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S.-H. Hong, T. Osborne, L. Ren, J. Briggs, C. Mazcko, S. S. Burkett
and C. Khanna "Protein kinase C regulates ezrin–radixin–moesin phosphorylation in canine osteosarcoma cells", September 2011, DOI: 10.1111/j.1476-5829.2010.00249.x
The development of metastasis is the most significant cause of death for both canine and human patients with osteosarcoma (OS). Ezrin has been associated with tumour progression and metastasis in human, canine and murine OS. Ezrin activation is dynamically regulated by protein kinase C (PKC) during metastatic progression in human and murine OS. To include the dog in the development of therapeutics that target ezrin biology, we characterized four new canine OS cell lines and confirmed the relationship between PKC and ezrin in these cells. Three of four cell lines formed tumours in mice that were histologically consistent with OS. All cell lines were markedly aneuploid and expressed ezrin and PKC. Finally, both ezrin phosphorylation and cell migration were inhibited using a PKC inhibitor. These data suggest that an association between PKC-mediated activation of ezrin and the metastatic phenotype in canine OS cells.
Osteosarcoma (OS) is the most common malignant primary bone tumour in dogs as well as humans.1–4 Spontaneously occurring canine OS, unlike induced tumours of rodent model systems, closely resembles human OS in its histological appearance, biological behaviour and response to therapy.2,4 – 6 Recent gene expression studies further suggest strong similarities between dog and human OS.7,8 Despite effective management of the primary tumour, the development of metastasis continues to be the most significant cause of death in both species.
We have previously reported that ezrin, a membrane -actin cytoskeleton linker protein, is necessary for the metastatic phenotype by providing an early survival advantage for OS cells that reach the lung.9
Ezrin plays a key role in
the coordination of signals and cellular complexes
that are required for the successful metastasis of
many tumour types.10,11 Ezrin is a member of
the ezrin–radixin–moesin (ERM) protein family, within the protein 4.1 superfamily.12 Ezrin is
involved in the determination of cell shape, cell
adhesion, motility and signal transduction. Phosphorylation of a critical threonine residue (T567) in
the c-terminus of ezrin results in a conformational
opening of its protein structure that then allows
the c-terminus of ezrin to bind the actin cytoskeleton and the n-terminus of ezrin to bind to the
cell membrane or membrane-associated proteins.
It is believed that this linkage allows for a physical connection between the cell membrane and the
actin cytoskeleton that is necessary for metastasis.
The linkage also promotes coordination of signalling events from membrane-associated receptors, resulting in increased efficiency and/or amplification of signal transduction.12 Indeed, mutation of T567, in both OS and rhabdomyosarcoma, abrogates the ezrin-dependent effects on metastasis.13,14 We have found ezrin expression in most human cancers, with aberrant expression most notably evident in cancers of mesenchymal origin.15 The linkage between ezrin expression and cancer progression and metastasis has been demonstrated in a variety of cancers including rhabdomyosarcoma, Ewings sarcoma, soft tissue sarcoma, melanoma and brain tumours.14,16 – 19 Recently, we reported that ezrin is not maintained in an active phosphorylated form throughout metastatic progression.13
Phosphorylation of ezrin is seen in cancer cells early
after their arrival in the lung and late as metastatic
lesions progress into the surrounding microenvironment. In addition, ezrin phosphorylation is
dynamically regulated during murine and human
OS metastasis by PKC. We hypothesize that targeting PKC activation of ezrin during specific times in
metastatic progression may be considered a means
to improve outcome for patients with metastasis.13
Such translational development of a therapeutic
approach targeting metastatic progression in OS,
would be optimized by the inclusion of dogs with
naturally occurring OS.
In this study, we report on the characterization
of four novel cell lines from dogs with OS.
We found ezrin expression in all four canine
OS cells lines. Furthermore, as in other species,
the phosphorylation of ezrin was regulated by
PKC. Finally, pharmacological inhibition of PKC
resulted in decreased motility of these cell lines.
These data support an association between PKC-mediated activation of ezrin in the metastatic
phenotype of canine OS cells and suggest the
continued opportunity to include canine OS in
the development of novel therapeutic approaches
targeting the activation of ezrin in OS metastasis.
Materials and methods
Establishment of canine OS cell lines
The canine OS cell lines were derived at the
time of definitive resection (amputation) of
the primary tumour (Table 1). Primary tumour
tissues were obtained from the amputated limb
under aseptic conditions. The tissues were washed
three times with Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen, Carlsbad, CA,
USA) culture media containing 10% foetal bovine
serum, penicillin (100 units/mL) and streptomycin
(100 μg/mL, Invitrogen, Carlsbad, CA, USA) and
minced into small fragments. Tumour tissues were
placed into filtered screw-top culture flasks (Nunc,
Rochester, NY, USA) moistened with medium.
The flasks were cultured at 37 ◦
C in a 5% CO2
incubator for 2 h, allowing cell adherence. Then a
small amount of DMEM containing 10% foetal
bovine serum, L-glutamine (2 mmol), penicillin
(100 units/mL) and streptomycin (100 μg/mL,
Invitrogen, Carlsbad, CA, USA) was added. The
culture medium was changed every other day
until flasks were confluent. The cells were split
into new culture flasks using 0.05% trypsin–EDTA
(Invitrogen, Carlsbad, CA, USA).
Canine OS cell line characterization
In vitro cell proliferation assay
Cell growth was analysed using a CCK-8 assay
according to manufacturers instructions (Dojindo
Molecular Technologies, Rockville, MD, USA).
Briefly, cells were plated in pentuplicate in 96-well
plates (2500 cells/well). At the indicated time points
the CCK-8 reagent was added to the medium at
a ratio with 1:10. The cells were incubated for
2 h at 37 ◦C in incubator. Optical density values
were measured at 450 nm in a microplate reader
(Molecular Devices, Sunnyvale, CA, USA).
Karyotype analysis was completed as previously
described.20 Briefly, cultured cells were arrested in
the metaphase stage of cellular division by incubating with Colcemid (KaryoMax® Colcemid Solution, Invitrogen, Carlsbad, CA, USA) (10 μg/mL)
for 2 h prior to harvest. Cells were dissociated
with 0.05% trypsin/EDTA (Invitrogen, Carlsbad,
CA, USA), treated with hypotonic solution (KCL
0.075 M) for 15 min at 37 ◦C and fixed with methanol: acetic acid 3:1. Five metaphase cells were
analysed for each cell line. Chromosomes were
stained with a trypsin-Giemsa staining technique
and karyotyped using an Axioplan 2 microscope
(Carl Zeiss MicroImaging, Thornwood, NY, USA)
coupled with a charge-coupled device camera (Photometrics, Tucson, AZ, USA), and images were
captured with Band View 5.5 karyotyping software
(Applied Spectral Imaging, Vista, CA, USA).
In vivo primary tumour growth, experimental
and spontaneous metastasis assay
For assessment of primary tumour growth and
spontaneous metastasis, two million canine tumour
cells were injected to a paraoseous location, adjacent to the left proximal tibia in 4-week-old female
severe combined immune-deficient (SCID)/Beige
mice (Charles River Laboratories International,
Wilmington, MA, USA).21 For assessment of experimental metastasis, one million cells were introduced by tail vein injection in 4-week-old female
SCID/Beige mice.21 Primary tumour endpoints
included: time to detection of tumour, percent
tumour take and tumour growth. When primary
tumours reached 1.5 cm, the tumour-bearing limb
was surgically resected. Mice were then monitored
for evidence of spontaneous metastasis and morbidity associated with pulmonary metastasis. Mice
were euthanized based on the development of these
symptoms. Necropsies were performed on all mice
to confirm the presence of metastatic lung disease.
Endpoints for experimental metastasis assays were
similar. Animal care and use were in accordance
with the guidelines of the NIH Animal Care and
Histopathology and immunohistochemistry
Primary tumour tissues were obtained at the time of
amputation and the entire lung was harvested from
mice at the time of euthanization. Primary tumour
tissues were fixed in 10% formalin in neutral buffer
overnight, no longer than 24 h, and then transferred
to 80% ethanol. Lungs were insufflated by tracheobronchial injection of 1 mL neutral buffered 10%
formalin fixed for 24 h, and then transferred to 80%
ethanol. The tissues were embedded in paraffin, sectioned at 5 μm thickness and mounted on the glass
slides. Slides were deparaffinized and rehydrated as
previously described.22 The tumour tissue sections
were stained with haematoxylin and eosin (H&E)
and examined with a light microscope. Immunohistochemistry for Alkaline Phosphatase (ALP) was undertaken as previously described.13 Briefly, slides were incubated in preheated target retrieval solution (Dako, Carpinteria, CA, USA), pH 6, in a steam
cooker for 20 min. Anti-ALP antibody (Abcam,
Cambridge, MA, USA) was used at 1:50 dilution.
The samples were counter-stained with haematoxylin (Dako, Carpinteria, CA, USA) for 30 s, mounted and examined by light microscopy.
Western blot analysis
Cells were lysed in either sodium dodecyl sulfate
(SDS) or radio-immuno precipitation assay buffer
(150 mM NaCl, 50 mM Tris, pH 8.0, 0.1% SDS,
0.5% deoxycholate, 1% NP-40) with proteinase
inhibitor cocktail (Roche Diagnostics, Indianapolis,
IN, USA). Protein lysates (20–40 μg/lane), as determined by DC protein assay (Bio-Rad Life Science,
Hercules, CA, USA), were separated by SDS-PAGE
using 4–20% Tris–glycine gels and transferred to
a nitrocellulose membrane (Invitrogen, Carlsbad,
CA, USA). The membranes were blocked with 5%
nonfat dried milk in TBS-Tween-20 (20 mmol/L
Tris–HCl, pH 7.5, 8 g/L of sodium chloride, 0.1%
Tween-20) and then incubated with anti-ezrin
(1:4000 dilution) (Sigma, St Louis, MO, USA), anti-ERM (1:1000 dilution) (Cell Signaling, Beverly,
MA, USA), anti-phosphorylated ERM (1:1000 dilution) (Cell Signaling, Beverly, MA, USA), anti-PKCα (1:1000 dilution) (Upstate, Swampscott, MA, USA), anti-PKCγ (1:1000 dilution) (BD Biosciences,
Palo Alto, CA, USA), anti-PKCι (1:250 dilution) (BD Biosciences, Palo Alto, CA, USA), anti-phosphorylated Akt (1:1000 dilution) (Cell
Signaling, Beverly, MA, USA), anti-Akt (1:1000
dilution) (Cell Signaling, Beverly, MA, USA) or
anit-β-actin (1:10 000 dilution) (Sigma, St Louis,
MO, USA) overnight at 4 ◦C. After incubation, the
membranes were washed and antibody binding was
visualized by exposure to a 1:20 000 dilution of
an anti-rabbit IgG HRP-linked antibody (Pierce,
Rockford, IL, USA) or anti-mouse IgG HRP-linked
antibody (Pierce, Rockford, IL, USA) for 1 h at
37 ◦C and developed by SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Rockford, IL,
USA) detection and subsequent exposure to film.
Kinase inhibitor treatments
An equal number of cells were plated in six-well
tissue culture plates, grown to 70% confluence and
then treated with the pharmacological inhibitor
for PKC, Ro31-8220 (Alexis Biochemicals, Lausen,
Switzerland). Dose titration (0, 0.1, 1, 5, 10 μM) at
1 h and time course study (0, 10, 30, 60, 120 min)
at 5 μM were performed. Dimethylsulphoxide was
used as the control for the treatments. The treated
cells were lysed in 200 μl of 1X Laemmli’s buffer.
Western blot analysis was performed on cell lysates
from these treated cells using anti-phosphorylated
ERM (1:1000 dilution) and anti-phosphorylated
Akt (Ser473) antibodies.
Wound-healing cell migration assay
Each canine OS cell line was plated in six-well
tissue culture dishes (Nunc, Rochester, NY, USA)
at near confluence in complete medium. A ‘wound’
was made by scraping with a P200 pipette tip in
the middle of the cell monolayer. Floating cells
were removed by washing with phosphate-buffered
saline and fresh complete medium containing
dimethylsulphoxide, 0.1 or 1 μM Ro31-8220 (Alexis
Biochemicals, Lausen, Switzerland) was added.
Cells were incubated at 37 ◦C for 13 h (KOS-
001, KOS-003 and KOS-004) or 24 h (KOS-002).
Phase contrast images were then taken using a Leica
DMIRB inverted microscope.
Morphology and in vitro cell proliferation
KOS-001 cells were variably sized spindloid to
elongate cells that contained small amounts of
cytoplasm and had angular borders (Fig. 1A). These
cells contained a single large, eccentric, round to
oval nucleus with finely stippled chromatin and one
to two prominent nucleoli. In addition, many cells
had long cytoplasmic extensions. KOS-002 cells
were variably sized mostly angular to stellate cells
interspersed with some spindloid cells (Fig. 1B). A
single large, round, centrally located nucleus was
present with finely stippled chromatin and a single
central nucleolus. These cells often had multiple
short cytoplasmic extensions. KOS-003 and KOS-
004 cells were variably sized spindloid to elongate
cells with fewer angular cells (Fig. 1C,D). These cells
had either a single long cytoplasmic extension or
multiple short cytoplasmic extensions. Nuclei were
large, round to oval, eccentrically located, with
finely stippled chromatin. Occasionally centrally
located nucleoli were present. Rarely KOS-003 cells
had multiple nuclei. Using CCK8 enumeration
of cells, growth curves for all canine OS cells
were developed (Fig. 1E) and doubling times
determined. The doubling times of KOS-001, KOS-
002, KOS-003 and KOS-004 were 19.8, 24.7, 16.0
and 12.1 h respectively.
The karyotype of all canine OS cell lines was
analysed by Giemsa trypsin banding. The karyotype
of each canine OS cell line was complex and
bizarre with multiple translocations and other
cytogenetic abnormalities (Fig. 1F). The number
of chromosomes of KOS-001 cells ranged from
97 to 105 (mode number was 101). KOS-002,
KOS-003 and KOS-004 were also abnormal with
chromosome range from 87 to 95 (mode number
was 95), from 81 to 82 (mode number was 81)
and from 96 to 103 (mode number was 103),
Figure 1. Canine cell lines have morphological appearance and karyotypic features of osteosarcoma (OS). (A–D) In vitro mesenchymal morphologies of canine cell lines. (E) The doubling times of KOS-001, KOS-002, KOS-003 and KOS-004 were 19.8, 24.7, 16.0 and 12.1 h, respectively. (F) Representative G-banding karyotype of canine (KOS-002) OS cells reveals marked aneuploidy. Similar complex and bizarre karyotypes were seen in all other cells. KOS-001, KOS-002, KOS-003 and KOS-004 cell line showed karyotype range 97–105 (mode number was 101), 87–95 (mode number was 95), 81–82 (mode number was 81) and 96–103 (mode number was 103), respectively. Scale bar = 200 μm.
In vivo primary tumour growth, experimental
and spontaneous metastasis
KOS-001, KOS-003 and KOS-004 cells developed
primary tumours after paraoseous injection in
immunocompromised mice (Fig. 2). Mice receiving
KOS-002 did not develop primary tumours
followed 1 year after injection (Table 2). On
histological examination hind limbs contained
Figure 2. Canine osteosarcoma (OS) cell lines are tumorigenic in mice and yield tumours with histological descriptors
characteristic of OS. (A,D,G) Primary tumour growth patterns of canine OS cells in Beige-SCID mice. Each line represents growth of an individual mouse. H&E staining (B,E,H) of primary tumour resulting from orthotopic injection of KOS-001, KOS-003 and KOS-004 cell lines showed streams of closely packed spindloid tumour cells within a fibrous stroma and small islands of osteoid (see arrow heads) are scattered between the tumour cells. (C,F,I) Primary tumours of KOS-001, KOS-003 and KOS-004 cell lines showed positive ALP staining. Scale bar = 100 μm.
unencapsulated, but well-demarcated masses that
invaded and replaced the musculature and adjacent
bone in all three canine OS cells (Fig. 2B,E,H).
Neoplastic cells were arranged in closely packed
streams and bundles supported by a fibrous
stroma. Individual cells were mainly elongated
spindle cells with some round to polygonal
cells scattered throughout. Cell borders were
often indistinct and contained scant to moderate
amounts of eosinophilic to amphophilic cytoplasm.
Nuclei were eccentric, round to oval with a
hypochromatic to euchromatic chromatin staining
pattern and finely stippled to coarsely clumped
chromatin distribution. One to three prominent,
round, basophilic nucleoli were present in most
cells. Small to moderate amounts of faintly to
intensely eosinophilic, hyaline material (osteoid)
was arranged in narrow ribbons to small irregular
islands between malignant cells. Strong ALP
expression was detected in primary tumours (KOS-
001, KOS-004 and KOS-003, Fig. 2C,F,I).
Following resection of the primary tumour bearing
limb in mice receiving KOS-003 cells,
pulmonary metastasis developed within 30 days
Figure 3. Assessment of in vivo metastatic phenotype of canine osteosarcoma (OS) cell lines. Overall survival of canine OS cells in xenograft metastasis model. (A) Kaplan–Meier survival curves demonstrate progression to spontaneous metastasis in KOS-003 cells following orthotopic delivery of tumour cells to mice. No spontaneous pulmonary metastases were seen in KOS-001 and KOS-004 over 1-year observation period. (B) H&E staining of the resultant metastases were consistent with OS. (C) Spontaneous lung metastasis tumours showed positive ALP staining. (D) The metastatic biology of the KOS-003 was confirmed using experimental metastasis via tail vein injection. (E) H&E staining of experimental metastasis again show osteoid production (arrow head) and (F) ALP staining. Scale bar = 100 μm.
(Table 2 and Fig. 3A). None of the mice receiving
KOS-001 and KOS-004 developed metastasis
following tumour-bearing limb resection through a
1-year period of observation (Table 2 and Fig. 3A).
Consistent with results for spontaneous metastasis, experimental metastases were present only in the
KOS-003 cells. The median survival (death because
of morbidity of pulmonary metastasis) for these
mice was 49 days (Fig. 3B). Pulmonary metastases
from both experimental and spontaneous metastasis were diffuse and widespread. Lung metastases
had a similar histo-morphological appearance to
the primary tumours, including islands of osteoid
and strong ALP expression (KOS-003, Fig. 3C,F).
Ezrin and PKC isoforms α, γ and ι are
expressed in canine OS
To determine the protein expression of ezrin and
PKC isoforms α, γ and ι in canine OS cells, Western blot analysis was performed including K7M2
murine OS cells lysates as a positive control.13 As
shown in Fig. 4A,B, ezrin and PKC isoforms α, γ
and ι were expressed in all canine OS cell lines
PKC regulates ezrin Thr567 phosphorylation
(activation) in canine OS cells
To test the hypothesis that PKC phosphorylates
ezrin at threonine 567 in canine OS cells, a
pharmacological inhibitor of PKC, Ro31-8220, was
used. As shown in Fig. 5, Ro31-8220 suppressed
c-terminal phosphorylation of ERM proteins in a
dose- and time-dependent manner. In contrast, Akt
(Ser473) phosphorylation was not affected by PKC
inhibitor treatment even at the highest dose–time
exposures of 5 μM over 120 min.
PKC-mediated tumour cell migration
in wound-healing cell migration assay
To verify the biological significance of PKC
regulation on ERM phosphorylation, we examined
the effects of PKC inhibition on cell motility using
a standard wound-healing assay for cell migration.
Cellular motility was assessed over 13 h (KOS-
001, KOS-003 and KOS-004) or 24 h (KOS-002).
Dose and time-point combinations were selected
to minimize any potential effects of PKC inhibition
Figure 4. Ezrin and PKC were expressed in all of canine
osteosarcoma (OS) cells. (A) Ezrin was expressed in all of
canine OS cells. (B) PKC isoforms α, γ and ι were expressed in all of canine OS cells. K7M2 murine OS cells were used as a positive control.13
on cell proliferation and viability. Images of the
‘wound’ were taken immediately (time 0) and 13
or 24 h post-wounding. As shown in Fig. 6, the
cells treated with the PKC inhibitor, Ro31-8220,
had a significant delay in migration compared with
We have established and characterized four canine
OS cell lines using both vitro and in vivo assessments
of tumour biology. The morphological phenotype
of these cells was mesenchymal. The karyotypic
features supported their canine origin, and the
complex and markedly aneuploid chromosomal
number and arrangement were characteristic of
OS.23 Histological features of xenograft tumours
were consistent with OS with the presence of
atypical sarcoma cells producing tumour osteoid
and expressing ALP. Following orthotopic growth
in mice, one of the established canine OS cell
lines formed spontaneous pulmonary metastasis
within 1 month of resection of the tumour-bearing
limb. This panel of cell lines will provide important
opportunities to study the complexity of OS biology
and therapy. Indeed, in this report we specifically
applied these cell lines to study the connection
between the metastasis-associated protein, ezrin
and PKC. All four OS cell lines expressed
ezrin and classical PKC family members. The
phosphorylation of ezrin and cellular motility was
inhibited using a specific and previously described
inhibitor of PKC in these canine cells. These data
support the continued inclusion of canine OS in the
development of novel therapeutics that target the
ezrin–PKC phenotype in metastatic progression.
We have previously published that ezrin
phosphorylation is dynamically regulated during
metastasis in murine and human OS.13 Ezrin is
expressed in a phosphorylated form early after
metastatic cells arrive in the lung. This active
conformation of ezrin is believed to be necessary for
OS cells to overcome the inefficiency of metastasis
that occurs early after metastatic cells arrive at a
secondary site. We hypothesize that ezrin protects
cells from the stresses experienced by cells as
they arrive in this foreign microenvironment. After
successful metastatic lesion progress from the single
cell to multiple cell level, we were surprised to find
that ezrin, although consistently expressed, was no
longer phosphorylated. As the metastatic lesions
progressed and cells began to engage the lung
parenchyma, ezrin again became phosphorylated
most notably at the periphery of the metastatic
lesion. Through a number of techniques we have
demonstrated that this dynamic regulation of ezrin
phosphorylation is the result of classical PKC family
members.13 Finally, some of the recognized effects
of PKC on cell motility and invasion were shown to
be dependent on ezrin activation.24 – 26 These data
suggested an opportunity to develop therapeutic
strategies for cancer metastasis that target the
An important hurdle that exists in the development of PKC-based therapeutics for cancer has
been toxicity.27 This problem has been managed
through increasingly selective inhibitors of PKC
and the development of treatment schedules that
are tolerable in patients. Small molecule inhibitors
of PKC have now successfully moved into phase
II clinical trials in human patients for a number
Figure 5. Ezrin (T567) phosphorylation is dependent on protein kinase C (PKC). (A) Canine osteosarcoma (OS) cells were incubated with various concentrations of PKC inhibitor Ro31-8220. Phospho-ERM expression is shown by western blot analysis (phosphorylated ERM is comprised of a phospho-ezrin/radixin band ∗ and a phospho-moesin band ∗∗). (B) Canine OS cells were treated with PKC inhibitors Ro 31-8220 for indicated times. The level of phospho-ERM was analysed by Western blotting. The blots were probed for β-actin as the loading control, and phospho-Akt (Ser473).
Figure 6. PKC inhibitors suppress in vitro cell migration of canine osteosarcoma cells. Nearly confluent cells were
‘wounded’ (Scratched) using a P-200 pipette, and images of the denuded area were taken immediately following wounding (0 h) and either 13 or 24 h (KOS-002) after the wound. Migration of KOS-001, KOS-003 and KOS-004 is evident by their migration across the wounded area (Control Migration). Inhibition of this migration is seen following exposure to 0.1 and 1 μM PKC inhibitor, Ro 31-8220. The specific effects of PKC inhibition on cell migration were assured through the selection of drug exposures that did not influence cell viability or proliferation (data not shown) and at times points less than the doubling times of each cell line. The native migration rate of KOS-002 even after 24 h was very low and consistent with its observed low-aggressive phenotype. Scale bar = 200 μm.
of indications.28,29 On the basis of our interest in
PKC, ezrin and metastasis, it is reasonable that
some of the toxicity concerns linked to long-term
high-dose exposures to PKC inhibitors may be
managed through intermittent dosing that may
effectively interrupt the dynamic regulation of PKC
on ezrin seen during metastasis. An optimal drug
development path based for PKC inhibitors used
in this manner requires the development of informative pharmacodynamic biomakers, and a means
to model the complexity of metastatic progression
from a period of minimal residual disease to gross
metastasis. The inclusion of pet dogs with OS into
such a development path would provide an opportunity to model this biology and answer questions
that are difficult to answer in conventional preclinical models or in human patients. The outcome of
such a successful development path could be more
effective treatments for both canine and human
patients with OS.
To extend our understanding of PKC-mediated
ezrin phosphorylation to canine OS, we report
herein on the expression of ezrin and PKC
family members in canine OS cells and the
regulation of ezrin phorphorylation by PKC. All
four canine OS cell lines expressed ezrin, other
ERM proteins and PKC α, γ and ι. The levels
of expression were comparable to a previously
characterized and metastatic murine model of OS
(K7M2). It is interesting to note that the, highly
aggressive KOS-003 showed lower expression of
some PKC isoforms, compared with other cells.
It is unclear what threshold of PKC isoforms
expression is necessary to convey a ‘PKC functional
status’ to a cell. These data further support
the recognized concept that the expression of a
single protein is not sufficient to account for the
phenotype of a metastatic cancer cell. Using a
small molecule PKC inhibitor, we found down
regulation of phosphorylated ERM in dose- and
time-dependent manner. The specificity of this
effect was confirmed by lack of effect on the
phosphorylation status of the Akt pathway. In
addition, using wound-healing migration assay as
an example of a PKC-related metastatic phenotype,
we found significant inhibition of cellular migration
following identical exposure to the small molecule
inhibitor of PKC.
In conclusion, using four newly established
canine OS cell lines we have demonstrated that
the connection between PKC and ezrin is linked
to the biology of canine OS. Specifically, PKC is
responsible for the phosphorylation of ezrin and cell
migration in canine OS cells. These data support the
continued study of novel therapeutic approaches
that target the ezrin–PKC phenotype in canine OS.
This project was supported by a grant (1046-A)
from the American Kennel Club Canine Health
Foundation. The contents of this publication are
solely the responsibility of the authors and do not
necessarily represent the views of the Foundation.
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