Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo.

Abstract

The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear.
In the present study, we showed by qRT-PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton.
Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.

Full text

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Full Paper
Targeting heat shock protein 27 (HspB1) interferes with bone
metastasis and tumour formation in vivo
B Gibert*,1,2, B Eckel3, V Gonin3, D Goldschneider2, J Fombonne2, B Deux3, P Mehlen2, A-P Arrigo2,
P Cle ´zardin3and C Diaz-Latoud*,1,3
1Centre de Ge ´ne ´tique Mole ´culaire et Cellulaire, CNRS UMR5534, Universite ´ Lyon 1, Universite ´ de Lyon, 43 Bd 11 Novembre 1918, 69622 Villeurbanne
Cedex, France;2Apoptosis, Cancer and Development Laboratory- Equipe labellise ´e ‘La Ligue’, LabEx DEVweCAN, Centre de Cance ´rologie de Lyon,
INSERM U1052-CNRS UMR5286, Universite ´ de Lyon, Centre Le ´on Be ´rard, 69008 Lyon, France;3INSERM, UMR 1033, LabEx DEVweCAN, Faculte ´ de
Me ´decine RTH Lae ¨nnec, Universite ´ Lyon 1, Universite ´ de Lyon, 43 Bd 11 Novembre 1918, 69622 Villeurbanne, Cedex, France
BACKGROUND: The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27
upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs
inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is
associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and,
more specifically, in the later and most deadly stage of tumour metastasis is still unclear.
METHODS/RESULTS: In the present study, we showed by qRT–PCR that Hsp27 gene is overexpressed in a large fraction of the
metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and
establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that
Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer
cells to metastasise and grow in the skeleton.
CONCLUSION: Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal
tumour growth.
British Journal of Cancer advance online publication, 24 May 2012; doi:10.1038/bjc.2012.188
& 2012 Cancer Research UK
www.bjcancer.com
Keywords: Hsp27; bone; metastasis; apoptosis; therapeutic target; breast
? ???????????????????????????????????????????????????
Heat shock protein 27 (Hsp27), also called HspB1, is a member of
the human small heat shock protein family characterised by a
highly conserved a-crystalline domain. This protein has a complex
structural organisation that modulates its chaperone activity (Paul
et al, 2010). Thus, like other small heat shock proteins, Hsp27 can
sequester damaged proteins and prevent their aggregation
(Wyttenbach et al, 2002; Arrigo, 2011). This property of molecular
sponge leads to increased cell survival, and is correlated with
complex patterns of Hsp27 oligomerisation and phosphorylation
on three serine residues (Jaya et al, 2009; Paul et al, 2010).
Hsp27 is also well characterised to counteract apoptotic cell
death induced by different inducers (Mehlen et al, 1996; Paul et al,
2010). It modulates cell death negatively by blocking the apoptotic
cascade at different levels. For example, Hsp27 binds and
inactivates cytochrome-c released from mitochondria during
apoptotic cell death (Bruey et al, 2000), as well as the pro-domain
of procaspase-3, and inhibits the activation process of this caspase,
two crucial events for apoptosis execution (Voss et al, 2007).
Moreover, Hsp27 is able to directly stabilise actin filaments to
protect cell integrity in response to heat, reactive oxygen species,
cytotoxic agents (Venkatakrishnan et al, 2006; Pivovarova et al,
2007) and some apoptotic inducers, as for example staurosporine
(Paul et al, 2002, 2010). Hsp27 is also implicated in radio- and
chemo-resistance and can negatively modulate cell death induced
by anticancerous cytotoxic agents like cisplatin, adriamycin,
etoposide or g-rays (Zhang and Shen, 2007; Aloy et al, 2008).
g-Ray resistance may be related to the ability of this protein to
counteract oxidative stress (Arrigo, 1998).
In murine vivo models, a direct demonstration of the
tumourigenic and metastatic potential of this protein has been
done (Garrido et al, 1998; Gibert et al, 2011). Of interest, Hsp27 is
highly expressed in many types of human tumours, particularly in
those of carcinoma origin, including ovarian, breast, head and
neck cancer. Hsp27 overexpression is considered a bad prognostic
for patient survival (Ciocca and Calderwood, 2005). This increase
in Hsp27 level is probably due to the pro-survival effect of the
protein allowing the proliferative potential of cancerous cells in
adverse in vivo conditions. All these faculties implicate Hsp27 as a
major therapeutic target in cancer (Arrigo et al, 2007; Gibert et al,
2011).
The role and clinical outcome of Hsp27 in primary tumours has
been well studied and documented (Ciocca and Calderwood, 2005;
Calderwood et al, 2006; Tsuruta et al, 2008; Huang et al, 2010).
However, its function in metastasis invasion has been less studied,
even though Hsp27 gene has been shown by proteomic analysis to
be overexpressed in tumour cells of patients that are able to
metastasise (Song et al, 2006; Tian et al, 2007). It is also interesting
to note that surface expression of murine Hsp27 stimulates tumour
growth and metastasis of the highly metastatic murine 4T1 breast
*Correspondence: Dr B Gibert or Dr C Diaz-Latoud;
E-mail: benjamin.gibert@lyon.unicancer.fr or chantal.diaz@univ-lyon1.fr
Received 7 March 2012; revised 3 April 2012; accepted 11 April 2012
British Journal of Cancer (2012), 1–8
& 2012 Cancer Research UKAll rights reserved 0007– 0920/12
www.bjcancer.com

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adenocarcinoma cells, whereas silencing of this protein eliminates
migration capability (Bausero et al, 2004; Kaur et al, 2011). In the
present study, we showed that Hsp27 is overexpressed in tumours
that are able to metastasise, in 53 human breast cancer patients.
We further investigated the role of human Hsp27 during bone
metastasis formation and tumour growth in mice. Bone metastasis
invasion is very often a dramatic stage of tumour progression,
which frequently occurs and generally leads to patient death. Bone
metastases in breast cancer patients are also associated with bone
destruction that correlates with hypercalcaemia and fractures.
We used the breast MDA-MB231/B02 metastatic cell line as a
model to analyse the effect of Hsp27 silencing (Pecheur et al, 2002;
Peyruchaud et al, 2003). This cell line displays a unique tropism
for bone dissemination and constitutively expresses a high level
of Hsp27. The aim of this work was to investigate the role of
Hsp27 in bone colonisation by breast cancer cells. We provide here
experimental evidence for a role of Hsp27 in migration and
invasion of bone tissue by metastatic breast cancer cells.
MATERIALS AND METHODS
Cell culture and transfections
The MDA-MB-231/B02 luciferase human breast cancer cell line is a
subpopulation of the MDA-MB-231 cancer cell line that was
selected for its high efficiency to metastasise to bones after
intravenous inoculation (Pecheur et al, 2002; Peyruchaud et al,
2003).
All cells were grown at 371C in a humidified atmosphere
containing 5% CO2. MDA-MD231/B02 cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10%
heat-inactivated fetal calf serum. Cells were transfected according
to the jet prime reagent procedure (Poly plus transfection,
Pontoise, France). For transient transfection assays, DNA vector
was left on cells for 6h; thereafter, cells were washed with
phosphate-buffered saline before being further incubated in fresh
medium.
Gel electrophoresis and immunoblotting
Cultured cells were immediately rinsed twice in ice-cold phos-
phate-buffered saline and scraped off the dish. At this point,
aliquots were withdrawn for determination of protein concentra-
tion. Thereafter, cells were directly lysed and denatured in
SDS buffer (62.5mM Tris-HCl, pH 6.8; 0.1 M dithioerythritol; 1%
SDS; 0.001% bromophenol blue; and 10% glycerol). Cell lysates
were subjected to SDS–polyacrylamide gel electrophoresis (SDS–
PAGE) performed as previously described (Javouhey et al, 2008).
Immunoblots probed with different specific antibodies were
revealed with the ECL system (Amersham Life Science, Pantin,
France). Autoradiographs were recorded on X-Omat LS films
(Eastman Kodak Co, Rochester, NY, USA).
ShRNA construction
The pCIstricke plasmid (Promega, Charbonnie `res, France) was
used for DNA vector-based shRNA construction. The Hsp27
targetingsequence of thedesigned
50-GCTGCAAAATCCGATGAG-30. After annealing, ligation and
transformation of the resulting DNA vector into competent DH5a
bacteria (Invitrogen, Cergy Pontoise, France), antibiotic resistance
was used to select the positive bacterial colonies. The correct
sequences of the final DNA preparations for pCIstrike-ShRNA27
(Sh27) were confirmed by sequencing (GenomExpress, Meylan,
France). pCIstrike-MsRNA27 (Ms27) vectors were designed as a
degenerated control from the above sequence of pCIstrike-
ShRNA27.
oligonucleotides was
Generation of stable MDA-MB231/B02 cells containing
reduced levels of Hsp27
Four cell culture dishes were seeded at 1?106cells per 78cm2.
Transfection of MDA-MB231/B02 luciferase (B02) cells was
performed with pCIstrike-ShRNA27 or pCIstrike-MsRNA27 DNA
vector (Pecheur et al, 2002; Peyruchaud et al, 2003). One day
later, B02 cells were incubated in the presence of neomycin
at a concentration of 1mgml?1. Neomycin-resistant clones were
tested for their Hsp27 levels by western blot. Three independent
clones expressing minimal endogenous Hsp27 (named: B02-Sh27)
and three independent control clones expressing normal levels of
Hsp27 (named: B02-Ms27) were selected, propagated and further
analysed.
Determination of cellular proliferation
Cell proliferation was determined using the WST-1 test (Promega,
Charbonnie `res, France). The B02 cells were seeded in 96-well
plates (7.5?103per well), incubated with 10ml per well for 4h in
tetrazolium WST-1 salt (4-[3-(4-iodophenyl)-2-(4-bonitrophenyl)-
2H-5-tetrazolio]-1,3-benzene disulphonate) and followed by absor-
bance measurement at 450nm with a Wallac 1420 Multilabel
Counter (PerkinElmer, Courtabœuf, France). The percentage of
cellular proliferation was calculated based on a control absorbance.
Chemotaxis assay
Cell migration and invasion assays were performed in 24-well cell
culture chambers with 8-mm-diameter pore-size inserts (Becton
Dickinson, Pont de Claix, France), as previously described
(Zhao et al, 2007). Inserts were coated with 100ml basement
membrane Matrigel (3mgml?1; Becton Dickinson) for 90min at
371C in order to perform cell invasion experiments. B02 cancer
cells (1.5?105cells per ml) were re-suspended in Dulbecco’s
modified Eagle’s medium containing 0.1% (w/v) bovine serum
albumin, and 300ml of this cell suspension was loaded into each
insert (upper chamber). The chemo-attractant (10% (v/v) fetal calf
serum) was placed in the lower chamber (750ml per well). The
plates were incubated for 6h at 371C in a 5% CO2incubator. After
incubation, the inserts were collected carefully, the non-migrating
cells were removed, and the migrating cells on the under-surface of
the inserts were fixed and stained with crystal violet. The
membranes were mounted on glass slides and cells were counted
under microscope.
Animal studies
Animal studies were performed according to ethics recommenda-
tions and animal welfare (Workman et al, 2010). Bone metastasis
experiments were conducted in Nude mice, as described previously
(Pecheur et al, 2002; Peyruchaud et al, 2003; Zhao et al, 2007).
MDA-MB-231/B02 cells that had been stably transfected with
shRNAs directed against Hsp27 or control mismatches (5?105in
100ml of phosphate-buffered saline) were injected into the tail vein
of nude mice anaesthetised with 130mg/kg ketamin and
8.8mgkg?1xylazin. Radiographs of anaesthetised animals were
taken weekly with the use of MIN-R2000 films (Eastman Kodak
Co) in an MX-20 cabinet X-ray system (Faxitron X-ray Corpora-
tion, Wheeling, IL, USA). Osteolytic lesions were identified on
radiographs as radiolucent lesions in the bone. The area of
osteolytic lesions was measured using a Visiolab 2000 compu-
terised image analysis system (Explora Nova, La Rochelle, France),
and the extent of bone destruction per leg was expressed in square
millimetres. Anaesthetised mice were killed by cervical dislocation
after radiography on day 28 (Workman et al, 2010).
Intra-osseous tumour xenograft experiments were conducted in
nude mice, as described previously (Zhao et al, 2007). Briefly,
Hsp27 enhances bone metastasis and bone tumour development
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a small hole was drilled with a 30-gauge sterile needle through the
tibial plateau with the knee flexed. Using a new sterile needle fitted
to a 50-ml sterile Hamilton syringe (Hamilton Co., Reno, NV, USA),
a single-cell suspension (1?105cells in 30ml of phosphate-
buffered saline) was injected into the bone marrow cavity. The
progression of osteolytic lesions was monitored by radiography as
described above (Zhao et al, 2007). Anaesthetised animals were
killed by cervical dislocation 7 weeks after tumour cell inoculation.
Bone histology and histomorphometry
Bone histology and histomorphometry analysis of bone lesions
were performed as previously described (Pecheur et al, 2002;
Peyruchaud et al, 2003; Zhao et al, 2007). Following killing of
metastatic animals, both hind limbs from each animal were
dissected, fixed in 80% (v/v) alcohol, dehydrated and embedded in
methylmethacrylate. A microtome (Microm, Micron Microtech,
Francheville, France, HM350S) was used to cut 7–9-mm thick
sections of undecalcified long bones, and the sections were stained
with Goldner’s trichrome. Histologic and histomorphometric analyses
were performed on Goldner’s trichrome-stained longitudinal medial
sections of the tibial metaphysis using a computerised image analysis
system (Visiolab 2000, Explora Nova). Histomorphometric measure-
ments (i.e., bone volume/tissue volume (BV/TV) and tumour burden/
soft tissue volume (TB/ STV) ratios) were performed in a standard
zone of the tibial metaphysis, situated at 0.5mm distance from the
growth plate, including cortical and trabecular bone. The BV/TV
ratio represents the percentage of bone tissue. The TV/STV ratio
represents the percentage of tumour tissue.
Statistics
The statistical significance of differences between groups was
evaluated by the Mann–Whitney U-test. All statistical tests were
two-sided, and P values o0.05 were considered to be statistically
significant: *Po0.05, **Po0.01.
Human breast tumour samples, quantitative PCR
Fifty-three human breast cancer samples were provided by the
tumour bank of the Centre Le ´on Be ´rard after obtaining patients’
consent. Fresh tissue of the tumour was obtained during breast
surgery before any systemic therapy and snap-frozen in liquid
nitrogen. To assay Hsp27 expression, total RNA was extracted from
sampleswith theNucleospin
Du ¨ren, Germany) and 1mg of RNA was reverse transcribed using
the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). Real-
time qRT–PCR was performed on a LightCycler 2.0 using the
LightCycler TaqMan Master kit (Roche Applied Science, Basel,
Switzerland) according to the manufacturer’s instructions. The
ubiquitously expressed TBP gene was used as internal control and
the same results have been confirmed with HPRT and HMBS genes
(de Kok et al, 2005). Primers and probe were given by Universal
Probe Library Assay Design Center website and are available upon
request (Roche Applied Science). All patient tissue specimens were
used according to French laws and regulations.
RNAII kit(Macherey-Nagel,
RESULTS
Hsp27 is a marker for human metastatic breast cancer
We first analysed Hsp27 expression by quantitative-PCR in a panel
of 53 human breast primary tumours (Figure 1A). These tumours
were from lymph node-negative patients (M0–N0, 13 patients),
lymph node-positive patients (M0–N1, 29 patients) or metastatic
patients at the time of diagnosis (M1, 11 patients). Hsp27 is
significantly more expressed in M0–N1 tumours than in M0–N0
tumours (median 2.95 vs 6.85; P¼0.0119). An even more striking
difference is observed when comparing Hsp27 expression in
M0–N0 with that in M1 tumours (median 2.95 vs 14.58;
P¼0.0019). A significant difference exists between M0–N1 and
M1 (median 6.85 vs 14.58; P¼0.026). As shown in Figure 1B,
Hsp27 is overexpressed in 79.30% of patients in the M0–N1 and
90.90% in the M1 group (based on the average of the M0–N0
group). Altogether, these data prove that Hsp27 is a marker of
the aggressive forms of human breast tumours, especially the
metastatic ones.
Establishment of Hsp27-depleted clones
The human breast cancer cell line MDA-MB231 is a well-
characterised model of metastasis invasion in vivo. To assess the
role of Hsp27 in breast cancer metastasis, we used a sub-clone of
this cell line, called MDA-MB231/B02 (B02), that has been
characterised to specifically metastasise in the bone marrow and
not in other tissues when injected in mice (Pecheur et al, 2002;
Peyruchaud et al, 2003). This cell line can be efficiently tracked
in vivo and its metastatic aggressiveness could be quantified easily
by radiographies. We first compared Hsp27 levels in B02 and
parental MDA-MB231. These cells have rather similar levels of
Hsp27. Using HeLa cells as control (about 4ngmg?1of total
proteins; Paul et al, 2010), their Hsp27 level was estimated at about
3ngmg?1. B02 cells expressed almost the same Hsp27 level as
parental cells (Figure 2A). The fact that Hsp27 expression level is
similar between MDA-MB231 and B02 cells suggests that Hsp27
expression is not related to B02 cells’ osteotropism.
To investigate the pro-oncogenic role of Hsp27 during
metastasis process, we established independent clones of B02 cells
transfected with shRNA directed against Hsp27 or with mis-
matches of shRNA. Assessment of Hsp27 levels in B02 cells, by
western blotting analysis, revealed a 90% inhibition of Hsp27
protein expression in hsp27 shRNA-transfected clones (Figure 2B).
0
M0–N0
patients
5
10
15
20
25
30
35
40
45
50
M0–N1
patients
M1
patients
HSP27 mRNA expression
**
Hsp27 median
expression
1
M0 N0M0 N1 M1
Hsp27
overexpression
2.57
2.95
30.80%79.30%90.90%
**
Figure 1
(A) Expression profile of Hsp27 examined by quantitative real-time
PCR by using mRNA extracted from 53 tumour biopsy specimens
were obtained from patients with tumours localised to the breast
(M0–N0), with only node involvement (M0–N1), and with distant
metastases at diagnosis (M1) (*Pp0.05; **Pp0.01). TBP gene (TATAA-
box-binding protein) was used as a reference here because it shows a weak
variability in the breast (de Kok et al, 2005). Identical results were obtained
with HBMS and HPRT housekeeping genes. (B) Hsp27 expression is given
as the ratio between Hsp27 expression in each sample and the average of
Hsp27 expression in the M0–N0 samples, shown by a horizontal bar in (A).
Hsp27 overexpression: percentages of M0–N0, M0–N1 and M1 of samples
showing a superior average as compared with the M0–N0 group average.
Hsp27 relative expression in metastatic breast tumours.
Hsp27 enhances bone metastasis and bone tumour development
B Gibert et al
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Two shRNA clones, as well as two negative control mismatch
clones, were selected for further studies.
Hsp27 silencing inhibits both cellular migration and
invasion but not proliferation
Metastatic tumour cells usually show enhanced migratory capacity
and this is often associated with a decreased adhesion. In order to
determine whether Hsp27 downregulation could impact this
mechanism, we tested the cellular migration of the genetically
modified B02 cell clones by performing chemotaxis assays in cell
culture chambers with pore inserts. As shown in Figure 2C,
migration of Hsp27-depleted cells was dramatically reduced down
to 70% as compared with control scramble cells (Pp0.01).
Invasion of tumour cells into surrounding tissues is also known
as a crucial event leading to development of distant metastases.
Therefore, we evaluated the invasion potential using inserts that
were coated with Matrigel basement membrane to evaluate
whether matrix degradation was occurring. Matrix invasion by
Hsp27-depleted clones was significantly decreased compared with
control cells (Figure 2D), but in the same proportions as in the
migration assay performed without Matrigel, suggesting that
Hsp27 depletion does not affect the ability of B02 cells to degrade
Matrigel.
In addition, we have tested the effect of Hsp27 downregulation
on cellular proliferation state (Park et al, 2002; Lee et al, 2004). In
previous studies using HeLa cells, we have shown that Hsp27
depletion decreased proliferation by interfering with G2/M phase
(Gibert et al, 2012). In contrast, analysis of the WST-1 proliferation
assay of B02 cells revealed that Hsp27 depletion was devoid of
significant effect (Figure 2D).
Hsp27 inhibition of expression reduces metastasis
formation in vivo
Metastasis invasion is correlated with the transport of cancer cells
by a circulatory system to distant sites. We next wanted to assess
whether the Hsp27 depletion in B02 cells inhibited bone metastasis
formation in vivo. Pools of B02-Sh27 or B02-Ms27 clones were
therefore injected in anaesthetised nude mice by intravenous
injection in the tail. B02 cells have been transfected to stably
express a luciferase enzyme, which allows in vivo B02 cell detection
by injection of luciferin. Luciferase activity as bioluminescence was
then quantified. It correlated with metastasis expansion and
dissemination, and was represented by an indicative colouration
inlay in mice pictures (Figure 3A). Metastasis development was
only detected in the bone marrow thigh of mice, confirming the
specificity of B02 cells to form bone metastasis in vivo, in contrast
to the MDA-MB-231 parental cells. Luciferase activity was
statistically decreased by about 50% during the first 3 weeks
(Pp0.01) in the group of B02-Sh27-inoculated mice as compared
with the ones injected with B02-Ms27 cells. These results suggest
that Hsp27 depletion delays metastasis formation by B02 cells
(Figure 3B).
Bone metastasis progression is highly correlated with bone
demineralisation and fractures due to cancer progression in this
environment. Hence, in addition to the in vivo analysis of B02 cells’
luciferase activity described above, radiographies of each mouse
were performed to analyse metastasis expansion. Measurement of
osteolytic lesions was achieved by quantification of bone destruc-
tion on hind limb radiograph (Figure 4A). As shown in Figure 4C,
the extent of osteolytic lesions was dramatically decreased when
animals were injected with B02 cells depleted in Hsp27 compared
with control cells. There was a significant difference between the
groups during all the experiments, including at day 28, when the
luciferase activity was similar between mice injected with B02-Sh27
and those injected with B02-Ms27 cells.
Histomorphometric studies were performed at day 28 in order
to quantify bone destruction and tumour burden in the animals.
Similar to quantitative-PCR on tumour biopsies, Sh27 still
invalidated Hsp27 expression after 4 weeks of xenografts
(Supplementary Figure 1). As shown in Figure 4C, the results
confirm that bone destruction is diminished in animals injected
with Hsp27-depleted B02 cells compared with control B02-Ms27
Migration level (cells mm–2)
BO2Ms27-1.2
20
40
60
Invasion potential (%)
**
**
**
**
Proliferation index
1
0.5
25
50
Actin
Hsp27
Actin
Hsp27
BO2Ms27-2.1BO2Ms27-1.2BO2 BO2Sh27-1.3BO2Sh27-2.1 BO2Sh27-3.3
BO2
MDA-MB231
HeLa
BO2Ms27-1.2
BO2Ms27-2.1
98.4
103.5
BO2
BO2Sh27-1.3
BO2SH27-2.1
BO2SH27-3.3
8.4
7.7
4.5
100
Hsp27 level
(%)
BO2Ms27-2.1
BO2Sh27-1.3BO2Sh27-2.1
BO2Ms27-1.2BO2Ms27-2.1
BO2Sh27-1.3 BO2Sh27-2.1
BO2Ms27-1.2
BO2Ms27-2.1
BO2Sh27-1.3BO2Sh27-2.1
Figure 2
Materials and Methods. Samples were collected 48h after transfection and Hsp27 level was analysed by western blot. Level of actin was used as a loading
control. (B) pCIstrike-MsRNA27 (Ms27) and pCIstrike-ShRNA27 (Sh27) vectors were constitutively expressed in B02 cells. Immunoblots were revealed by
the corresponding antibodies. (C) Hsp27 depletion reduces migration and invasion potential but not proliferation of B02 cells. The modified Boyden
chamber technique was used for cell migration assessment. B02 cells were cultured without serum in the insert chambers with pore inserts. Cells able to
migrate through the membrane were stained with crystal violet and counted (**Pp0.01). (D) Invasiveness of cells was studied by addition of a Matrigel layer
in the upper chambers, and like in the migration assay, clones were quantified as described in Materials and Methods (**Pp0.01). (E) WST-1 tests were
performed every 12h during 5 days. Proliferation index was determined and reported for each clone.
Characterisation of Hsp27-depleted B02 clones. (A) HeLa, MDA-MB231 and MDA-MB231 B02 (B02) cells were cultured as described in
Hsp27 enhances bone metastasis and bone tumour development
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cells. It should be noted that trabecular bone was only partially
destroyed in tibiae of mice injected with B02-Sh27 cells
(BV/TV¼23.5%; TB/STV¼26.5%), whereas it had completely
disappeared in the metaphysic tibiae of animals injected with
B02-Ms27 cells (BV/TV¼9.4%; TB/STV¼40.0%).
This study also showed that at day 28, Sh27 tumour size is
dramatically decreased in comparison with Ms27 tumours. This
result is not in accord with the luciferase activity measured in these
animals (see above; Figure 3), a discrepancy that could be the
result of the fact that at day 28 the large size of the tumours
Day 28Day 21 Day 14 Day 7
BO2Sh27
BO2Ms27
Luciferase activity (photons mm–2)
0
500
1000
1500
2000
*
BO2Sh27
BO2Ms27
+
–
Luciferase activity (photons mm–2)
Time (days)
7 142128
**
**
Figure 3
(B02Sh27) were injected intravenously into nude mice. (A) Luciferase activity is quantified weekly using the night owl technology, as described in Materials
and Methods, after a subcutaneous injection of luciferin. Luciferase activity is inlaid in the mice picture with the corresponding ladder. (B) Luciferase
expression, weekly quantified in B02-SH27 clones and control cells, is reported in the graph. (**Pp0.01).
Effect of Hsp27 silencing on breast cancer bone metastasis formation in animals. B02Ms27 cells and cells silenced for Hsp27 expression
BO2Ms27
BO2Sh27
BO2Ms27 BO2Sh27
BO2Sh27 BO2Ms27
0
1
3
2
BO2Sh27 BO2Ms27
4
3
2
0
1
5
6
BO2Sh27 BO2Ms27
6
4
0
2
8
10
12
16
14
Osteolytic lesions (mm2)
Histomorphometry
BO2-Sh27
BO2-Ms27
**
**
Week 2Week 3Week 4
**
Week 2 Week 3
Week 4
BV/TV (in %) TB/STV (in %)
26.5 ± 6.0*
40.7 ± 11.8 9.4 ± 3.2
23.5 ± 4.1*
Figure 4
tumour cell inoculation in nude mice. (A) Osteolytic lesions on representative radiographs are indicated by white arrows. Scale bar¼0.5cm.
(B) Representative bone histology of Goldner’s trichrome-stained tibial metaphysis from metastatic animals. The bone is coloured in green; bone marrow
and tumour cells are coloured in red. Scale bar¼0.5mm. BV/TV: bone volume relative to tissue volume. TB/STV: skeletal tumour burden relative to soft
tissue volume ratio (arrows: bone decalcification) (**Pp0.01). (C) Bone decalcification was quantified by software using Visiolab 2000, and the results are
reported in the graph (**Pp0.01).
Effect of Hsp27 depletion on bone lesion formation. Radiographic, immunostaining and histomorphometric data were obtained on day 31 after
Hsp27 enhances bone metastasis and bone tumour development
B Gibert et al
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interfered with luciferase bioluminescence activity, which was then
not precise enough to detect the differences observed in the
histomorphometric study (El Hilali et al, 2002).
Hsp27 depletion inhibits bone tumour formation
In order to determine whether the decrease in bone metastasis
formation observed in response to Hsp27 inhibition was only due
to the inhibition of migration and invasion of the cells, we next
injected the cells directly into the tibiae of nude mice. In this
experiment, most of the mechanisms of migration could be
ignored because the cells were already on the metastatic site.
Xenograft development was evaluated weekly during 2 months by
bioluminescence quantification and radiography. Luminescence
activity was only detected in the injected tibiae, showing that
the cells once injected in the tibiae did not disseminate in
the organism (Figure 5A). Quantification of luciferase activity
revealed a decrease in tumour burden in animals injected with
B02 cells depleted of Hsp27 (Figure 5B). Therefore, this result was
confirmed by histomorphometric analysis. Osteolytic lesions were
also dramatically reduced in case of the inhibition of Hsp27, as
also confirmed by histomorphometric analysis (Figure 5C and D).
Moreover, quantification of bone destruction confirmed that
osteolytic lesions were reduced after injection of the B02-Sh27
(BV/TV¼23.5%; TB/STV¼23.5%) cells compared with the B02-
Ms27 cells (BV/TV¼9.4%; TB/STV¼40.7%).
Taken together, these experiments show that Hsp27 is not only
important for increased migration and invasion properties of
metastatic cancer cell, but it also has a major role in formation of
the metastatic lesion per se at the secondary bone settings.
DISCUSSION
Hsp27 has been shown to be a major therapeutic target for cancer
therapies against primary tumour formation by protecting cells
against apoptosis. This stress protein is upregulated in many
tumour types, including breast, ovarian, colon, head and neck
cancer (Ciocca and Calderwood, 2005; Calderwood et al, 2006;
Tsuruta et al, 2008; Huang et al, 2010), but was not well
characterised during in vivo metastasis development. In our study,
we show that Hsp27 is overexpressed in the aggressive forms of 53
human breast tumours. Thus, in tumours positive for lymph node
invasion, the increase is 2.57-fold over the median expression,
whereas it is 2.95-fold over the median in the metastatic ones
(Figure 1A). This increased expression may have some correlation
with the results presented in a recent study highlighting the role of
Hsp27 in epithelial–mesenchymal transition (Wei et al, 2011).
To understand whether this overexpression is important in the
development of metastasis in distant organs, we analysed the
consequences of Hsp27 silencing in a mice xenograft model. We
used the breast cancer cell line MDA-MB231/B02, characterised for
its tropism for bone invasion (Pecheur et al, 2002). In breast
cancer, bone metastases have been correlated with bone alteration-
like demineralisation and fractures. The role of Hsp27 in breast
cancer metastases, particularly in bone metastasis, is not clearly
elucidated. We show here using two different techniques that
Hsp27 silencing induces a decrease in metastasis formation
(Figure 3 and 4). Luminescence detection was highly correlated
with bone decalcification as revealed by radiographies, except in
one case (at day 28, Figure 3A), probably because luciferase
activity is altered in important metastases, leading to quantifica-
tion defects as proven by other studies (El Hilali et al, 2002; Paroo
et al, 2004).
Lemieux et al (1999) have shown that overexpression of Hsp27
leads to a decrease in cellular migration of MDA-MB231 cells, and
that in the MDA-MB231 cells, overexpression of Hsp27 did not
show a significant effect on bone dissemination and alteration
in vivo. In our study, we silenced the expression of Hsp27 using
a ShRNA approach and we characterised clones presenting
a decrease of about 90% in Hsp27 expression. These depleted
clones showed a decreased ability to migrate and invade in vitro.
BO2Sh27BO2Ms27
BO2Sh27 BO2Ms27
0
1
3
2
4
Week 5
BO2Sh27BO2Ms27
0
1
3
2
4
5
Week 6
BO2Sh27
BO2Ms27
Week 5Week 6
BO2Sh27
BO2Ms27
Osteolytic lesions (mm2)
Luciferase activity
(photons mm–2)
Luciferase activity (photons mm–2)
+
–
Time (days)
21 28
Week 6
3542
1000
2000
3000
BO2Sh27
BO2Ms27
Histomorphometry
BO2-Sh27
BO2-Ms27
BV/TV (%) TB/STV (%)
**
**
**
**
16.9 ± 6.5*
39.0 ± 5.211.8 ± 2.2
17.9 ± 1.2*
Figure 5
(**Pp0.01). (A) Luciferase activity was quantified using the night owl technology as described in Materials and Methods. Photographs presented are
representative of the results for each animal group. (B) Luciferase activity was measured weekly during 2 months and are reported on graph. (C) Bone
lesions were weekly quantified by radiography of the injected tibiae. Demineralisation is depicted with white arrows. Scale bar¼0.5mm.
(D) Histomorphometric data of tibiae were obtained 2 months after cell inoculation. The bone is coloured in green, whereas bone marrow and
tumour cells (stars) are coloured in red. BV/TV: bone volume relative to tissue volume. TB/STV: skeletal tumour burden relative to soft tissue volume ratio.
Scale bar¼0.5mm (arrows: bone decalcification). (E) Quantification of osteolysis is performed on radiographs and is presented in a graph for each group of
mice.
Hsp27 depletion decreases tumour formation in bone. B02-Ms27 cells and B02-Sh27 cells were injected in the left tibiae of female nude mice
Hsp27 enhances bone metastasis and bone tumour development
B Gibert et al
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Page 7

Hence, it can be concluded that a decrease in bone metastasis
formation can be observed following a depletion (our study) or
overexpression (Lemieux et al, 1999) of Hsp27. These apparently
conflicting results may suggest that the intracellular level of
Hsp27 has a crucial role in this phenomenon. Consequently,
a negative or positive change in Hsp27 balance level may alter
the heterogenic structures formed by this protein (Paul et al,
2010) and destabilise its interactions with pro-metastatic client
protein targets. In this regard, Hsp27 has been shown to stabilise
F-actin filaments during cell migration (Pivovarova et al, 2007;
Lee et al, 2008; Chen et al, 2009), confirming our results obtained
with antisense strategy. Overexpression of Hsp27 could also have
drastic effects on cytoskeleton architecture that may also lead
to a decreased ability of cells to migrate. Future experiments are
needed to test this hypothesis.
Our results also suggest that the decrease in cellular migration
and invasion are not the only mechanisms responsible for the low
level of bone metastasis formation. Indeed, we have observed that
silencing of Hsp27 is associated with a decreased amount of
tumour cells in the bone microenvironment (Figure 5). One
possible hint for the effect is the fact that it is known that the bone
microenvironment is a very stressful environment, owing mostly
to intense mechanical stresses. We have previously shown that
Hsp27 functional inhibition, by interfering peptides called
aptamers, could modify the in vivo proliferation of tumours,
whereas proliferation was not modified in cell culture conditions
(Gibert et al, 2011). These results are in correlation with our
presented data because proliferation is not modified in the in vitro
context, whereas B02-Sh27 tumour growth was altered in vivo. It is
thus tempting to propose that Hsp27 may have an important role
in protecting bone cells against these kinds of stress (Muraoka
et al, 2010).
Altogether, these results support the view that Hsp27 has a
crucial role during metastasis and tumour implementation and
expansion in the bone. Targeting Hsp27 may then prove to be a
promising strategy to prevent bone metastasis, which is a terrible
step towards patient death.
ACKNOWLEDGEMENTS
This work was supported by a research grant from the
Comite ´ du Rho ˆne of la Ligue Contre le Cancer and by the
Region Rho ˆne-Alpes. BG post doctoral fellowship is supported
by ARC.
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on British
Journal of Cancer website (http://www.nature.com/bjc)
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Hsp27 enhances bone metastasis and bone tumour development
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