metastasis - top 5% under-expressed(TCGA breast)

1019

TCGA breast

Under-expressed5

Oncomine gene expression
signatures

Breast cancer - metastasis - top 10% copy number loss
(TCGA breast 2)

1881

TCGA breast 2

Copy number loss

10

Oncomine gene expression
signatures

Breast cancer - metastasis - top 10% under-expressed(TCGA breast)

2039

TCGA breast

Under-expressed10

in patient samples [39, 50], we predict that loss of SIM2s
may be important for progression of in situ lesions to invasive disease via upregulation of COX-2 expression andactivity. Consistent with this hypothesis, in the DCIS.COM model, loss of SIM2s is associated with increased
invasiveness and enhanced tumor aggressiveness andprogression, all of which are also observed with gain of
COX-2 [22, 23, 37–40, 50]. Specifically, upon loss ofSIM2s in tumors, increased co-localization of keratin 5
and vimentin has been observed [39], which is indicativeof mesenchymal and invasive phenotypes; furthermore,gain of COX-2 results in increased collagen depositionin the tumor microenvironment, which tumor cells
utilize to invade the surrounding tissue and access thevasculature to form metastasis [22, 52, 53]. Matrix metalloproteinases (MMPs), which are associated withbasement membrane degradation during mammary
gland development and cancer, are also significantly increased with loss of SIM2s [54–56]. These changes likely
promote an increased potential for progression of DCIS
to IDC and furthermore for tumor cell escape from theprimary site. Interestingly, COX-2 has been shown topromote angiogenesis [57] and to inhibit anoikis via

Fig. 5 A model depicting SIM2s and NFκB cross-talk regulated to COX-2

activation of Akt [58], suggesting that this pathway canalso promote dissemination and survival in circulation.
Furthermore, increased COX-2 and increased proliferation are associated with subsequent recurrence of DCIS
in patients [21]. Here, we show that cells with low invasive potential exhibit increased expression of COX-2
upon knockdown of SIM2s and endogenously express
moderate levels of SIM2s compared with the low level ofSIM2s observed in the more invasive cells [37]. Likewise,
overexpression of SIM2s in invasive cells decreases
COX-2 expression. Coincidently, SIM2s overexpression
also significantly decreased COX-2 staining in tumor
sections and all point toward a role for SIM2 in preventing metastasis. This is consistent with data from the
TCGA showing downregulation of SIM2s in a metastasis
gene signature. Since it is well known in the literaturethat COX-2 inhibition is associated with better prognosis for breast cancer patients [59, 60], further studies on
strategies for re-expression of SIM2s may be beneficial
in improving prognosis of breast cancer patients. Furthermore, an additional implication is that SIM2s could
be utilized as a marker to identify DCIS patients that areof low risk for acquisition of COX-2 expression andWyatt et al. Breast Cancer Research(2019) 21:131

Page 10 of 12

progression to IDC and/or metastatic disease. However,relevance for this mechanism beyond local invasion,
such as in metastatic spread, remains an unansweredquestion that we will address with future studies.

Conclusions
These findings support a role for SIM2s in the preventionof breast cancer progression through its ability to repress
PTGS2 expression via modulating the NFκB signaling pathway. It has long been established that NFκB regulates genes
involved in cell proliferation and cell survival. Specifically,
blocking NFκB in tumor cells can lead to susceptibility to
anti-cancer agents. However, due to the complexity of thetumor microenvironment, NFκB signaling also has beenfound to have anti-cancer effects in various cancer cells.
Thus, it is important to investigate a mechanism, specifically in mammary tissue, in which the targeted pathways arehighly involved with cell proliferation, survival, migration,and invasion. Due to elevated COX-2 expression correlating with poor prognosis, it is imperative to investigate reducing COX-2/PTGS2 expression. In the data provided here,
we have demonstrated an integral role for SIM2s involvement in mediating NFκB signaling to decrease expressionof COX-2/PTGS2, which could lead to an improved prognosis for breast cancer patients.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s13058-019-1224-y.
Additional file 1: Figure S1: Verification of effective transduction
knockdown of SIM2 via sh-RNA. Infection of MCF7 cells with the SIM2shRNA construct results in decreased SIM2 protein levels in comparison
to a nonspecific scrambled shRNA construct. Figure S2: Verification of
NFkB promoter luciferase and SIM2 promoter luciferase assays in the
HEK293 cell line. A. Luciferase activity in HEK293T cells co-transfected with
5x kB binding sites upstream of the luciferase gene (5x NFkB-luc) and
NFkB p65 and/or SIM2s. (Diagram of promoter construct is shown above
for reference.) B. Luciferase activity in HEK293T cells co-transfected with
5x NFkB-luc and NFkB p65 and/or SIM2s with its repression domain deleted (SIM2sΔR). C. SIM2 promoter activity in HEK293T cells co-transfectedwith SIM2 promoter upstream of the luciferase gene and increasing
amounts of NFκB p65 (50ng,100ng, and 150ng). D. SIM2 promoter activity
in HEK293T cells after co-transfection of promoter with control vector(pcDNA3), NFκB p65, and/or IκB-SR. E. SIM2 promoter activity in HEK293T
cells co-transfected with SIM2 promoter upstream of the luciferase geneand 150ng NFκB p65 compared with the SIM2 promoter activity in
HEK293T cells co-transfected with NFκB double mutant SIM2 promoter
upstream of the luciferase gene. ANOVA and Student’s t-test was performed to test significance. A, B, C all significant at p<0.05, *p<0.05. Figure S3: Verification of PTGS2 in various breast cancer cell lines. PTGS2
expression in MCF7, DCIS.COM, and SUM159 parental breast cancer celllines. ANOVA and Student’s t-test was performed to test significance. A, B,
C all significant at p<0.05. Figure S4: No correlation between Tumor size
and SIM2s or COX-2 gene expression. A. %SIM2s positive nuclei compared to tumor size (cm2). B. %COX-2 (M+S) compared to tumor size(cm2). Prism7 was utilized for statistical significance analysis. Two-tailed ttest was performed to test significance. Figure S5: SIM2 expression in
TCGA Breast primary site and metastasis. Oncomine analysis of SIM2 expression in TCGA Breast primary site (n=529) compared to metastasis (n=3). t-Test performed to test significance, p-value=0.006. The Oncomine™

Platform (Thermo Fisher, Ann Arbor, MI) was used for analysis andvisualization. For further information, refer to the terms of use. Table S1:
Antibodies used in study. Table S2: Primer sequences used in study.
Abbreviations
ChIP: Chromatin immunoprecipitation; COX-2: Cyclooxygenase 2;
DCIS: Ductal carcinoma in situ; DHA: Docosahexaenoic acid; EMT: Epithelial
mesenchymal transition; IDC: Invasive ductal carcinoma; IKKα: Inhibitor kappab kinase alpha; IKKβ: Inhibitor kappa b kinase beta; IκB: Inhibitor kappa b;IκBα: Inhibitor kappa b alpha; IκB-SR: Inhibitor kappa b alpha super repressor;
NFκB: Nuclear factor kappa b; PUFA: Polyunsaturated fatty acids;
SIM2s: Singleminded-2s
Acknowledgements
We gratefully acknowledge Kornelia Polyak and Andriy Marusyk for providing
the shCOX-2 DCIS cell lines and Pepper Schedin for providing the shCOX2 tumors and controls from the in vivo studies. Sarah Tarullo is acknowledged
for independent/blinded confirmation of the invasion analysis.
Authors’ contributions
GW performed the in vitro experiments, formal data analysis, interpretationof the data, production of figures, writing of the original draft, and reviewing
and editing the final draft of the manuscript. CY and LSC were involved inanimal husbandry, tumor immunostaining, and immunoblot analysis. VW was
involved in designing and performing immunostaining of the xenograftstudies. CMM and SWW performed treated cells, harvested proteins,
harvested RNA, and assisted in the experimental design and western blots.
TG was involved in animal husbandry, in vivo experiments, and ChIP analysis.
YF and RC provided the polyunsaturated fatty acid resources. WP and TL
oversaw all the experiments, experimental design, and interpretation of thedata; provided the resources; and contributed to the writing as well as
reviewing and editing of the manuscript. All authors read and approved thefinal manuscript.
Funding
This research is supported by an NRSA F31CA236140 (LSC/TL), the Universityof Colorado Department of Medicine Outstanding Early Career Scholars
Program (TL), and the National Cancer Institute R21CA197896 (WP),
R01HD083952 (CO-PI WP, MR), R21CA185226 (TL), and R01CA211696 (TL).
Availability of data and materials
Not applicable
Ethics approval and consent to participate
Animal studies were approved by the Institution’s laboratory care committee.
Consent for publication
Not applicableCompeting interests
The authors declare that they have no competing interests.
Author details
Department of Integrative Biosciences, College of Veterinary Medicine, Texas
A&M University, College Station, TX, USA. 2Department of Medicine, Divisionof Medical Oncology, University of Colorado Anschutz Medical Campus,
Aurora, USA. 3The University of Colorado Cancer Center Young Women’s
Breast Cancer Translational Program, Aurora, CO, USA. 4Department of
Nutrition, Texas A&M University, College Station, TX, USA.
1

Received: 9 May 2019 Accepted: 7 November 2019

References
1. Cowell CF, et al. Progression from ductal carcinoma in situ to invasive
breast cancer: revisited. Mol Oncol. 2013;7(5):859–69.
2. Schedin P, et al. Microenvironment of the involuting mammary gland
mediates mammary cancer progression. J Mammary Gland Biol Neoplasia.
2007;12(1):71–82.

Wyatt et al. Breast Cancer Research

3.

4.
5.
6.

7.
8.

9.
10.
11.

12.

13.
14.

15.
16.

17.

18.
19.

20.
21.

22.

23.

24.
25.

26.
27.
28.
29.
30.

(2019) 21:131

Pensa S, Watson CJ, Poli V. Stat3 and the inflammation/acute phase
response in involution and breast cancer. J Mammary Gland Biol Neoplasia.
2009;14(2):121–9.
Clarkson RW, Watson CJ. Microarray analysis of the involution switch. J
Mammary Gland Biol Neoplasia. 2003;8(3):309–19.
Ernster VL, et al. Detection of ductal carcinoma in situ in women undergoing
screening mammography. J Natl Cancer Inst. 2002;94(20):1546–54.
ACS. American Cancer Society: Cancer Facts and Figures 2013. American
Cancer Society 2013 January 6, 2012; Available from: http://www.cancer.
org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-figures-2012. Accessed Aug 2018.
Narod SA, et al. Breast cancer mortality after a diagnosis of ductal
carcinoma in situ. JAMA Oncol. 2015;1(7):888–96.
Kerlikowske K, et al. Characteristics associated with recurrence among
women with ductal carcinoma in situ treated by lumpectomy. J Natl Cancer
Inst. 2003;95(22):1692–702.
Esserman L, Yau C. Rethinking the standard for ductal carcinoma in situ
treatment. JAMA Oncol. 2015;1(7):881–3.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell.
2011;144(5):646–74.
Fitzpatrick SF, et al. An intact canonical NF-kappaB pathway is required for
inflammatory gene expression in response to hypoxia. J Immunol. 2011;
186(2):1091–6.
Schmedtje JF Jr, et al. Hypoxia induces cyclooxygenase-2 via the NF-kappaB
p65 transcription factor in human vascular endothelial cells. J Biol Chem.
1997;272(1):601–8.
Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors.
Oncogene. 1999;18(49):6853–66.
Newton R, et al. Evidence for involvement of NF-kappaB in thetranscriptional control of COX-2 gene expression by IL-1beta. Biochem
Biophys Res Commun. 1997;237(1):28–32.
Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell. 2008;
132(3):344–62.
Hugo HJ, et al. New insights on COX-2 in chronic inflammation driving
breast cancer growth and metastasis. J Mammary Gland Biol Neoplasia.
2015;20(3):109–19.
Griswold DE, Adams JL. Constitutive cyclooxygenase (COX-1) and inducible
cyclooxygenase (COX-2): rationale for selective inhibition and progress todate. Med Res Rev. 1996;16(2):181–206.
Ristimaki A, et al. Prognostic significance of elevated cyclooxygenase-2
expression in breast cancer. Cancer Res. 2002;62(3):632–5.
Denkert C, et al. Elevated expression of cyclooxygenase-2 is a negative
prognostic factor for disease free survival and overall survival in patientswith breast carcinoma. Cancer. 2003;97(12):2978–87.
Spizzo G, et al. Correlation of COX-2 and Ep-CAM overexpression in humaninvasive breast cancer and its impact on survival. Br J Cancer. 2003;88(4):574–8.
Gauthier ML, et al. Abrogated response to cellular stress identifies DCIS
associated with subsequent tumor events and defines basal-like breast
tumors. Cancer Cell. 2007;12(5):479–91.
Lyons TR, et al. Postpartum mammary gland involution drives progressionof ductal carcinoma in situ through collagen and COX-2. Nat Med. 2011;17(9):1109–15.
Lyons TR, et al. Cyclooxygenase-2-dependent lymphangiogenesis promotes
nodal metastasis of postpartum breast cancer. J Clin Invest. 2014;124(9):3901–12.
Black SA, et al. Semaphorin 7a exerts pleiotropic effects to promote breast
tumor progression. Oncogene. 2016;35(39):5170–8.
Costa C, et al. Cyclo-oxygenase 2 expression is associated with angiogenesis
and lymph node metastasis in human breast cancer. J Clin Pathol. 2002;
55(6):429–34.
Esbona K, et al. COX-2 modulates mammary tumor progression in response
to collagen density. Breast Cancer Res. 2016;18(1):35.
Harris RE, Casto BC, Harris ZM. Cyclooxygenase-2 and the inflammogenesisof breast cancer. World J Clin Oncol. 2014;5(4):677–92.
Mann JR, Backlund MG, DuBois RN. Mechanisms of disease: inflammatory
mediators and cancer prevention. Nat Clin Pract Oncol. 2005;2(4):202–10.
Sheehan KM, et al. The relationship between cyclooxygenase-2 expression
and colorectal cancer. Jama. 1999;282(13):1254–7.
Visscher DW, et al. Association between cyclooxygenase-2 expression in
atypical hyperplasia and risk of breast cancer. J Natl Cancer Inst. 2008;
100(6):421–7.

Page 11 of 12

31. Bourn J, Cekanova M. Cyclooxygenase inhibitors potentiate receptor
tyrosine kinase therapies in bladder cancer cells in vitro. Drug Des Devel
Ther. 2018;12:1727–42.
32. Buttar NS, et al. Chemoprevention of esophageal adenocarcinoma by COX2 inhibitors in an animal model of Barrett’s esophagus. Gastroenterology.
2002;122(4):1101–12.
33. Wang W, Wang J. Toll-like receptor 4 (TLR4)/cyclooxygenase-2 (COX-2)
regulates prostate cancer cell proliferation, migration, and invasion by NFkappaB activation. Med Sci Monit. 2018;24:5588–97.
34. Wu C, et al. IL-1beta-mediated up-regulation of WT1D via miR-144-3p andtheir synergistic effect with NF-kappaB/COX-2/HIF-1alpha pathway on cellproliferation in LUAD. Cell Physiol Biochem. 2018;48(6):2493–502.
35. Zeng X, Yi S. Cyclooxygenase inhibitors in epithelial ovarian Cancer
treatment. Int J Gynecol Cancer. 2018;28(6):1085–9.
36. Gustafson TL, et al. Ha-Ras transformation of MCF10A cells leads torepression of Singleminded-2s through NOTCH and C/EBPbeta. Oncogene.
2009;28:1561-8.
37. Kwak HI, et al. Inhibition of breast cancer growth and invasion by
single-minded 2s. Carcinogenesis. 2007;28(2):259–66.
38. Laffin B, et al. Loss of Singleminded-2s in the mouse mammary gland
induces an epithelial-mesenchymal transition associated with up-regulationof slug and matrix metalloprotease 2. Mol Cell Biol. 2008;28(6):1936–46.
39. Scribner KC, Behbod F, Porter WW. Regulation of DCIS to invasive breast
cancer progression by Singleminded-2s (SIM2s). Oncogene. 2013;32(21):2631–9.
40. Scribner KC, et al. Singleminded-2s (Sim2s) promotes delayed involution of
the mouse mammary gland through suppression of Stat3 and NFkappaB.
Mol Endocrinol. 2011;25(4):635–44.
41. Kane LP, et al. Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol.
1999;9(11):601–4.
42. Romashkova JA, Makarov SS. NF-kappaB is a target of AKT in anti-apoptotic
PDGF signalling. Nature. 1999;401(6748):86–90.
43. Miller FR, et al. MCF10DCIS.com xenograft model of human comedo ductal
carcinoma in situ. J Natl Cancer Inst. 2000;92(14):1185–6.
44. Hu M, et al. Role of COX-2 in epithelial-stromal cell interactions andprogression of ductal carcinoma in situ of the breast. Proc Natl Acad Sci U S
A. 2009;106(9):3372–7.
45. Bouwens M, et al. Fish-oil supplementation induces antiinflammatory gene
expression profiles in human blood mononuclear cells. Am J Clin Nutr.
2009;90(2):415–24.
46. Calviello G, et al. n-3 PUFAs reduce VEGF expression in human colon cancer
cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alphainduction pathway. Carcinogenesis. 2004;25(12):2303–10.
47. Lee SA, et al. DHA and EPA down-regulate COX-2 expression throughsuppression of NF-kappaB activity in LPS-treated human umbilical vein
endothelial cells. Korean J Physiol Pharmacol. 2009;13(4):301–7.
48. Al-Jawadi A, et al. Protective properties of n-3 fatty acids and implications in
obesity-associated breast cancer. J Nutr Biochem. 2018;53:1–8.
49. Straka S, et al. Incorporation of eicosapentaenioic and docosahexaenoic
acids into breast adipose tissue of women at high risk of breast cancer: arandomized clinical trial of dietary fish and n-3 fatty acid capsules. Mol Nutr
Food Res. 2015;59(9):1780–90.
50. Pearson SJ, et al. ATM-dependent activation of SIM2s regulates homologous
recombination and epithelial-mesenchymal transition. Oncogene. 2019;38(14):2611–26.
51. St-Germain ME, et al. Regulation of COX-2 protein expression by Akt inendometrial cancer cells is mediated through NF-kappaB/IkappaB pathway.
Mol Cancer. 2004;3:7.
52. Provenzano PP, et al. Collagen density promotes mammary tumor initiationand progression. BMC Med. 2008;6:11.
53. Provenzano PP, et al. Collagen reorganization at the tumor-stromal interface
facilitates local invasion. BMC Med. 2006;4(1):38.
54. Liu D, et al. Association between polymorphisms in the promoter regions ofmatrix metalloproteinases (MMPs) and risk of cancer metastasis: a metaanalysis. PLoS One. 2012;7(2):e31251.
55. Mendes O, Kim HT, Stoica G. Expression of MMP2, MMP9 and MMP3 in breast
cancer brain metastasis in a rat model. Clin Exp Metastasis. 2005;22(3):237–46.
56. Barajas-Castaneda LM, et al. Overexpression of MMP-3 and uPA withdiminished PAI-1 related to metastasis in ductal breast Cancer patients
attending a public hospital in Mexico City. J Immunol Res. 2016;2016:
8519648.

Wyatt et al. Breast Cancer Research(2019) 21:131

57. Gately S, Li WW. Multiple roles of COX-2 in tumor angiogenesis: a target for
antiangiogenic therapy. Semin Oncol. 2004;31(2 Suppl 7):2–11.
58. Choi EM, et al. COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway
in human bladder cancer cells. Exp Mol Med. 2005;37(3):199–203.
59. Kwan ML, et al. NSAIDs and breast cancer recurrence in a prospective
cohort study. Cancer Causes Control. 2007;18(6):613–20.
60. Li J, et al. Celecoxib in breast cancer prevention and therapy. Cancer ManagRes. 2018;10:4653–67.

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