Mangone et al. Molecular Cancer 2010, 9:106
http://www.molecular-cancer.com/content/9/1/106
Open Access
RESEARCH
Smad2 and Smad6 as predictors of overall survival
in oral squamous cell carcinoma patients
Research
Flavia RR Mangone*†1, Fernando Walder†2,3, Simone Maistro1, Fátima S Pasini1, Carlos N Lehn3, Marcos B Carvalho3, M
Mitzi Brentani1, Igor Snitcovsky1 and Miriam HH Federico*1
Abstract
Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous
cell carcinoma (SCC).
Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection
Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched
adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or
negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR
(QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC.
Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC
patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good
prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition,
simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas
the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we
found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight
TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients
were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological
characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.
Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after
curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the
future.
Background
The Smad family of proteins, Smads 1 to 8, are key molecules in Transforming Growth Factor-β (TGFβ) signaling,
eventually modulating both TGFβ tumor suppressive and
oncogenic effects [1]. Among them, Smad2 and Smad3
are known as receptor regulated Smads (R-Smads) and
are phosphorylated in response to TGFβ itself. The phosphorylated protein, in conjunction with the common
Smad (Co-Smad), Smad4, translocates to the nucleus eliciting the transcription of other genes [2-4]. The Inhibi* Correspondence: flavia@lim24.fm.usp.br, federico@usp.br
Disciplina de Oncologia, Departamento de Radiologia, LIM 24, Hospital das
Clínicas da Faculdade de Medicina da Universidade de São Paulo, Avenida Dr
Arnaldo 455, São Paulo, Brasil
† Contributed equally
tory Smads (I-Smads) Smad6 and 7, on the other hand,
prevent the activation of R-Smad by phosphorylation
and/or interfering with its nuclear translocation [5-7].
Smad signaling seems to be relevant to the pathogenesis of several epithelial cancers. Smad4 and Smad2 functions are disrupted in pancreatic, esophageal, gastric,
colon and lung cancer [8-12]. Over-expression of inhibitory Smad6 and Smad7 was described in pancreatic cancer and in pancreatic cancer cell lines [13,14]. Smad2 and
3 present different targets and have distinctive roles, as
shown in skin tumors of transgenic mice [15].
Concerning head and neck squamous cell carcinoma
(HNSCC), however, data on Smads are still scarce. Studies done with HNSCC samples have shown alterations of
individual Smad expression as measured by immunohis-
Full list of author information is available at the end of the article
© 2010 Mangone et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Mangone et al. Molecular Cancer 2010, 9:106
http://www.molecular-cancer.com/content/9/1/106
Page 2 of 10
tochemistry [16,17]. In addition, evidence obtained in in
vitro studies indicates that Smad signaling may enhance
invasiveness in HNSCC [18].
We have previously suggested that, in oral SCC but not
in other HNSCC sites, the tumor supressive effect of
TGFβ was absent in lymph node positive (pN+) but still
present in lymph node negative (pN0) patients [19].
Therefore, we assumed that the extent of expression of
individual Smad mRNAs might reflect the degree of
TGFβ resistance, and in this way, correlate with progression in oral SCC and consequently with survival. In this
work, we found that Smad family mRNA expression was
globally increased in HNSCC as compared to adjacent
tissue. In addition, among all Smads, Smad2 and Smad6
were suggested to be prognostic markers, correlating
with overall survival.
10 μg of total RNA were hybridized with α32P-UTP (GE
Healthcare Biosciences - formerly Amersham-Biosciences, St. Giles, UK) labelled riboprobes (8.0 × 105 cpm
per sample) for 16 hours at 56°C, subjected to RNAse
A+T1 digestion followed by a phenol-chloroform extraction and ethanol precipitation. The protected double
strand RNAs were eletrophoresed in a 5% acrylamide/
bis-acrylamide (29:1) urea-containing gel, then the gel
was dried and subjected to autoradiography (Hyperfilm,
Amersham Biosciences) for 48 hs at -70°C. Specific bands
were identified by their distinctive migration pattern as
compared to the pattern of undigested probes. Densitometric analysis (ImageMaster VDS software, version 2.0 Amersham Biosciences) was used for quantification. Each
Smad mRNA expression was normalized to L32 housekeeping gene.
Patients and Methods
Real-Time Quantitative Reverse Transcriptase PCR (real
time QRT PCR)
Patients
Surgical specimens of primary oral SCC were prospectively and sequentially obtained from 48 patients (median
age 55 years, range 30 - 86; 43 male and 5 female) with
previously untreated, operable HNSCC admitted at the
Department of Head and Neck Surgery, Hospital
Heliópolis - São Paulo - SP - Brazil. Matched adjacent
mucosa, from the resection margin, was obtained from
40 patients. The Smad 1-8 mRNA expression of these and
other 35 samples from different head and neck sites was
evaluated. The general characteristics of patients are presented in Table 1.
All specimens were snap-frozen and stored in liquid
nitrogen until analysis. Tumor staging was performed
according to the Fifth Edition of the UICC TNM Classification of malignant tumors. Patient follow-up ranged
from 14.0 to 53.0 months (median 33.0 months). At the
last follow-up, among the 83 patients, 30 had local recurrences, 17 had regional recurrences, 44 patients had died
and 6 patients were lost to follow-up.
The protocol was approved by the human review
boards at the participating institutions and registered at
Brazilian National Research Committee (CONEP). All
patients provided voluntary written informed consent
before enrolment in compliance with the Declaration of
Helsinki and its amendments.
RNA Extraction and Ribonuclease Protection Assay (RPA)
Frozen tissue samples were pulverized and total RNA was
obtained by using TRIzol Reagent (Invitrogen, Life Technologies) following the manufacturer's instructions.
Detection and quantification of the Smad family members and of the ribosomal protein L32 were carried out
with hSmad multiprobe template set (PharMingen's
RiboQuant™ Mult-Probe Ribonuclease Protection Assay
System) according to the manufacturer's protocol. Briefly,
Five micrograms of total RNA were reverse-transcribed
using Random Hexamer primer pre-hit for 10 minutes at
70°C and incubated 10 minutes at room temperature
before the addition of the reaction mix (1× Buffer Super
Script III, 20 μM of each deoxynucleotide triphosphate,
10 U Super Script III and 0.02 M DTT - Invitrogen, CA,
USA). The reaction was performed at 55°C per 50 minutes and interrupted by 15 minutes incubation at 70°C.
Real-time QRT PCR was carried out with SYBR Green
dye in a Rotor Gene - RG300 (Corbett Research, DE). Oligonucleotide primers were designed for human TGFβ
isoforms and β-actin house keeping using the Primer3
program (Whitehead Institute for Biomedical Research,
http://www.bioinformatics.nl/cgi-bin/primer3/
primer3_www.cgi), based on its mRNA sequences. The
synthesized forward and reverse primer sequences were
(IDT, Integrated DNA Technologies, IA, USA): β-actin
(NM_001101.3: fw 5'AGAAAATCTGGCACCACACC3'
and rev 5'AGAGGCGTACAGGGATAGCA3'); TGFβ1
(NM_000660.4: fw 5'CCCTGGACACCAACTATTGC3'
and rev 5'TGCGGAAGTCAATGTACAGC3'; TGFβ2
(NM_003238.2: fw 5'GAGTGCCTGAACAACGGATT3'
and rev 5'TTCACAACTTTGCTGTCGATG3'); TGFβ3
(NM_003239.2: fw 5'TGATCCAGGGGCTGGCGGAG3'
and rev 5'GGGTTGGGCACCCGCAAGA3').
The PCR reaction mixture, performed with 100 ng of
cDNA, was: 1.5× SYBR Green I nucleic acid gel stain
(Molecular Probes, OR, USA), 1× PCR Buffer, 2 mM
Magnesium Chloride, 0.2 mM of each deoxynucleotide
triphosphate, 1.5 U of Platinum Taq DNA Polymerase, 0.5
mg/mL Bovine Serum Albumin Acetylated (Promega,
WI, USA), 5% of DMSO (Sigma, CA, USA), 0.2 μM of
each primer in a total volume of 20 mL in Molecular Biology Grade Water (Invitrogen, Life Technologies, CA,
USA). These experiments were performed in duplicate.
Mangone et al. Molecular Cancer 2010, 9:106
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Table 1: Clinical Pathological characteristics of studied population.
Oral Cavity
Larynx
Oropharynx
Hypopharynx
total
pN0
23
11
2
2
38
pN+
25
10
8
2
45
pT1/T2
17
4
1
0
22
pT3/T4
31
17
9
4
61
I/II
13
4
0
0
17
III/IV
35
17
10
4
66
48
21
10
4
83
Lymph node status
Tumor size
Clinical Staging
total
Oral cavity: 25 mouth floor, 5 lower gum, 5 retromolar area, 10 tongue border, 1 hard palate, 1 tongue ventricular surface, 1 mouth anterior
floor; Larynx: 6 aryepiglottic folds, 10 vocal cords, 3 epiglottis, 2 false cord; and Oropharynx: 2 glossotonsilar sulch, 6 tonsil, 1 soft palate, 1
vallecula. All 4 Hypopharynx tumors were from pyriform sinus.
The thermal cycling included an initial denaturation step
of 5 minutes at 95°C followed by 35 cycles of 15 seconds
at 95°C, 1 minute at 60°C and 1 minute at 72°C. Melting
analysis was performed by heating the reaction mixture
from 74 to 99°C at a rate of 0.2°C/second. Threshold cycle
(Ct) and melting curves were acquired by using the
"quantitation" and "melting curve" program of the Rotor
gene 6 software version 6.0 Corbett Research (Corbett
Research, DE). Only genes with clear and single melting
peaks were taken for further data analysis. Samples with
irregular melting peaks were excluded from the calculation. The threshold was set manually, using identical
threshold levels for one gene in all analyzed samples.
Reaction efficiency was established for each set of primers, after quantification of four different dilutions of a reference cDNA.
The Ct value of three targets genes (TGFβ1, TGFβ2 and
TGFβ3) was normalized to the reference gene Ct (βactin) and the relative quantification was performed
according to Pfaff mathematical model [20].
Statistical analysis
Comparisons between groups were performed by the
paired Wilcoxon test, when appropriate. For survival and
Spearman's correlation analysis, patients were categorized as positive (Smad+, TGFβ+) or negative (Smad-,
TGFβ-) according to the median relative expression of
each (above or equal/below the correspondent median
tumor expression). Overall survival (OS) and disease free
survival (DFS) were considered from the day of the surgery to date of death or the date in which recurrence was
detected, by means of physical examination or imaging.
Survival curves were estimated using the Kaplan Meier
method and compared using the univariate Log Rank
test. A Cox multivariate analysis was performed to identify independent predictors of survival. All statistics were
done using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL). Differences were considered statistically significant for P value ≤ 0.05.
Results
Smad mRNA expression in oral SCC
In this study, we determined Smads (Smad1 to 8) mRNA
expression in 48 primary tumors (Table 1) from patients
with oral SCC submitted to curative ressection (see representative assay in figure 1A). Quantification of the RPA
signals, normalized to L32 mRNA, revealed that, up to
91% of the tumors expressed all Smad mRNAs, except for
Smad8 mRNA, detected in 73% of the tumors. In parallel,
100% of the 40 available specimens of adjacent mucosa
expressed Smads 1 to 7, and Smad8 mRNA expression
was detected in 52.5%. The distribution and median of
Smads expression are shown in Figure 1B.
Paired analysis revealed that oral SCC express more
Smad2, 3, 5 and 7 mRNAs than matched mucosas (P <
0.05, Table 2). When clinical-pathological features such
as lymph node status, tumor size, differentiation degree,
pathological staging, age, gender and smoking degree
were considered, no statistically significant differences
were found.
In survival analysis, Smad2- patients presented longer
median OS as compared to Smad2+ patients (median OS
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A
B
Smad 1
Smad 2
Smad 3
Smad 4
Smad 5
Smad 6
Smad 7
Smad 8
L32
T
M
1
T
M
2
T
M
3
Figure 1 Smad mRNA expression in oral SCC: A. RPA representative assay. Lane 1: riboprobe; represented as pairs: T-tumor M- matched adjacent
mucosa; L32: housekeeping gene. B. Boxplot representing Smad1-8 mRNA expression. Boxes: 25th, 50th and 75th percentiles; bars: 10th and 90th percentiles; °: outlier values (1.5-3 box-lengths from 75th percentiles); *: extreme values (>3 box-lengths from 75th percentiles).
not reached and 14.6 months, respectively), while the
mOS for the entire oral SCC group was 31 months, a similar behaviour being found for mDFS (Figure 2A and 2B).
Similarly, Smad6+ patients presented a median OS three
times longer than Smad6- patients (52 and 14.6 months,
respectively). Also, the Smad6+ mDFS was 52 months, six
times longer than that of Smad6- patients (Figure 2C and
2D). According to lymph node status, the mOS was 37
months in pN0 patients and 14 months in pN+ patients,
with survival rates of 61% in pN0 patients (n = 23) and 36
in pN+ patients (n = 25, p < 0.05).
By Cox regression multivariate analysis, those two
markers, Smad2 and Smad6, were shown to be prognostic
factors in oral SCC, independent of lymph nodal status
(Table 3). In line with this, even with a small population,
pN+ patients who were Smad6+ presented 31 months
mOS, three times longer than that presented by Smad6patients (11 months). Accordingly, in the pN0 subgroup,
Smad6- patients had 23 month mOS, shorter than that of
Smad6+ patients who did not reach mOS (P = 0.009). The
same seems to occur with simultaneously Smad2+ and
pN+ patients who presented a poorer prognosis as compared to Smad2-/pN+ patients who did not reach mOS.
Consonant with this, patients who were Smad2-/Smad6+
simultaneously did not reach mOS and mDFS whereas
Smad2+/Smad6- patients presented mOS 12 months and
mDFS 6 months (P = 0.0044, P = 0.0012, respectively, Figure 2E, F).
Smad mRNA expression in larynx SCC and in other HNSCC
subsites
Except for Smad6 (90%) and Smad8 (86%), Smads were
expressed in 100% of larynx SCC. In matched adjacent
mucosa, Smad6 was expressed in 73%, Smad7 in 93% and
Smad8 in 67%, the others being expressed in 100% of
samples.
Paired analysis revealed that only Smad7 was overexpressed in tumors (0.08 ± 0.06) as compared to adjacent
mucosa (0.03 ± 0.04, P = 0.002). This marker was the only
one to correlate with survival in this subset of patients,
with a survival advantage observed in Smad7- patients
(mOS not reached) over those who were Smad7+ (mOS
6.97 months, P = 0.04, Figure 3). The survival rate of
Smad7- and Smad7+ patients was 54.5% and 30% respectively. When clinical-pathological features as lymph node
status, tumor size, differentiation degree, pathological
staging, age, gender and smoking degree were considered, no statistical difference was found related to Smad7
status.
In oropharynx SCC, even with a small sample size,
paired analysis showed Smad overexpression for Smad2
to 5, while in the entire HNSCC population, a significant
difference was not achieved only for Smad8, confirming
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Table 2: Smad mRNA expression in head and neck SCC and adjacent mucosa.
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7
Smad8
All patients n = 65
Oral Cavity n = 40
Larynx n = 15
Oropharynx n = 8
T
0.06 ± 0.06
0.06 ± 0.05
0.07 ± 0.06
0.06 ± 0.10
M
0.05 ± 0.05
0.06 ± 0.05
0.05 ± 0.04
0.03 ± 0.05
P = 0.017*
P = 0.209
P = 0.256
P = 0.012
T
0.15 ± 0.10
0.16 ± 0.10
0.17 ± 0.10
0.13 ± 0.10
M
0.11 ± 0.09
0.11 ± 0.08
0.15 ± 0.10
0.08 ± 0.11
P = 0.002*
P = 0.020*
P = 0.532
P = 0.036*
T
0.15 ± 0.11
0.13 ± 0.08
0.18 ± 0.13
0.18 ± 0.16
M
0.11 ± 0.09
0.10 ± 0.08
0.15 ± 0.12
0.06 ± 0.08
P = 0.001*
P = 0.028*
P = 0.047*
P = 0.017*
T
0.13 ± 0.09
0.13 ± 0.08
0.15 ± 0.09
0.12 ± 0.12
M
0.11 ± 0.08
0.12 ± 0.07
0.13 ± 0.08
0.09 ± 0.11
P = 0.051*
P = 0.202
P = 0.733
P = 0.017*
T
0.10 ± 0.06
0.10 ± 0.05
0.12 ± 0.08
0.08 ± 0.07
M
0.08 ± 0.06
0.08 ± 0.05
0.01 ± 0.07
0.04 ± 0.06
P = 0.001*
P = 0.006*
P = 0.798
P = 0.036*
T
0.03 ± 0.04
0.04 ± 0.04
0.03 ± 0.03
0.01 ± 0.01
M
0.02 ± 0.02
0.03 ± 0.03
0.10 ± 0.01
0.01 ± 0.02
P = 0.072*
P = 0.132
P = 0.334
P = 0.833
T
0.08 ± 0.06
0.09 ± 0.06
0.08 ± 0.06
0.04 ± 0.03
M
0.04 ± 0.03
0.05 ± 0.03
0.03 ± 0.04
0.03 ± 0.03
P<0.0001*
P<0.0001*
P = 0.002*
P = 0.123
T
0.02 ± 0.03
0.02 ± 0.03
0.02 ± 0.03
0.01 ± 0.01
M
0.01 ± 0.02
0.01 ± 0.01
0.02 ± 0.03
0.01 ± 0.01
P = 0.378
P = 0.389
P = 0.875
P = 0.496
T: tumor, M: adjacent mucosa. Values are presented as median ± standard deviation, * significant differences by Wilcoxon test.
that differences between HNSCC subsites must be considered (Table 2).
Concerning Smad expression, OS and DFS, no correlations were found as revealed by Log Rank analysis, in the
HNSCC population as a whole.
TGFβ isoforms mRNA expression profile in Oral SCC
We assessed the three TGFβ isoforms using real-time
QRT PCR in 35 available oral SCC. TGFβ1 mRNA
expression was observed in 100% of samples (mean ±
standard deviation: 0.82 ± 0.68, median: 0.57), while
TGFβ2 (94%) and TGFβ3 (97%) isoforms were detected
in almost all tumors (TGFβ2:1.24 ± 1.82, 0.72; TGFβ3:
2.12 ± 3.58, 1.15).
No statistically significant associations were found
between TGFβ isoforms and clinical-pathological characteristics such as pT, pN, pathological staging and histological differentiation. After categorization according to
median mRNA expression, the observed positivity of
each isoform was 23%, 37% 49% for TGFβ1, TGFβ2 and
TGFβ3, respectively.
The correlations among categorized Smad2, Smad6,
TGFβ1, TGFβ2 and TGFβ3 were tested by Spearman's
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Figure 2 Kaplan-Meier survival curves of oral cavity SCC patients grouped according to Smad expression. Patients were categorized as positive (above) or negative (equal or below) according to median Smad expression in tumors. Log Rank test was performed for curves comparison. In E
and F, patients were grouped according to the co-expression of Smad2 and Smad6. Smad2+/Smad6-: n = 13; Smad2+/Smad6+: n = 11; Smad2-/Smad6: n = 13; Smad2-/Smad6+: n = 11.
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Table 3: Cox multivariate analyses of survival prediction in oral SCC.
Variable
Hazard ratio (95% CI)
P value
Smad2
0.374 (0.155 - 0.906)
0.029
Smad6
4.153 (1.613 - 10.692)
0.003
pN
0.598 (0.233 - 1.531)
0.284
pT
0.557 (0.197 - 1.575)
0.270
CI - confidence interval
rho test. Results revealed that Smad2 was inversely correlated to TGFβ1 (P = 0.05, R = -0.33). Among the eight
TGFβ1+ tumors, seven (87.5%) were also Smad2-, while in
TGFβ1- subgroup an equal distribution between Smad2(48%) and Smad2+ (52%) was observed.
Discussion
In this study, we provide evidence that Smads may have a
key role in head and neck cancer, influencing survival.
Specifically, we show, prospectively, that high Smad6
mRNA expression and low Smad2 mRNA correlate with
better survival in oral SCC patients submitted to curative
surgery, independent of nodal status. In addition, Smad7
high mRNA expression correlated with shorter survival
in patients with larynx SCC submitted to curative surgery. The fact that these correlations were restricted to
specific tumor locations supports the hypothesis that
Figure 3 Kaplan-Meier survival curves of larynx SCC patients
grouped according to Smad expression. Patients were categorized
as positive (above) or negative (equal or bellow) according to median
tumor expression. Log Rank test was performed for curves comparison.
underlying biological heterogeneity exists between different subsites within head and neck.
TGFβ is known as a potent tumor supressor in normal
epithelial cells and in early-stage tumors but during
tumor progression it becomes an oncogenic factor,
mainly in advanced tumors [21]. Since Smad6 has a
blocking effect on TGFβ signaling either by direct binding to the TGFβ receptor, by competing with Co-Smad
for R-Smad complex formation or by targeting receptor
for degradation [6,7,22,23], we can speculate that this
effect on survival occurs because Smad6 would inhibit
the TGFβ tumorigenic signaling, thus favouring a better
outcome. The favorable prognosis presented by Smad6+
oral SCC patients agrees with that described in 115
esophageal SCC using immunohistochemistry [24]. Reinforcing this idea, we found that even in the poor prognosis group, pN+, the presence of Smad6+ was associated
with a survival advantage similar to that of pN0 patients.
Smad2 is the classically R-Smad of the TGFβ pathway
[1], our own data suggest Smad2 negativity reinforces the
effect of Smad6 on survival of oral SCC. Smad2 may be a
key factor for the interruption of TGFβ tumorigenic signaling in this group of patients, together with Smad6. In
accordance with that, we found TGFβ1 positive tumors
were also Smad2 negative. Our finding regarding the
association between lack of Smad2 expression and a
favourable clinical outcome is in disagreement with previous studies done in head and neck cancer as well as
other tumor types. In oral squamous cell carcinoma, a
study involving 125 patients suggested a link between
decreased expression of both activated Smad2 (p-Smad2)
and TGFβ receptor II (TβR-II), with aggressive tumor
features, which suggests TGFβ signaling exerts a protective role possibly through Smad 2 [25]. In accordance, the
Smad expression profile assessed by tissue array in 170
head and neck squamous cell carcinoma pointed to the
loss of TGFβ/Smad2 signaling as a possible cause of
adverse outcome [17]. A protective role of Smad 2 was
also suggested for esophageal, colon and breast cancer
[26-28]. In line with this effect, others have identified a
missense mutation of Smad2 in the squamous cell line
SCC-15 suggesting that the loss of Smad 2 may be part of
head and neck carcinogenesis [29].
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There are, however, several studies in line with our
data. Matrix metalloproteinases play roles in cancer progression by degrading the extracellular matrix and basement membrane. TGFβ1 signaling induces MMP-9
expression via Smad 2/3 [30]. Accordingly, the TGFβ1/
Smad 2/3 axis regulates MMP-9 expression through the
transcriptional factors Snail and Ets-1, contributing to
oral cancer progression [31]. In addition, the metastasisassociated protein metastatin can physically and functionally interact with Smad2/3, and enhance TGFβ mediated MMP-9 induction [32]. Activation of Smad 2/3
signaling has also been linked to enhancement of MMP13 expression and invasion of head and neck squamous
carcinoma cells [18].
Smad 2 accumulation seems to parallel an elevation of
H-ras, both of which are essential for epithelial mesenchymal transition (EMT), an essential step during carcinogenesis. Having undergone EMT, others have shown
that fibroblastoid carcinoma cells with elevated levels of
activated Smad2 gain the capability to spread to a wide
variety of tissues by a further increase in Smad2 expression [33]. In addition, a prominent expression of
alpha(v)beta(6) integrin at tumor stroma interface in
xenograft model, which resembles human head and neck
carcinomas, has been connected with cancer progression.
Alpha(v)beta(6) interacts with TGFβ, as an
Alpha(v)beta(6) blocking antibody can inhibit TGFβ
mediated Smad2/3 phosphorylation, which leads to inhibition of tumor growth in vivo, suggesting a role for the
microenvironment for this effect [34].
It is not easy to reconcile these conflicting results
regarding the role of Smad2, in head and neck tumors,
since they reflect the dual roles of TGFβ itself, acting both
as a tumor suppressor and a tumor enhancer, depending
on the context. What is not clear, however, is what constitutes this context [35]. In our head and neck patient
series, Smad2 seems to be a TGFβ signaling node that
enhances tumor aggressiveness (Figure 4). An intriguing
possibility, that merits further study, is that HPV infection may be interacting with TGFβ network as shown in
cervical carcinoma [36].
Taking into account that Smad6 is at the crossroad of
many signaling pathways, being regulated not only by
family members of bone morphogenetic protein (BMP)
and TGFβ, but also by epidermal growth factor and Ras/
MAPK [6,7,37], its expression may be key to shift signaling from oncogenesis to tumor supression, towards or
against proliferation, independent of Smad3 and Smad4,
at least in oral SCC (Figure 4).
If we consider Smad6 and Smad7 as interchangeable
TGFβ blocking Smads, our data in oral SCC is also in
keeping with others, showing that a combination of
increased immunohistochemical expression of Smad7
and decreased Smad4 expression are markers of good
Page 8 of 10
TGFE
?
Smad2
Smad6
(+)
(-)
Tumor Aggressiveness
(-)
(+)
Survival
Figure 4 Hypothesis scheme of Smad influence on survival. TGFβ1
upregulation is associated with tumor agressiveness and poor patient
survival. Smad2 and Smad6 have opposite roles in this process. Full
and dashed lines represent our own findings and previous published
data, respectively.
prognosis in gastric cancer, with 67.5% survival rate versus 52.2%, (P = 0.0011) [38]. However, data concerning
Smad7 seems to be more controversial since our own
present data indicates that Smad7 mRNA low expression
correlates with shorter survival in larynx SCC (Figure 3).
In agreement with our results, patients with colon and
gastric cancers, with Smad7 gene deletion or low Smad7
protein expression, were described as having prolonged
survival as compared to patients with higher Smad7
expression [39,38].
Concerning the lack of influence of Smad4 mRNA
expression on patient survival in our study, data in the literature suggests that there is no single rule for this. Low
Smad4 expression, detected by immunohistochemistry,
and poorer five-year survival was shown in 249 patients
with advanced gastric cancer and in 258 esophageal
squamous cell carcinomas (P <0.05) [9,10]. In colon cancer, such an influence was not shown, which is in accordance with our present data [39]. Taking into account
that Smad4 mRNA was the most ubiquitous among
Smads we can argue that perhaps the amount of Smad4
was not a crucial element for these patients.
Finally, we found higher Smad mRNA expression in
SCC primary tumors as compared to adjacent tissue in
Mangone et al. Molecular Cancer 2010, 9:106
http://www.molecular-cancer.com/content/9/1/106
agreement with previously published data, showing that
HNSCC cell lines present multiple defects in TGF-beta
signaling [40]. In HNSCC samples, data obtained from
170 tumors using tissue array technology, shows expression of Smad2, Smad3 and Smad4 proteins [17]. Another
small study comparing the expression between tumor
and adjacent tissue did not find differences at least with
respect to Smad4, Smad6 and Smad7 protein expression
in 13 head and neck SCC tumors [16].
To sum up, our results suggest that increased Smad6
mRNA expression and low Smad2 mRNA expression
might be markers of better outcome in oral SCC but not
in larynx cancer submitted to curative surgery. These
effects may be linked to aberrant TGFβ signalling. In larynx cancer, a similar relationship was found for Smad7
mRNA low expression. Our findings need to be validated
in larger prospective studies, and may in the future, help
to stratify candidate patients for adjuvant treatment in
head and neck cancer.
Conclusions
TGFβ is classically known as tumor supressor in normal
epithelial cells that turns into a malignant factor during
tumor progression favoring tumor growth and metastasis. Here we propose that the interruption in TGFβ tumorigenicity by Smad2 dowregulation or by Smad6
overexpression confers a better outcome. Although both
might be considered as prognostic markers, the molecular mechanism envolved in this process is not clear. Further studies are warranted to explore the mechanisms
involved.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FRRM carried out RNAse protection assay and Real-Time QRTPCR, was involved
in the study design, data management, statistical analysis and manuscript
preparation, FSP responsible for primer designing, real-time QRTPCR support,
helped in statistical analysis and revised the manuscript SM helped in statistical
analysis and revised the manuscript; SI oversaw the statistical analysis and
helped draft the manuscript; CL, FW and MBC were the head and neck surgeons in the team, who collected samples, all clinical data and were involved
with data management; MMB helped to draft the manuscript; MHHF conceived and was responsible for coordination of the study, and was responsible
for the study funding through FAPESP. All authors read and approved the final
manuscript.
Acknowledgements
This study was supported by FAPESP 02/01738-9 and CNPq
Author Details
1Disciplina de Oncologia, Departamento de Radiologia, LIM 24, Hospital das
Clínicas da Faculdade de Medicina da Universidade de São Paulo, Avenida Dr
Arnaldo 455, São Paulo, Brasil, 2Departamento de Otorrinolaringologia,
Universidade Federal de São Paulo, São Paulo, Brasil and 3Serviço de Cirurgia
de Cabeça e Pescoço, Hospital Heliópolis, São Paulo, Brasil
Received: 8 May 2009 Accepted: 12 May 2010
Published: 12 May 2010
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Cite this article as: Mangone et al., Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients Molecular Cancer 2010,
9:106
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