Promotion of Cancer Cell Invasiveness and Metastasis Emergence Caused by Olfactory Receptor Stimulation

Abstract

Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by β-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase γ dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist β-ionone significantly enhanced metastasis emergence and spreading.

Full text

Promotion of Cancer Cell Invasiveness and Metastasis
Emergence Caused by Olfactory Receptor Stimulation
Guenhae
¨
l Sanz
1,2
*, Isabelle Leray
3,4
, Aure
´
lie Dewaele
1,2
, Julien Sobilo
5
, Ste
´
phanie Lerondel
5
,
Ste
´
phan Bouet
6,7,8
, Denise Gre
´
bert
1,2
,Re
´
gine Monnerie
1,2
, Edith Pajot-Augy
1,2
, Lluis M. Mir
3,4
*
1 INRA, UR1197 Neurobiologie de l’Olfaction et Mode
´
lisation en Imagerie, Jouy-en-Josas, France, 2 IFR 144 NeuroSud Paris, Gif-sur-Yvette, France, 3 CNRS, UMR8203
Vectorologie et the
´
rapeutiques anti-cance
´
reuses, Institut Gustave-Roussy, Villejuif, France, 4 Univ. Paris-Sud, UMR8203, Orsay, France, 5 CNRS-TAAM, UPS44, Centre
d’Imagerie du Petit Animal, Orle
´
ans, France, 6 INRA, UMR 1313, Ge
´
ne
´
tique Animale et Biologie Inte
´
grative, Jouy-en-Josas, France, 7 CEA, DSV, IRCM, SREIT, Laboratoire de
Radiobiologie et Etude du Ge
´
nome, Jouy-en-Josas, France, 8 AgroParisTech, UMR 1313, Ge
´
ne
´
tique Animale et Biologie Inte
´
grative, Jouy-en-Josas, France
Abstract
Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues
with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in
enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors
identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of
the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells,
expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists
promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G
protein-coupled Receptor), an endogenously overexpressed OR, by b-ionone, its odorant agonist, resulted in the same
phenotypic change. We also showed the involvement of a PI3 kinase c dependent signaling pathway in this promotion of
tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG
immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist b-ionone significantly enhanced metastasis
emergence and spreading.
Citation: Sanz G, Leray I, Dewaele A, Sobilo J, Lerondel S, et al. (2014) Promotion of Cancer Cell Invasiveness and Metastasis Emergence Caused by Olfactory
Receptor Stimulation. PLoS ONE 9(1): e85110. doi:10.1371/journal.pone.0085110
Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America
Received April 24, 2013; Accepted December 1, 2013; Published January 8, 2014
Copyright: ß 2014 Sanz et al. This is an open-access arti cle distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by INRA and CNRS (The National Centre for Scientific Research (France). This research was conducted in the scope of the LEA
EBAM (The European Associated Laboratory (LEA) entitled ‘‘Pulsed Electric Fields Applications in Biology and Medicine"). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Guenhael.Sanz@jouy.inra.fr. (GS); Luis.MIR@gustaveroussy.fr (LMM)
Introduction
Olfactory receptors (ORs) are G protein-coupled receptors
mainly expressed in olfactory sensory neurons (OSNs) of the
olfactory epithelium, where they detect and discriminate myriads
of odorants according to a combinatorial code in which an OR
can be activated by various odorants and an odorant can stimulate
various ORs [1,2]. Moreover, ORs are expressed in non-olfactory
tissues [3–5] where they can play additional roles. They notably
govern sperm chemotaxis, regulate migration and adhesion of
muscle cells, and control serotonin secretion by enterochromaffin
(EC) cells [6–10]. Several studies also reported that some ORs can
be tumor marker, one of them modifying in vitro the proliferation
of LNCaP prostate cancer cells [11,12,13,14].
In particular, EC cells can acquire a tumoral phenotype and
differentially express ORs depending on the neuroendocrine
carcinoma evolution [15]. The BON cells, a human EC cell line
derived from a metastasis of a pancreatic carcinoma [16,17], were
described to endogenously express ORs [8] which could be tumor
markers when overexpressed [15]. Because BON cells were
derived from a metastasis, we explored whether activation of
ORs by agonist odorants could have a role in tumor progression.
To this end, we decided to identify the ORs expressed in BON
cells. However the agonist or antagonist odorants specific of BON
cells ORs are unknown, like for most of the hundreds of identified
human ORs. We thus tried to develop a model by transfecting
these cells with deorphanized ORs. The heterologous expression
achieved allowed us to assess the in vitro invasiveness of these cells
upon stimulation with the odorant ligand of the transfected
receptor. Furthermore, we identified PI3 kinase cPI3Kc as a
component of the signaling pathway induced by OR stimulation
and promoting cell invasiveness. A more physiological model was
also used in vitro, the LNCaP prostate cancer cells which
overexpress the PSGR (Prostate Specific G protein-coupled
Receptor), an endogenous and deorphanized OR considered as
a tumor marker, and wich was described to inhibit the
proliferation of these cells in vitro [12]. This model was then used
in vivo to analyze the role of ORs stimulation in tumor progression,
that is in metastasis emergence and spreading.
Materials and Methods
Ethics Statement
The animals were handled in conformity with the Guidelines of
the French government regarding operative procedures and animal
care. Protocol was approved by the ethics comity for experiments
with animals named ‘‘Comite´ d’Ethique en Expe´rimentation
Animale de l’IRCIV’’ CEEA-26 (protocol number 2012-043).
PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e85110

Reverse transcription (RT)-PCR, cloning and sequencing
Total RNAs were extracted using TRIzol reagent (Invitrogen)
and treated with DNase I. RT was performed with the «
SuperScript First-StrandH, Synthesis System for RT-PCR» kit
(Invitrogen).
For single cell RT-PCR, single cells were collected by aspiration
into a glass pipette and RT was performed using the « Single Cell
Superscript
TM
III Cells Direct cDNA Synthesis System » kit
(Invitrogen) after cell disruption, protein denaturation and DNAse
treatment.
Nested PCR was carried out starting from 1
mL of RT products
and using degenerate primers targeting OR conserved regions, or
primers specifically targeting OR identified with the degenerate
primers. Degenerate primers sequences were kindly provided by
Stephan Bieri (Givaudan, Switzerland). Absence of genomic DNA
was controlled using human GAPDH or b-actin primers on
DNase I-treated RNAs without reverse transcriptase.
PCR products amplified with degenerate primers were cloned
into the pGEM-T vector (Promega) and sequenced by Beckman
Coulter Genomics. PCR products amplified with specific primers
were directly sequenced (Beckman Coulter Genomics).
Chemicals
Odorants, DMSO and mineral oil (M3516) were purchased
from Sigma-Aldrich, Fluka or Acros Organics at the highest purity
available. AS605240 was purchased from Euromedex (Selleck,
S1410) and gallein from TOCRIS bioscience. Paraffin (CellWax)
was obtained from CML, and hemalun, eosin, and safran from
RAL.
Mammalian expression vectors
OR1G1 or OR17-40 coding sequences were introduced into
the pCMV-Tag3B mammalian expression vector (Stratagene) in a
way resulting in the fusion of a cmyc epitope at the receptor N-
terminus. The resulting vectors were named pCMV-TagOR1G1
and pCMV-TagOR17-40.
Cell culture and transfection
BON cells (subclone #7) were kindly provided by Dr Courtney
M. Townsend (Department of Surgery UTMB, Galveston, TX
77551, USA) [16,17]. They were grown in DMEM/F-12 (Ham)
without phenol red (GIBCO, Invitrogen Corporation) supple-
mented with 10% fetal bovine serum (Hyclone, Perbio) and
antibiotics (100 U penicillin/mL and 100
mg streptomycin/mL,
Invitrogen), at 37uC in a humidified incubator with 5% CO
2
. Cells
were transiently transfected with pCMV-TagOR1G1 or pCMV-
TagOR17-40 using jetPEI
TM
(Polyplus-transfection) according to
the manufacturer’s instructions. OR expression at the cell surface
was checked by immunofluorescence microscopy using the
monoclonal anti-cmyc-Cy3 antibody (C6594, Sigma-Aldrich) on
non-permeabilised cells.
LNCaP cells were purchased from ATCC (Clone FGC, No.
CRL-1740
TM
) at passage 19, and grown in RPMI 1640 medium
(ATCC, No. 30-2001) supplemented with 10% fetal bovine serum
(ATCC, No. 30-2021), at 37uC in a humidified incubator with 5%
CO
2
.
Calcium imaging
BON and LNCaP cells were seeded onto a 96-well culture plate
(black microtiter plate, Greiner Bio-one), respectively at a density
of 10
5
and 0.5610
5
cells per well. 24 hours later, cells were loaded
with 2.5
mM of fluo-4 acetoxymethyl ester (Molecular Probes), as
previously described [18]. Calcium imaging was performed using
an inverted epifluorescence microscope (CK40 Olympus)
equipped with a digital camera (ORCA-ER, Hamamatsu
Photonics). Ca
2+
reponses were observed at 460–490 nm excita-
tion and $ 515 nm emission wavelengths. Data acquisition and
analysis was performed using the SimplePCI software (Hama-
matsu, Compix). Odorants and mineral oil were prepared
extemporaneously by a first dilution into DMSO and then serial
dilutions into Hanks’ salt solution (Eurobio) supplemented with
20 mM Hepes, pH 7.2. Stimuli were tested at concentrations that
do not elicit calcium responses in mock-transfected cells. 1
mM
isoproterenol (Sigma-Aldrich) was applied as a positive control.
The Ca
2+
signal was measured as the relative change in
fluorescence intensity DF/F = (F–F
0
)/F
0
, where F
0
is the
fluorescence level before stimulation. Results were expressed as
the mean of the DF/F of at least twenty cells.
In vitro assessment of cell invasion
Collagen type I gels were prepared as described by De Wever
[19]. Cells were cultured for 48 hours before seeding them as a
suspension of single cells deposited on top of the collagen type I
gels. To stimulate ORs, odorants and mineral oil were first diluted
into DMSO and then into the collagen I solution or the culture
medium. The effects of specific inhibitors of PI3Kc (AS605240)
and bc subunits of G proteins (gallein) were also assessed by
adding them to the collagen gel and culture medium. For controls,
DMSO was added at the same final concentration used to dilute
these chemicals. The amount of added DMSO did not exceed
0.2% and did not modify the number of invasive cells compared to
tests without DMSO (data not shown). 24 hours after their
seeding, invasive cells presenting invasive extensions into the
collagen gel and non-invading cells were counted in 10–15
microscope fields randomly selected. Results were expressed as the
percentage of invasive cells (invasion index). Morerover, F-actin
cytoskeleton was observed using rhodamine-conjugated phalloidin
(Invitrogen). For this purpose, cells were fixed for 20 min with 3%
paraformaldehyde in PBS at room temperature, then permeabi-
lized for 15 min with 0.5% Triton X-100 in PBS and blocked for
30 min with 2% BSA, 1% glycine in PBS. Cells were incubated
with rhodamine-phalloidin (1:300) in PBS for 30 min at room
temperature and extensively washed with PBS before observation
with an inverted epifluorescence microscope.
Mice
Nod Scid Gamma (NSG) male mice were bred in the animal
housing facilities of the Institut Gustave Roussy, with free access to
food and water. Plastic cages were connected to controlled
ventilated racks. The cages with the animals exposed to the
odorant b-ionone were connected to a separated ventilation unit.
In vivo assessment of cell invasion and metastases
LNCaP cells at passage 25 were inoculated into 8 week-old
castrated male NSG mice (castration was performed two weeks
before cell inoculation). 10
6
cells were suspended in 75 mLof
RPMI 1640 plus 75
mL of Matrigel (BD Biosciences) and injected
with a needle (26G) into the subcutaneous space, at 2 sites in each
flank of the mice. The odorant b-ionone was first diluted into
DMSO at a concentration of 100 mM and then into the RPMI +
Matrigel mixture at the final concentration of 100
mM. DMSO
was also added at the same dose to the RPMI + Matrigel mixture
without odorant. A first group of 5 mice was inoculated with
LNCaP cells (in the presence of DMSO) and received no further
treatment. Five other mice were inoculated with LNCaP cells (in
the presence of DMSO) and brushed with mineral oil three times a
day during 6 weeks and then three times a week until sacrifice. A
Olfactory Receptors Promote Tumor Cell Invasion
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third group of 5 mice was inoculated with LNCaP cells in the
presence of b-ionone in DMSO. These mice were brushed with
1mMb-ionone directly diluted in mineral oil three times a day
during 6 weeks and then three times a week until sacrifice. Before
sacrifice, some animals were first examined by tomoscintigraphy
(SPECT, NanoSPECT/CT Bioscan) using
99m
Tc-MDP, a classi-
cal bone scintigraphy agent for functional imaging of the bone.
This analysis was not performed on all animals because it
appeared less informative than X rays in our study. Thus all mice
were explored in vivo by microcomputed tomography (
mCT)
(CT120, General Electric Healthcare) to detect bone metastasis.
360 X ray projections were collected in 1u increments (100 kVp,
50 mA, 20 msec exposure) for about 5 min total scan time. Images
were reconstructed into 3D volumes (50
mm resolution) on a
reconstruction cluster using a modified tent-FDK conebeam
algorithm (GE reconstruction software). 3D data were processed
using MicroView (GE Healthcare). Data analysis was performed
first on individual slices (axial, coronal, sagittal) then on
reconstructed volumes and MIP images (Maximum Intensity
Projection). Animals were sacrificed when tumor size exceeded
1,500 mm
3
. Upon autopsy, tumors and tissues known to harbor
metastases from prostate tumors such as lymph nodes, lungs and
spines, were sampled. Livers and Tyson glands were also sampled,
some livers appearing anomalous and some Tyson glands
surprisingly large. Tissues were fixed for 24 hours in formaldehyde
then stored in 70% ethanol at 4uC. For spines, decalcification was
realized by an additional incubation in 10% EDTA, pH 7.4, at
4uC during one week. All samples were dehydrated in ethanol and
included in paraffin. Serial sections of 5
mm thickness were
prepared and dewaxed in toluene and rehydrated in ethanol and
then water. Some sections were stained with hemalun (RAL), eosin
and safran (HES staining). Immunohistochemistry was performed
on other sections using anti-PSGR (LS-A6332, Cliniscience), anti-
PSA (ab9537, abcam), or rabbit serum as a negative control, the
Vectastain Elite ABC-Peroxidase Kits Rabbit IgG (Cliniscience),
and a DAB revelation (SK-4100, Vector).
Results
ORs endogenously expr essed by BON cells
Since BON cells display an heterogeneous morphology, we
isolated homogeneous subclones. OR expression was investigated
by nested PCR on cDNAs from nine clones using degenerate
primers targeting OR conserved regions, and PCR products
sequencing. We detected ORs transcripts in six of the clones
(Table S1). Among them, five displayed expression of more than
one OR gene or pseudogene, and the panel of ORs identified
varied from clone to clone. To confirm these results, we performed
nested PCR with primers specifically targeting the previously
identified ORs. Actually all nine clones expressed ORs transcripts
(Table 1) and some of them (OR7D2, OR1F1) were found in most
of the clones. It must be highlighted that OR7A17, OR7D2 and
OR2A1 transcripts are also found in several tumors (ESTs listed in
the HORDE database).
To further assess that, contrary to OSNs, BON cells co-express
several ORs, we analyzed OR expression at the single-cell level.
We succeeded in amplifying cDNAs corresponding to GAPDH or
b-actin for most tested cells, but OR cDNAs could be amplified
only for a few cells, probably because of the very low level of OR
mRNAs at the single-cell level. Our data show that some single
BON cells do co-express more than one OR transcript (Figure
S1a).
Heterologous functional expression of ORs in BON cells
Since BON cells endogenously express ORs, we infered that
they could also heterologously express functional ORs after
transfection of the OR1G1 and OR17-40 genes. BON cells
appeared to express these heterologous receptors and to expose
them at the plasma membrane (Figure S1b). We also found in
BON cells the transcript of REEP1, a protein which facilitates OR
expression in OSNs [20] (Figure S1c). We then demonstrated that
the heterologously expressed receptors are functional, inducing a
calcium response when they are stimulated with their respective
ligand (1-nonanol for OR1G1 and helional for OR17-40 [18,21])
(Figure 1). The calcium response induced by stimulation of the
OR17-40 receptor is less pronounced than that induced by
stimulation of the OR1G1 receptor, but it remains significant.
Differences between OR response levels can be due to different
expression levels of the receptors, to a different coupling efficiency
with the endogenous G-proteins of heterologous cells, or to a
different efficiency of the ligands used. Mock-transfected cells did
not respond to nonanol nor helional, showing that the odorants
tested are not agonists of the ORs endogenously expressed in BON
cells.
OR-induced enhancement of cell invasiveness
Using BON cells heterologously expressing OR1G1 or OR17-
40 receptors, we assessed the invasiveness of collagen type I gels
[19] by these cells, stimulated or not with the odorant agonists of
these ORs. In absence of odorant stimulation, the invasiveness of
BON cells was not modified by heterologous expression of ORs
(the invasion index remains around 3%, Figure 2a). Nonanol
stimulation increased significantly the invasion index of OR1G1-
expressing cells (OR1G1 cells) by a factor of 2.7, whereas helional
stimulation increased the invasion index of OR17-40 cells by a
factor of 2.5 (Figure 2a). We observed that 10
26
and 10
27
Mof
nonanol induced the same invasion level, whereas 10
26
M
appeared more efficient in activating OR1G1 in calcium imaging
experiments. This may be due to the inability of BON cells to
reach larger invasion levels (around 10% invasive cells). Nonanol
and helional had no significant impact on mock-transfected
control cells. Nonanol had no significant effect on OR17-40
expressing cells, nor helional on OR1G1 expressing cells.
Furthermore, vanillin, an antagonist of the OR1G1 receptor
[22], was able to specifically counteract the invasiveness induced
by nonanol in OR1G1 cells. The invasion index of control cells
stimulated by nonanol alone or by a mixture of nonanol and
vanillin was unchanged (Figure 2a). Invasive cellular extensions
into collagen type I gels, characterizing the invasive cells, were also
observed after immunolabeling of the F-actin cystoskeleton (Figure
2b). All together, these results demonstrate that, in vitro, ORs
stimulation by odorants can specifically promote invasiveness of
the OR-expressing cancer cells.
We confirmed this result using the LNCaP prostate cancer cells
which endogenously express an OR, the PSGR. This receptor has
known agonist and antagonist odorants [12], respectively the b-
ionone and a-ionone. We used a 100
mM concentration of b-
ionone to stimulate LNCaP cells, since this dose was already
reported to stimulate the PSGR [12] and it induced the highest
invasiveness of LNCaP cells in our hands (data not shown). As
shown in Figure 2c, stimulation of PSGR with 100
mM b-ionone
increased invasiveness of LNCaP cells by a factor of 2.75 and this
effect was totally abrogated by the antagonist a-ionone. Alone, this
antagonist had no effect on LNCaP cells invasion level. While
there is no negative control with LNCaP cells that would not
express PSGR, the drastic pharmacological effect of a-ionone
argues in favor of a specific effect of b-ionone through PSGR
Olfactory Receptors Promote Tumor Cell Invasion
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stimulation. Exclusion of a non specific chemical effect of b-ionone
on LNCaP cells inducing invasiveness is also supported by the fact
that a-ionone, which is very similar to b-ionone and was applied at
twice the b-ionone dose, did not induce invasiveness of LNCaP
cells. Moreover, we tested the effect of 100
mM b-ionone on the
invasiveness of PC3 cells, other prostate cancer cells that do not
express the PSGR [12], and we did not observe an increased
invasiveness in these cells. Experimental results detailed below also
support the idea that LNCaP invasiveness can be enhanced
through PSGR stimulation.
Involvement of PI3Kc in cell invasiveness induced by ORs
PI3Kc activation through GPCRs can be involved in
transforming functions such as invasion [23], and a crosstalk
between odorant signaling and PI3Kc was described in olfactory
sensory neurons [24,25]. We thus explored whether PI3K c could
be part of the signaling pathway which is triggered by the odorant
activation of ORs and promotes cell invasiveness. First we showed
the expression of PI3Kc in BON and LNCaP cells by crude lysates
immunoblotting with an antibody targeting PI3Kc (data not
shown). We then assessed the invasiveness of BON cells
hetorologously expressing OR1G1 or of LNCaP cells upon
stimulation with agonists of OR1G1 or PSGR, in the presence
of a specific inhibitor of PI3Kc (AS605240). 10
26
M of AS605240
have been reported to completely inhibit PI3Kc [26]. Concerning
BON cells, using 10
26
M and 10
27
M of AS605240, we found a
similarly large (about 80%) but not total reduction of the cell
invasiveness promoted by OR1G1 upon nonanol stimulation
(Figure 3), indicating that the maximal effect is observed at 10
27
M
of AS605240. Thus, PI3Kc appears to play a major role in
mediating BON cell invasiveness promoted by the OR stimulation
by its specific odorant, even if other signaling pathways might also
be involved. Involvement of PI3Kc was confirmed for LNCaP
cells (Figure 3). However, contrary to BON cells, PI3Kc inhibitor
AS605240 induced a reduction of LNCaP invasiveness even in
absence of PSGR stimulation. Therefore PI3Kc seems to be also
involved in the basal invasiveness of LNCaP cells. Moreover, since
PI3Kc can be activated by the G
bc
subunit of the G proteins
through GPCR activation [27], we used gallein, a G
bc
subunits
inhibitor that interferes with the interaction of G
bc
subunits with
PI3Kc [28], and showed that it counteracted the enhancement of
LNCaP cell invasiveness induced by PSGR stimulation (Figure 3).
This result also supports the involvement of PI3Kc in the
invasiveness of tumor cells induced by OR stimulation.
OR activation-induced enhancement of cancer cell
invasiveness in vivo
Since in vitro enhancement of cell invasiveness by ORs activation
suggests a possible role of (at least some) ORs in metastasis
emergence in vivo, we inoculated LNCaP prostate tumor cells
subcutaneously into immunodeficient NSG (NOD scid gamma)
mice. Animals were either left untreated, or daily brushed on skin
with PSGR agonist b-ionone diluted in mineral oil (an oily
excipient needed to apply the lipophilic odorants over the mice
skin), or with mineral oil alone as a control. Tumor size was
measured and metastases were detected by in vivo imaging and by
post-mortem immunohistochemistry using antibodies targeting
PSGR or PSA (Prostate Specific Antigen) (examples of spine and
lung metastases are displayed in Figure 4). PSGR expression was
detected in primary tumors and in all metastases (see other
examples in Figure S2), confirming that this receptor was present
Table 1. Targeted search of ORs expressed by subclones of BON cells, using nested RT-PCR with primers specific to previously
identified ORs.
Clone OR4F16 OR13H1 OR7A17 OR10Q1 OR7D2 OR2A1 OR6V1 OR1F1 OR13A1
1X X X X X
3X X X
4X X X
8X X X X
9XXX
10 X X X X
14XXXXX X
19 X X X X
23XX XXXX
(X) indicates identified ORs.
doi:10.1371/journal.pone.0085110.t001
Figure 1. Functional response of ORs heterologously ex-
pressed in BON cells. BON cells were transiently transfected to
express OR1G1 or OR17-40 receptors. 72h later, cells were loaded with
fluo-4 and stimulated with the respective odorant ligands of the
transfected ORs (1-nonanol and helional). Calcium responses due to the
interaction between the OR and its specific odorant agonist are
expressed as the mean fluorescence variation DF/F (%). (open circles)
OR1G1 cells, 1-nonanol ; (filled diamonds) OR17-40 cells, helional ; bars
indicate standard deviation (n = 3). Mock-transfected cells did not
respond to 1-nonanol nor helional.
doi:10.1371/journal.pone.0085110.g001
Olfactory Receptors Promote Tumor Cell Invasion
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and possibly activated during our experiments. Without treatment,
metastases emerged mainly in the inguinal nodes and occasionn-
ally in spine and liver (Figure 4a). Metastases located in the
inguinal nodes were well developed while those located in spine
and liver were micrometastases. The number of metastases
increased upon treatment with mineral oil and their localization
was more diverse in the presence of b-ionone. Actually, metastases
appeared in lungs and Tyson glands only for mice treated with b-
ionone (3 out of 5 animals for Tyson glands and 2 out of 5 animals
for lungs). Moreover, metastases located in Tyson glands were
highly developed, with sizes approaching 1,000 mm
3
. In lungs,
only micrometastases were detected, like in spine and liver. Since
mice were not sacrificed at the same time, but depending on tumor
size, we show in Figures 5b and 5c the evolution with time of the
number of metastases according to the number of sacrificed mice
and the average number of metastases per mouse at the time of
sacrifice for each experimental group. We observed that mice
treated with mineral oil or b-ionone had to be sacrificed earlier,
due to faster tumor growth, and that they displayed a significant
increase in metastases number compared to untreated mice. In
parallel we also observed that tumors developed at 85% of the
inoculated sites in untreated mice, while they were less numerous
(50%) in mineral oil or b-ionone treated mice.
The results with the mineral oil were intriguing. Since ORs can
be activated by different molecules [2,12,18], we investigated
whether mineral oil can stimulate the PSGR in in vitro cell invasion
assays and calcium imaging experiments. Mineral oil increased
both the invasiveness and the calcic response of LNCaP cells, and
the PSGR antagonist a-ionone abrogated these effects (Figure 6
a,b). These results demonstrate that a mineral oil component
stimulated LNCaP cells via the PSGR, promoting their invasive-
ness. Moreover, LNCaP cells invasiveness induced by mineral oil
was inhibited by PI3Kc or G
bc
subunits inhibitors (respectively
AS605240 and gallein), demonstrating that PI3Kc is involved in
this process, as it is involved in cell invasiveness induced by b-
ionone. However, even if mineral oil enhanced metastasis
emergence, metastasis occurence was even more pronounced in
the presence of b-ionone, resulting in the cells spreading to lungs
and Tyson glands that occurred only in the presence of b-ionone.
All these data converge to demonstrate that stimulation of an OR,
Figure 2. Promotion of cancer cells invasiveness upon odorant stimulation. (a) BON cells were transiently transfected to express OR1G1 or
OR17-40 receptors or mock-transfected. Cells were seeded on collagen type I gels and stimulated by the respective odorant ligands of OR1G1 and
OR17-40 receptors (nonanol: OR1G1 agonist, vanillin: OR1G1 antagonist, helional: OR17-40 agonist). Invasive cells were counted 24 hours later.
Results are presented as the invasion index. (b) Modification of the F-actin cytoskeleton of BON cells in collagen type I matrices. F-actin was revealed
by rhodamine-conjugated phalloidin. Invasive extensions into collagen gels characterizing invasive cells are indicated by arrows. (c) LNCaP cells were
seeded onto collagen type I gels and stimulated by PSGR ligands (b-ionone: agonist, a-ionone: antagonist). Invasive cells were counted 24 hours later.
Results are presented as the invasion index relative to control cells without odorant stimulation. Standard deviation of the control was 13,42%.
Statistics were performed using a two-tailed Student test and bars indicate standard deviation (n = 3).
doi:10.1371/journal.pone.0085110.g002
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the PSGR, can contribute to the dissemination of prostate tumor
cells and in metastases generation.
Discussion
In this study, we showed that contrary to OSNs, individual
BON tumor cells (BON) co-express various ORs transcripts.
Genes encoding the co-expressed ORs are not located in the same
cluster nor on the same chromosomes, excluding a simultaneous
expression controlled by a common promoter. OR co-expression
by a same cell and differences in the set of expressed ORs between
cells could ensure a wide detection of luminal odorants in the
gastro-intestinal tract. Indeed normal and neoplastic EC cells were
previously reported to detect odorants through OR activation,
which participate in the control of their serotonin secretion [8,9].
Deregulation of serotonin release can induce pathological disor-
ders [29-32] and thus it could be expected that such disorders
could result from changes in the levels and variety of the ORs
expression. We also unexpectedly found that BON tumor cells
express OR pseudogenes at the mRNA level. Some of them
(OR7E38P and OR2A9P) and several functional receptors
expressed by BON cells (OR7A17, OR7D2 and OR2A1) have
been found in other tumors. Moreover, other ORs are overex-
pressed in EC tumor cells [15] and prostate tumor cells [12]. Thus,
the expression of some OR genes and pseudogenes in BON cells,
which originate from a pancreatic carcinoma metastasis, could be
part of their tumor phenotype.
Since BON cells endogenously express ORs, we also demon-
strated their ability to efficiently express transfected functional
heterologous ORs with known odorant ligands. In particular, we
found that the transcript of REEP1, a protein described to
improve OR expression [20], was present in BON cells. These
cells are thus a promising system for deorphanizing ORs, which
still remains a challenge due to the well-known low and variable
Figure 3. PI3Kc involvement in cell invasiveness promoted by
ORs. BON cells heterologously expressing the OR1G1 receptor were
seeded onto collagen type I gels and were stimulated by nonanol
(OR1G1 agonist) in the presence of a specific PI3Kc inhibitor
(AS605240). LNCaP cells were seeded onto collagen type I gels and
were stimulated by b-ionone (PSGR agonist) in the presence of a
specific PI3Kc inhibitor (AS605240) or an inhibitor of bc subunits of G
proteins (gallein). Invasive cells were counted 24 hours later. Results are
presented as the invasion index relative to that of control cells (with no
odorant nor AS605240 nor gallein). Standard deviation of the control
was 4,74% for the control BON cells and 13,86% for the control LNCaP
cells. Statistics were performed using a two-tailed Student test and bars
indicate standard deviation (n = 3).
doi:10.1371/journal.pone.0085110.g003
Figure 4. Examples of metastases found in the spine or the lungs of immunodeficient mice inoculated with LNCaP cells. Metastases
are indicated by arrows. In the spine, metastases were observed using microcomputed tomography (Maximum Intensity Projection) and confirmed
post-mortem by HES staining and immunohistology using anti-PSA (prostate specific antigen) and anti-PSGR antibodies (only one metastasis is
presented). In the lungs, metastases were characterized by HES staining and immunohistology using anti-PSA and anti-PSGR antibodies.
doi:10.1371/journal.pone.0085110.g004
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OR expression in heterologous cells. To date, even using HEK
293 cells engineered to improve OR expression, only 4% of 245
human ORs investigated were deorphanized [20,33,34]. Identify-
ing OR ligands and studying OR pharmacology is of increasing
interest given the high number of ORs (several hundreds in
humans) and their involvement in various physiological functions,
and possibly in physiopathological functions such as tumor
progression.
Furthermore, we demonstrate, for the first time, that ORs play a
role in tumor progression by promoting cell invasiveness and
metatasis emergence. Taking advantage of the ability of BON cells
to heterologously express ORs for which odorant ligands are
known, we showed that odorant stimulation of heterologously
expressed ORs enhanced BON cell invasiveness in vitro. We further
assess this result using LNCaP prostate cancer cells, which
overexpress an OR, the PSGR (also named OR51E2), described
as a prostate tumor marker [11,12]. Stimulation of LNCaP cells
with the PSGR agonist b-ionone promoted their invasiveness, and
this phenomenon was inhibited by a-ionone, a PSGR antagonist.
Recently, ORs were reported to participate in early cytokinesis by
exerting a regulatory role on actin cystoskeleton, and particularly
in cancer cell lines [35]. This suggests that ORs could favor cell
invasion by regulating actin cytoskeleton. Yet, the signaling
pathway triggered by OR stimulation and inducing cell invasive-
ness remains to be explored. Our results provide a first evidence
for a major role of PI3Kc, which is also supported by the following
data. The G
aolf
protein subunit, activated by ORs in OSNs, was
reported to promote invasion in human digestive and urogenital
epithelial cells, and in particular in LNCaP cells, through PI3
kinase, Rho GTPases and PKC dependent pathways [36]. Yet, in
our study, we found that PI3K is activated by bc subunits of G
proteins. Thus, in LNCaP cells, two PI3K dependent pathways
originating from PSGR activation could coexist. Moreover,
GPCRs activation of PI3Kc is involved in transformed cell
functions such as invasion and alteration of homotypic cell-cell
adhesion [23], and a crosstalk between odorant signaling and
PI3Kc was reported in OSNs [24,25]. All together, these data
suggest that an interplay between ORs, G proteins and PI3Kc in
tumor cells might promote the cell invasiveness phenotype.
We also demonstrate in vivo that stimulation of some ORs
expressed in tumor cells could facilitate cells dissemination and
metastasis generation. Indeed, we stimulated xenografted LNCaP
cells in NSG mice with a PSGR ligand. Without stimulation, mice
developed tumors at 85% of the inoculation sites, but also some
metastases in inguinal nodes, spine and liver, which is seldom
reported in the literature concerning prostate cancer models using
immunodeficient mice and LNCaP cells [37–39]. When treated
with mineral oil alone or containing b-ionone, mice showed a
significantly increased number of metastases, despite the small
number of animals in the experiment. Noteworthy, only the b-
ionone treated mice developed metastases in other tissues, namely
in lungs and Tyson glands, and those in Tyson glands were
particularly well developed. In addition, we showed that the
mineral oil used as an excipient in our experiments induces
LNCaP cell invasiveness by activating the PSGR and involving a
PI3Kc inhibitor pathway, like b-ionone. These results corroborate
that metastases emergence enhancement observed in mineral oil
treated mice can be due to PSGR activation, at least partly, since
we cannot exclude the presence of other ORs in LNCaP cells and
of their specific ligands in the mineral oil. It would be interesting to
identify the mineral oil component activating the PSGR, but this
appears difficult due to the complex and unavailable detailed
composition of mineral oil. Moreover, while in vitro there was no
additive effect of mineral oil and b-ionone in promoting cell
invasion, in vivo, addition of b-ionone to mineral oil not only
slightly boosted metastasis emergence in the same tissues but
moreover induced metastasis spreading to additional tissues. Also,
mice treated with b-ionone had to be sacrificed earlier due to
faster tumor growth, and the number of metastases detected in
Figure 5. Metastases detection in immunodeficient mice
inoculated with LNCaP cells. (a) The number of metastases
observed in various tissues after mice sacrifice (white: untreated control
mice, gray: mice treated with mineral oil, black: mice treated with 1 mM
b-ionone in mineral oil). (b) Cumulative number of metastases,
regardless of their location, as a function of the time elapsed between
LNCaP cells inoculation and mice sacrifice (white: untreated control
mice, gray: mice treated with mineral oil, black: mice treated with 1 mM
b-ionone in mineral oil). Mice were sacrificed when tumor size exceeded
1,500 mm
3
. The number of sacrificed mice is indicated in brackets for
each time point and each mice group. (c) Curves reporting the ratios of
the number of metastases over the number of sacrificed mice as a
function of the time (black dashed: untreated control mice, gray: mice
treated with mineral oil, black: mice treated with 1 mM b-ionone in
mineral oil).
doi:10.1371/journal.pone.0085110.g005
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those mice was sligthly higher. Thus, in the limit of our
experimental conditions, where the real amount of mineral oil
and b-ionone stimulating the LNCaP cells cannot be controlled,
the presence of b-ionone appeared to further promote metastasis
emergence. We cannot totally exclude that the observed effect on
metastases emergence could also be partly due to an increased
tumor growth rate in the presence of b-ionone. Indeed, b-ionone
seemed to accelerate tumor growth, as suggested by the early
sacrifice of some animals of the b-ionone treated group (actually,
all the animals were sacrificed when their largest lesion – the
inoculated tumor or a metastasis – reached 1500 mm
3
). However,
the treated animals sacrificed earlier presented more metastases
than untreated animals sacrificed later, whereas it could be
expected the contrary since the number of metastases usually
increase with age. Moreover, there were no significant differences
in tumor size between mice groups at sacrifice time. So, the
observed differences in the number and localization of the
metastases cannot be attributed just to differences in tumor
growth. We can also notice that the faster tumor growth observed
in vivo in the presence of b-ionone is not in agreement with the
results of Neuhaus et al. [12] demonstrating a reduced in vitro
proliferation of LNCaP cells in the presence of b-ionone. Finally,
we observed that tumor engraftement was less important in treated
mice than in untreated mice. This could be due to a greater
migration of the cells stimulated with mineral oil or b-ionone from
the inoculation site, detrimental to the local tumor development.
In humans, the PSGR can be activated by endogenous ligands
such as steroid hormones [12]. Nevertheless, we performed this
study in an androgen-depleted context (that is in castrated male
mice) and therefore our findings are of major relevance concerning
the androgen-independent progression of prostate cancer, which is
still poorly understood. Since mineral oil and b-ionone are present
in various products of our close environment, namely cosmetics,
food and beverages, it is conceivable that these exogenous
molecules could be found in the body and our findings might
help defining prostate cancer prevention measures. An important
prospect of future studies would be also to identify PSGR odorant
antagonists (other than a-ionone which is irritant and harmful,
side effects that would limit its use) as potential new anti-cancer
agents probably possessing low side effects since the PSGR is not
widely expressed in normal tissues.
Finally this study should be extended to other cancer types.
Indeed, not only a genomics approach has associated the olfactory
transduction pathway with an increased pancreas cancer risk [40]
but moreover overexpression of 34 ORs genes has been reported
in patients bearing breast tumors caused by CHEK2 1100delC-
mutation [41]. Hence, it appears of great importance to continue
addressing the role of ORs in tumor progression, in hormono-
dependent tumors as well as in non hormono-dependent ones.
Supporting Information
Figure S1 ORs expression in BON cells. (a) Example of
nested PCR performed on RT products obtained from a single
BON cell from clone 14, using two pairs of primers specifically
targeting OR10Q1 or OR4F16. Each amplification product was
was about 400 bp long and was verified by sequencing. Lane 1: 2-
Log DNA Ladder (BioLabs), lane 2: OR4F16 amplification, lane
3: OR10Q1 amplification, lanes 4 and 5: controls without cell with
each pair of primers. (b) Heterologous expression of ORs by BON
cells. BON cells were transiently transfected to express OR1G1 or
OR17-40 receptors tagged by the cmyc epitope at their
Figure 6. Mineral oil effect on LNCaP cells invasiveness and calcium response. (a) LNCaP cells were seeded onto collagen type I gels and
were stimulated by mineral oil or mineral oil mixed with 10
24
M b-ionone, 2.10
24
M a-ionone, 10
27
M AS605240 or 10
25
M gallein. Mineral oil was first
diluted 1/4 in DMSO (mineral oil was not totally solubilized at this dose, but this allowed us to expect a very large solubilization of the mineral oil
components) and the solution was then diluted 1/1000 in the culture medium or collagen I gel. Invasive cells were counted 24 hours later. Results are
presented as the invasion index relative to that of control cells (with only the DMSO). Bars indicate standard deviation. Standard deviation of the
control was 3,67%. Results obtained with mineral oil alone and with mineral oil containing b-ionone are not significantly different. Statistics were
performed using a two-tailed Student test (n = 3). (b) LNCaP cells were loaded with fluo-4 and stimulated with mineral oil or a mixture of mineral oil
and 10
24
M a-ionone. Calcium responses are expressed as the mean fluorescence variation DF/F relative to that of control, which corresponds to cells
stimulated with buffer containing the same DMSO concentration than the odorants and mineral oil samples. Bars indicate standard deviation.
Standard deviation of the control was 15,96%. Statistics were performed using a two-tailed Student test (n = 7).
doi:10.1371/journal.pone.0085110.g006
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extracellular N-terminal end. 72h later, non-permeabilised cells,
labeled with an anti-cmyc-Cy3 antibody, were observed by
immunofluorescence microscopy. Mock-transfected cells are
shown as a negative control. (c) REEP1 transcripts in BON cells.
RT-PCRs were performed using total RNA from BON cells and
primers targeting human RTP1, RTP2 or REEP1 cDNAs.
Expected PCR products lengths were 686 bp for RTP1, 564 bp
for RTP2 and 524 bp for REEP1. Negative controls were obtained
with the same primers on RNAs treated without reverse
transcriptase. Only REEP1 cDNA was amplified at the expected
length (boxed in white).
(TIF)
Figure S2 PSGR expression in various tissues of mice
inoculated with LNCaP cells. PSGR expression in primary
tumors, inguinal nodes, Tyson glands and livers was detected using
an anti-PSGR antibody (LS-A6332, Cliniscience) and negative
controls were performed using rabbit serum.
(TIF)
Table S1 Blind search of ORs expressed by subclones of
BON cells, using nested RT-PCR with degenerate
primers. Alternative ORs denominations are given in brackets.
« P » indicates pseudogenes.
(DOCX)
Acknowledgments
We thank Elena Legenre for her contribution to this work during a training
period, Patrice Congar (INRA, UR1197, Jouy-en-Josas) for his help in
collecting single BON cells, Christophe Calvet (CNRS, UMR8203, Institut
Gustave-Roussy) for taking part in mice treatment and tumor growth
evaluation, and Maryline Le Me´e (CNRS, TAAM-CIPA, UPS44, Orle´ans)
for performing mice necropsies. We also thank the staff of the Service
Commun d’Expe´rimentation Animale de Gustave Roussy for mice feeding
and nursing. This work was partly performed in the scope of the European
Associated Laboratory EBAM.
Author Contributions
Conceived and designed the experiments: GS IL JS SL SB LMM.
Performed the experiments: GS IL AD JS SB DG RM. Analyzed the data:
GS IL JS SL LMM. Wrote the paper: GS IL JS SL EPA LMM. Obtained
permission for use of cell line: EPA.
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