Summary:
Oral cancer is the sixth most common cancer worldwide and the incidence of new
cases indicates a continuing rise in developing countries. Several genetic and
epigenetic alterations underlie the progressive acquisition of a malignant
phenotype
in head and neck squamous cell carcinoma (HNSCC). The molecular dissection of
aberrant signaling networks, including EGFR, Ras, NF-κB, Wnt/β-catenin, TGF-β,
and PI3K-AKT-mTOR signaling pathways, has increased our understanding on the
basic mechanisms controlling HNSCC progression.
Signal transducer and activator of transcription (STAT) proteins constitute a
family of transcription factors, which exist in the cell as latent cytoplasmic
transcription factors and become activated in response to stimulation by
cytokines and growth factors, translocate to the nucleus, where they interact
with the promoters of target genes. Phosphorylation of STAT molecules on a
tyrosine residue is the first critical event for their activation and has been
convincingly shown to correlate with STAT DNA binding and transcriptional
activity. In contrast, STAT serine phosphorylation, which may also occur in
response to growth factor and cytokine stimulation, has been associated mainly
with negative regulation of STAT activity. STAT signaling has been found to be
involved in oncogenesis There is compelling evidence that STAT3 constitutive
activation, mainly associated with aberrant TGF-α/EGFR signaling, is linked to
HNSCC development and growth.
Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved
kinase modules that link extracellular signals to the machinery that controls
fundamental cellular processes such as growth, proliferation, differentiation,
migration and apoptosis. MAPKs phosphorylate serine and threonine residues of
specific target proteins. They are classified into three major subfamilies,
including extracellular signal-regulated kinases (ERKs), p38 MAPKs, and c-Jun
NH2-terminal kinases (JNKs)
Previous studies supported an association between activation of specific
members of the MAPK family and negative regulation of STAT3, manifested by
downregulation of STAT3 tyrosine phosphorylation and induction of STAT3 serine
phosphorylation, in various cell types including cancer cells.
PURPOSE
The aim of the present investigation was to evaluate whether oncogenic
constitutive STAT3 signaling in oral squamous cell carcinoma (OSCC) cells can
be modulated by regulation of specific MAPKs. The expression and activation
status of STAT3 and MAPKs in HNSCC cell lines were recorded and the effects of
selective MAPKs inhibition or activation on STAT3 signaling and cellular
proliferation were monitored, in an attempt to elucidate important molecular
aspects of oral cancer with potential therapeutic implications.
MATERIAL AND
METHODS
Cell lines and cell culture. Experiments were performed using established cell
lines of human HNSCC (SCC9 and SCC25) obtained from the American Type Culture
Collection (ATCC;Manassas, VA, USA).
Selective inhibition of MAPKs. Cells were treated either with the vehicle
alone(DMSO) or with the selective MAPK inhibitor (JNK1/2- SP600125,
p38-SB203580, Erk1/2-U0126) (Calbiochem, San Diego, CA, USA) at specific
concentrations for 24 h.
Selective induction of MAPKs. Cells were treated either with the vehicle alone
(DMSO) or with the selective MAPK (active Mek1/2-Erk1/2, active MKK7-JNK1/2)
inducers (ProSpec, Israel) at specific concentrations for 48 h.
siRNA transfection: JNK1, JNK2 and Erk1, Erk2 siRNAs were based on NCBI
Reference Sequences (GenBank:Erk1: NM_002746 and Erk2: NM_002745). All siRNA
and scrambled control siRNA (siControl) were purchased from Qiagen. All siRNA
transfections were performed using Lipofectamine 2000 (Invitrogen), according
to the manufacturer's protocol. After OSCC cells were grown, they were
transiently transfected with 50 μg of the empty vector or siRNA using
Nucleofector II reagents (Amaxa Biosystems-Lonza, Cologne, Germany).
Western blot experiments. Western blotting was performed using antibodies
against: total STAT3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), total
p44/42(Erk1/2) total p38, total JNK1/2, phospho-p38, phospho-c-Jun, cyclin D1
(Cell Signaling, Beverly, MA,USA), phospho-Erk1/2 (Upstate, Charlottesville,
VA, USA), cyclin D1 (Cell Signaling) and β-actin (Sigma Chemical).
Cell proliferation and viability. Cells were counted with a hemocytometer under
inverted microscope. Cell viability after treatment was determined by the
trypan blue dye exclusion test 24 h after each treatment. All assays were
performed in
quadruplicate and results are reported as the mean ± SD.
Immunohistochemistry. The material consisted of 60 specimens diagnosed as oral
SCC, selected from the Department of Oral Pathology and Medicine, Faculty of
Dentistry, University of Athens. The specimens were divided in 3 groups of 20
cases according to their differentiation level (high, medium, poor). Staining
was performed for the following antibodies: phospho-STAT3 (Tyr705)(1:100),
phospho-STAT3 (Ser727) (1:100), phospho-cJun (1:100), phospho-ERK1/2(1:100),
phospho-p38(1:100).
Statistical Analysis. The relationship between cell viability/cell number and
concentrations of chemical reagents was assessed with the t-test. The
association between immunohistochemical variables (intensity/ percentage of
positive cells, combined score) and differentiation levels was analyzed by
Fisher’s test. Differences were considered to be statistically significant
when: p< 0.05.
RESULTS
The results of this study are summarized as follows:
Erk1/2 inhibition: Erk1/2 inhibition by U0126 treatment was more potent in oral
SCC25 cells and was associated with a decrease in p-ser STAT3 and cyclin D1
levels without affecting p-tyr STAT3 levels.
Moreover, specific silencing of Erk1/2 resulted in a decrease in p-ser STAT3
and cyclin D1 levels in both cell lines and an increase in p-tyr-STAT3,
particularly in oral SCC9 cells. Both chemical inhibition with U0126 and
silencing Erk1/2 resulted in a dose-dependent reduction in cell growth and cell
viability in both cell lines.
Erk1/2 induction: Erk1/2 induction caused upregulation of p-ser STAT3 and
cyclin D1 levels in both cell lines, decrease in p-tyr-STAT3 and resulted in a
significant dose-dependent increase in cell growth, which was more prominent in
the oral SCC25 cell line. On the contrary, treatment of cells with active
MEK1/2 did not appear to induce notable changes in cell viability in either
cell line.
JNK1/2 inhibition: JNK1/2 inhibition by SP600125 treatment was detected in both
cell lines and was associated with increases in p-tyrr Stat3 and cyclin D1
levels as well as decreases in p-serr Stat3 levels at the highest concentration
used.
Specific silencing of JNK1/2 resulted in decreases in p-ser Stat3 and cyclin D1
levels in both cell lines and increases in p-tyr-Stat3 particularly in oral
SCC9 cells. Similar to chemical inhibition with SP600125 silencing of
JNK1/2 resulted in a dose-dependent rise in cell growth and cell viability in
both cell lines. JNK1/2 induction: JNK1/2 induction
caused upregulation of p-ser Stat3 levels in both cell lines and decreases in
p-tyr Stat3 and cyclin D1. Moreover, active MKK7 treatment at the highest
concentration resulted in significant dose-dependent decrease in cell growth
and cell viability, which was more prominent in oral SCC25 cell line.
P38 inhibition: p38 inhibition by SB203580 treatment was not associated with
any significant change in phosphorylated levels of STAT3 (ser/tyr) and cyclin
D1 in both cell lines. Furthermore treatment with SB203580 for 24h resulted in
no statistically significant changes in cell growth and cell viability in both
cell lines tested.
Immunohistochemistry
p-STAT3(ser): Eventhough poorly differentiated neoplasms showed the lowest mean
average score, no statistically significant correlation was found between the
three histopathological parameters (intensity/percentage of positive cells,
combined score) and differentiation level for
p-STAT3(ser).
p-STAT3(tyr): Statistically significant correlation (p<0.05) was found between
all three histopathological parameters and differentiation level of neoplasms
with poorly differentiated cases showing the highest mean average scores.
Furthermore statistically significant difference (p<0.05) was found between
poorly and well differentiated neoplasms for all tested
parameters. p-p38: No
statistically significant correlation was found between the three
histopathological parameters (intensity/percentage of positive cells, combined
score) and differentiation level for
p-p38.
p-Erk1/2: There is statistically significant correlation (p<0.05) between all
three histopathological parameters and differentiation level of neoplasms
with poorly differentiated cases showing the highest mean average scores.
Moreover statistically significant difference (p<0.05) was found between
poorly and well differentiated neoplasms for all the tested
parameters.
p-cJun: Poorly differentiated neoplasms showed the lowest mean average score
for all three histopathological parameters, but the only statistically
significant correlation (p<0.05) concerned intensity score between poorly and
medium differentiated neoplasms.
CONCLUSIONS
- Our data are supportive of the oncogenic potential of Erk1/2 in OSCC, which
appears to contribute to cell proliferation.
- The oncogenic STAT3 constitutive signaling in OSCC cells appears to be
negatively regulated by Erk1/2. The Erk1/2-STAT3 crosstalk appears to involve
mainly ERK-induced upregulation of STAT3(Ser727) phosphorylation while Tyr705
phosphorylation does not exhibit major changes.
- JNK1/2 oncosuppressor potential in OSCC is supported by the reduction of
cell proliferation and cell viability.
- The oncogenic Stat3 constitutive signaling in OSCC cells is negatively
regulated by JNK. The JNK-STAT3 crosstalk seems to respond mostly through the
downregulation of Stat3 phosphorylation on Ser727 residue and upregulation of
Tyr 705.
- P38 does not appear to play a significant role in STAT3 signaling, cell
proliferation and cell viability.
- ΕRK1/2, p38 and p-cJun are strongly expressed in OSCC indicating their
significance in this type of cancer.
- It is possible that the role of Erk1/2, JNK and p38 in STAT3 modulation
varies according to the type and status of the cells studied, indicating the
need to identify the role of MAPK activation in relation to STAT3 signaling in
specific cell types.
- Understanding MAPK pathway complexity and cross-talk between other major
molecules such as STAT3 will be helpful to pharmacologic therapies for cancer
prevention.
