Expression and in vitro assessment of tumorigenicity for NOD1 and NOD2 receptors in breast cancer cell lines
Velloso et al. BMC Res Notes
Expression and in vitro assessment of tumorigenicity for NOD1 and NOD2 receptors in breast cancer cell lines
Fernando J. Velloso 1
Mari Cleide Sogayar 1
Ricardo G. Correa 0
0 Sanford Burnham Prebys Medical Discovery Institute , 10901 North Torrey Pines Rd., La Jolla, CA 92037 , USA
1 Cell and Molecular Therapy Center (NUCEL-NETCEM), Internal Medicine Department, School of Medicine, University of São Paulo (USP) , São Paulo, SP 05360-130 , Brazil
Objective: Immune-related pathways have been frequently associated to tumorigenesis. NOD1 and NOD2 are innate immune receptors responsible for sensing a subset of bacterial-derived components, and to further translate these pathogenic signals through pro-inflammatory and survival pathways. NOD1 and NOD2 have been further associated with tumorigenesis, particularly in gastrointestinal cancers. NOD1 has also been suggested to be a tumor suppressor gene in a model of estrogen receptor-dependent breast cancer. Contrarily, NOD2 polymorphisms are associated with higher risk of breast cancer, with no tumor suppressor role being reported. To better delineate this issue, we investigated NOD1 and NOD2 expression in a panel of breast cancer cell lines, as well as their potential impact in breast tumorigenesis based on in vitro assays. Results: The highly invasive Hs578T breast cell line presented the second highest NOD1 expression and the lowest NOD2 expression in our panel. Therefore, we investigated whether NOD1 and/or NOD2 might act as a tumor suppressor in this cell model. Our studies indicate that overexpression of either NOD1 or NOD2 reduces cell proliferation and increases clonogenic potential in vitro. Elucidation of NOD1 and NOD2 effects on tumor cell viability and proliferation may unveil potential targets for future therapeutic intervention.
NOD1; NOD2; NLR; Hs578T; Breast cancer; γ-Tri-DAP; MDP
Breast cancer is the malignancy with the highest
incidence in women worldwide, accounting for 29% of
all diagnosed cancers in females [
]. Despite clinical
improvements in diagnosis and treatment, breast
cancer remains the leading cause of cancer mortality among
women, representing 14% of all deaths from cancer in
], mostly associated to metastatic tumors [
Breast cancer is classified according to
immunohistological detection of protein markers, including receptors
for estrogen (ER), progesterone (PR), androgen (AR), and
the amplified HER2 (Human Epidermal Growth Factor
Receptor 2) receptor [
]. Approximately 15% of all breast
tumors lack expression of ER, PR and amplification of
HER2, being therefore classified as triple negative breast
cancers (TNBC). The absence of well-defined molecular
targets, such as ERα and PR, prevents the use of selective
drug therapies, rendering TNBC the most lethal type of
breast cancer [
Inflammation is an important underlying factor for
cancer development [
]. In a number of tissues, including
breast, tumor onset and progression have been
associated to immune-related molecules, such as interleukins,
caspases and a set of cytosolic receptors called NLRs
(NACHT and Leucine Rich Repeat domain containing
proteins). NLRs are pattern recognition receptors (PRRs)
which recognize both pathogen-associated molecular
patterns (PAMPs) and danger associated molecular
patterns (DAMPs), thus acting as innate immunity
“sensors” towards pathogen-derived components and cellular
damage/stress. NOD1 and NOD2 (nucleotide-binding
oligomerization domain-containing protein 1 and 2) are
two major NLRs that directly modulate
pro-inflammatory pathways, including NF-κB and MAPK [
and NOD2 display variable tandem C-terminal
leucinerich repeat domains (LRRs), which are responsible for
ligand recognition and allow these receptors to detect
the bacterial peptidoglycan iE-DAP
(gamma-d-glutamylmeso-diaminopimelic acid) and MDP (muramyl
dipeptide), respectively [
]. Ligand-bound NOD1 and NOD2
recruit RIP2, which activates the IKK complex towards
NF-κB and stress kinase cascades through MAPKs [
Persistent activation and deficiency of NOD1 and
NOD2 receptors have been associated to
gastrointestinal cancers [
]. In other tissues, including breast, NOD1
and NOD2 knockdown models display increased
predisposition for tumorigenesis [
]. Additionally, NOD1
and NOD2 polymorphisms have been associated to
increased risk for several cancer types, including breast
]. In the estrogen-dependent MCF7 breast
cancer cell line, NOD1 activation was shown to promote
RIP2 and caspase 8-mediated apoptosis and to reduce
estrogen-induced proliferative responses in vitro .
Likewise, the absence of NOD1 leads to increased
sensitivity to estrogen-induced cell proliferation and a failure
to undergo NOD1-dependent apoptosis. Upon injection
into a severe combined immune deficiency (SCID) mouse
xenograft model, MCF7 cells lacking NOD1 displayed
increased estrogen-dependent tumor growth [
Moreover, NOD1 overexpression halted estrogen-dependent
tumor proliferation. Therefore, NOD1 has been proposed
to act as a tumor suppressor gene in ER-positive cells.
Contrarily, NOD2 activation does not induce apoptosis
in this cell line [
In order to determine whether NOD1 and/or NOD2
play significant roles in the onset and progression of
breast cancer, here we evaluated the expression of NOD1
and NOD2 in a panel of progressively invasive breast
cancer-derived cell lineages. In addition, we analyzed
the impact of NOD1 and NOD2 overexpression in breast
cancer, based on cell proliferation and clonogenic assays.
NOD1 and NOD2 expression profiling in breast cancer derived
Expression profiling was obtained for a panel of breast
cancer derived cell lines, including non-tumorigenic
MCF10A (ATCC®: CRL-10317™; ER−/PR−/AR−/
HER2−) and MCF12A (ATCC®: CRL-10782™; ER−/
PR−/AR+/HER2−), estrogen-positive MCF-7 (ATCC
® HTB-22™; ER+/PR+/AR+/HER2−) and ZR-75-1
(ATCC® CRL-1500™; ER+/PR+/AR+/HER2+), and
estrogen-negative SK-BR-3 (ATCC® HTB-30™; ER−/
PR−/AR+/HER2+), MDA-MB-231 (ATCC® HTB-26™;
ER−/PR−/AR+/HER2−) and Hs578T (ATCC®
HTB126™; ER−/PR−/AR+/HER2−). Cells lines were
obtained from ATCC (American Type Culture
Collection) and analyzed at low passages (3–6) to avoid genetic
drift aberrations. Replicated experiments were carried
out with cells at increasing sequential passages.
Total RNA was isolated using Trizol reagent (Thermo),
followed by DNAse I treatment (Thermo) for 20 min at
37 °C. Reverse transcription was performed using
Superscript III polymerase (Thermo), according to
manufacturer’s protocols. RT-qPCR was carried out using FAST
SYBR Green Master Mix (Thermo) in a ViiA 7 Real-Time
PCR System (Thermo). Transcript amount
quantification was calculated using the Comparative CT Method
]), based on three technical replicates, with the
QuantStudio™ Software V1.3 (Thermo). Graph design
and statistical analyses were performed in Graphpad
Prism V6 (Graphpad Software). Primers for the following
human genes were synthesized (IDT): NOD1 (forward:
(forward: 5′-AAATCAGGTTGCCGATCTTCA-3′; reverse:
5′-TGGACCTGGTTGTTCACTCCTT3′; reverse: 5′-CAACAGCATCATGAGGGTTTTC-3′).
Overexpression of NOD1 and NOD2 in Hs578T breast
NOD1 and NOD2 cDNAs were previously subcloned into
a lentiviral 6xHis-FLAG-containing vector, co-expressing
EGFP under an IRES sequence [
production followed as previously described [
]. Hs578T cells
were transduced by spinfection (MOI > 5). Transduced
GFP-positive cells were sorted using a FACS Aria II flow
cytometer (BD Biosciences).
Ectopic proteins were detected by immunoblotting
using mouse monoclonal anti-FLAG antibody (ab18230,
Abcam). Reversible Ponceau staining was used to control
the equal loading of protein lysates.
5 × 103 cells from each population were cultured in
3.8 cm2 wells (12-well plate) for 6 days. Cells were
harvested every 24 h and the total cell number was obtained
using the Accuri C6 Plus flow cytometry system (BD
Biosciences). Statistical analysis was carried out using
Graphpad Prism V6 (Graphpad Software), with two-way
repeated measures ANOVA test (n = 3; 0.05 alpha),
querying the cell populations as the source of variation.
Population doubling time (PDT), was calculated
from the equation Δt × [ln2/(lnNt − lnN0)], where Δt
is the duration of cell proliferation (exponential phase)
in hours, and N0 and Nt are the respective numbers of
cells at the beginning and end of this period [
Colony formation assay in solid substrate
2 × 102 cells from each population were cultured
in 9.5 cm2 wells (6-well plate) for 12 days. Colonies
were counted after fixation (4% formaldehyde; Sigma
Aldrich) and staining (0.05% crystal violet).
Statistical analysis was performed using two-tailed, unpaired,
student’s t-test (n = 3; 0.05 alpha) (Graphpad Prism
Colony formation assay in soft‑agar substrate
1 × 104 cells from each population were seeded in
1.9 cm2 wells (24-well plate) previously covered with
0.5 mL DMEM growth medium containing 0.6% agar.
After seeding, a layer of 0.5 mL 0.3% agar DMEM was
added and allowed to gellify before addition of 0.5 mL
DMEM per well. Cultures were maintained for 14 days.
Colonies were fixed in 4% formaldehyde and counted.
Statistical analysis was performed using two-tailed,
unpaired, student’s t-test (n = 4; 0.05 alpha) (Graphpad
NOD1 and NOD2 are differentially expressed in breast cancer
Figure 1 shows the expression profiles of NOD1 and
NOD2 genes in a panel of breast cancer-derived cell lines,
including estrogen-positive (MCF-7 and ZR-75-1) and
estrogen-negative (MCF10A, MCF12A, SK-BR-3,
MDAMB-231 and Hs578T) cell lines. We found that NOD1
and NOD2 expression varies among these cell lines, with
no clear pattern discriminating estrogen
receptor-positive or negative groups. Interestingly, the Hs578T cell line
presented the highest expression of NOD1 and the
lowest expression of NOD2 in our panel. Due to its specific
characteristics, namely, its tumorigenic potential and
TNBC origin [
], we decided to overexpress NOD1 or
NOD2 in Hs578T cells for further functional studies.
Overexpression status of NOD1/2 in transduced Hs578T cells
Hs578T/NOD1 and Hs578T/NOD2 populations were
generated, overexpressing high amounts of ectopic
NOD1 and NOD2 at both mRNA and protein levels
(Fig. 2a, b). A control population, expressing only GFP,
displayed NOD1 and NOD2 expression levels similar to
those of the wild-type Hs578T cell line (Fig. 2a). Since
Hs578T/NOD cells co-express GFP [
], we used
flow cytometry to ensure over 97% enrichment in
GFPpositive cell content (Fig. 2c).
Ponceau S Anti-FLAG
Fig. 2 Hs578T/NOD1 and Hs578T/NOD2 populations highly express respective transgenes and display a lower proliferative rate. a Relative NOD1
and NOD2 transcripts detection by RT-qPCR in Hs578T cell populations after transduction with lentiviruses for overexpression of GFP, NOD1
or NOD2. Normalization was performed as indicated in Fig. 1 (mean + standard deviation, n = 3). b Ponceau S staining and anti-FLAG immunoblot
detection of overexpressed GFP (arrowhead), NOD1 and NOD2 (arrow) proteins in transduced H578T populations. c Detection of GFP-positive
cells in Hs578T populations overexpressing NOD genes by flow cytometry (FSC-A vs FITC). Positive cells are gated inside the upper right quadrant,
representing over 97% of all transduced populations. d Growth curves comparing the proliferative rate of the wild-type versus transduced Hs578T
populations overexpressing GFP, NOD1 or NOD2. 5x103 cells were seeded in 3.8 cm2 wells (12-well plate) and cultured for six days. Total cell number
per well is presented at each time point (two-way ANOVA, n=3)
Hs578T/NOD1 and Hs578T/NOD2 populations display
a lower proliferation rate
In vitro assays were carried out, with cell populations
overexpressing NOD1/2, to assess their proliferative
potential and viability. Hs578T/NOD1 and Hs578T/
NOD2 displayed decreased proliferative rates when
compared to wild-type (Hs578T) and GFP-only control cells
(two-way ANOVA, P ≤ 0.005, n = 3). Control populations
(wild-type and GFP-transduced cells) displayed
statistically identical proliferative rates, with doubling times of
24.9 and 23.9 h, respectively. Intriguingly, the Hs578T/
NOD2 population displayed the lowest growth rate, even
when compared to Hs578T/NOD1 cells (33.4 vs. 28.9 h,
respectively) (Fig. 2d).
Hs578T/NOD1 and Hs578T/NOD2 cell populations display
higher cellular viability
We employed in vitro colony formation assays to infer
tumor growth and viability of the
NOD1/2-overexpressing populations. Upon seeding on solid substrate,
the control populations presented statistically similar
number of colonies per well. However, Hs578T/NOD1
cells showed an increased number of colonies formed per
well (unpaired student’s t-test; P < 0.05; n = 3) when
compared to the controls. This effect was further enhanced by
treatment with 5 μg/mL γ-tri-DAP, a NOD1-specific
agonist (unpaired student’s t-test; P ≤ 0.01; n = 3). Hs578T/
NOD2 displayed a non-statistical tendency for increased
number of colonies in the absence of any treatment, and
a statistically significant increase in colony number upon
treatment with 5 μg/mL MDP, a NOD2-specific agonist
(unpaired student’s t-test; P ≤ 0.01; n = 3) (Fig. 3a, b).
Hs578T/NOD1 and Hs578T/NOD2 display higher anchorage‑independent growth
The ability to form colonies in soft agar, a close in vitro
approximation of the in vivo tumorigenic potential, was
also investigated. As shown in Fig. 3c, both Hs578T/
NOD1 and Hs578T/NOD2 cells displayed an increased
number of colonies in semi-solid substrate (unpaired
student’s t-test; P < 0.01; n = 4; unpaired student’s t-test;
P ≤ 0.001; n = 4). Again, this effect was further enhanced
by treatment with 5 μg/mL γ-tri-DAP (unpaired student’s
t-test; P ≤ 0.05; n = 4) or 5 μg/mL MDP (unpaired
student’s t-test; P ≤ 0.0001; n = 4) (Fig. 3c).
Here, we show that NOD1 and NOD2 display variable
expression levels in a panel of estrogen receptor-positive
and estrogen receptor-negative breast cancer cell lines.
NOD1 was previously described as a possible tumor
suppressor gene in an estrogen receptor-positive
cellular model, acting through the blockage of ERα [
elected the triple-negative Hs578T cell line to investigate
whether the same effect would be observed in an
estrogen receptor-negative cell line, by generating transduced
cells overexpressing NOD1 or NOD2 receptors. In the
aforementioned report on the estrogen
receptor-positive model, NOD1 overexpression had no effect on cell
proliferation, whereas silencing of this receptor
actually increased the estrogen-dependent cell proliferation.
In our model, the NOD1 overexpressing cells displayed
a decreased proliferative rate in vitro. Remarkably,
NOD2 overexpression caused an even more pronounced
decrease in cell proliferation. Therefore, our data
suggest that both NOD1 and NOD2 signals may regulate cell
proliferation in this TNBC model, through a presumptive
Our in vitro colony formation assays indicated an
increased viability and cell growth rate in NOD1- and
NOD2-overexpressing Hs578T populations. An even
more pronounced effect was found for NOD2 towards
cell proliferation and colony formation. The impact of
NOD1/2 overexpression in colony formation was also
increased by specific agonists, further indicating that
activation of these receptors is directly linked to the
effects observed. The apparent contradiction between
proliferation and colony formation in our model may
be explained by the variety of signals triggered by
NOD receptors in downstream pathways. The effects
observed in cell proliferation may be a direct result of
signals towards MAPK pathway, thus modulating cell
cycle checkpoints. On the other hand, colony formation
may be affected by signaling pathways related to NF-κB,
increasing cellular viability through apoptotic escape or
promoting independence from contact and anchorage
signals such as ECAD (E-cadherin) [
(Intercellular adhesion molecule 1) [
] and VCAM1 (Vascular
cell adhesion Molecule 1) [
Given the incidence and high mortality rate of TNBC,
decoding sub-pathways through which NOD receptors
modulate cell proliferation may offer potential new
targets for future therapeutic interventions.
A more conclusive assessment of NOD1/2 levels could
benefit from western blot detection. However, due to
their typically limited protein levels in a vast number of
cell models, endogenous NOD1 and NOD2 amounts are
usually evaluated by RT-qPCR [
15, 17, 25, 26
]. As an
example, immunoblot detection of NOD1 in HCT-116
cells, a bonafide cell model for NOD1/2 signaling, can
only be achieved after immunoprecipitation and
enrichment with a second NOD1 specific antibody .
Additionally, generating Hs578T populations with null
expression of NOD1 and/or NOD2 would provide a
valuable functional corroboration model to complement our
overexpression results. Also, determining the sensitivity
to apoptotic induction in our overexpressing populations
could allow a better understanding of the pathways
participating in the cellular effects observed.
AR: Androgen receptor; DAMP: Danger-associated molecular patterns; ECAD:
E-cadherin; ER: Estrogen receptor; ERα: Estrogen receptor alpha; HER2: Human
epidermal growth factor receptor 2; iE-DAP:
Gamma-d-glutamyl-mesodiaminopimelic acid; IKK: Inhibitor of κB (IκB) kinase; LRR: Leucine-rich repeat
domain; MAPK: Mitogen-activated protein kinase; MDP: Muramyl dipeptide;
NLR: NACHT and Leucine-Rich Repeat domain containing protein; NOD1:
Nucleotide-binding Oligomerization Domain-containing protein 1; NOD2:
Nucleotide-binding Oligomerization Domain-containing protein 2; PAMP:
Pathogen-associated molecular pattern; PR: Progesterone receptor; PRR:
Pattern recognition receptor; SCID: Severe combined immune deficiency; TNBC:
Triple negative breast cancer; γ-tri-DAP: l -Ala-y-d-Glu-mDAP; VCAM1: Vascular
cell adhesion molecule 1.
FJV performed most of the wet-lab and statistical analyses, under RGC
coordination. FJV and RGC wrote the manuscript. MCS and RGC outlined the original
research proposal, discussed the results and revised the manuscript. All
authors participated in manuscript preparation. All authors read and approved
the final manuscript.
The authors thank Dr. Carlos D. Pereira (Butantan Institute, Sao Paulo, Brazil) for
sharing cell lineages used in this study.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The present work was fully supported by the “Science without Borders”
Program from CAPES (Federal Agency for Superior Education and Training,
Brazil). F.J.V was also supported by grants from CAPES. M.C.S. was additionally
supported by grants from FAPESP (São Paulo State Foundation for Research),
CNPq (National Research Council), BNDES (Brazilian National Bank for
Economic and Social Development), FINEP (Funding Authority for Studies and
Projects), MCTI (Science, Technology and Innovation Ministry) and MS-DECIT
(Science and Technology Department of the Health Ministry). R.G.C. was
supported by a Special Visiting Researcher (PVE) grant from the “Science without
Borders” Program (CAPES).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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