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The elevated level of ERα36 is correlated with nodal metastasis and poor prognosis in lung adenocarcinoma
Accepted Manuscript The Elevated Level of ERα36 is correlated with Nodal Metastasis and Poor Prognosis in Lung Adenocarcinoma Shuming Zhang, Chen Qiu, Lei Wang, Qi Liu, Jiajun Du PII: DOI: Reference:
S0039-128X(14)00134-2 http://dx.doi.org/10.1016/j.steroids.2014.05.021 STE 7583
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
11 March 2014 18 May 2014 23 May 2014
Please cite this article as: Zhang, S., Qiu, C., Wang, L., Liu, Q., Du, J., The Elevated Level of ERα36 is correlated with Nodal Metastasis and Poor Prognosis in Lung Adenocarcinoma, Steroids (2014), doi: http://dx.doi.org/10.1016/ j.steroids.2014.05.021
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1
The Elevated Level of ERα36 is correlated with Nodal
2
Metastasis and Poor Prognosis in Lung Adenocarcinoma
3
*1 2 Shuming Zhang, *1Chen Qiu, 3Lei Wang ↑1 Qi Liu, ↑ 3Jiajun Du
4 5
1. Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong
6
University, Shandong University. 324 Jingwu Road, Jinan 250021, P.R. China.
7
2. State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital,
8
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
9
100871, P.R. China
10
3. Department of Thoracic surgery, Shandong Provincial Hospital Affiliated to
11
Shandong University, Shandong University. 324 Jingwu Road, Jinan 250021, P.R.
12
China.
13
* These authors equally contributed to this work.
14
↑
15
Email: [email protected], [email protected]
16
Tel: +86-15964524862
17
Fax: +86-0531-68777100
Corresponding author: Jiajun Du, Qi Liu
18 19 20 21 22 23 1
1
Abstract
2
Introduction: ERα36 is a recently cloned variant of estrogen receptor-alpha which
3
has been proved to play an active role in a series of malignant diseases.
4
Method: ERα36 expression was examined using immunohistochemical methods with
5
sections from 126 resected NSCLC specimens. The immunoreactivity of ERα66 was
6
also studied as a comparison. Kaplan-Meier method and multivariable Cox
7
proportional hazards regression analyses were used to examine the relationship
8
between ERα36 and survival.
9
Result: ERα36 was more highly expressed in NSCLC patients compared to ERα66.
10
ERα36 expression has a strong correlation with histology (AC: 53/70, SCC: 16/56,
11
P<0.000) and had a significantly positive correlation with lymphatic metastasis
12
(P=0.014) in adenocarcinoma. High ERα36 expression was correlated with poorer
13
overall survival (OS) (P = 0.020) and disease-free survival (DFS) (P= 0.024) in
14
adenocarcinoma. Furthermore, ERα36 status was a significant independent prognostic
15
factor of OS (P = 0.018, HR: 3.142, 95% CI: 1.215-8.128) and DFS (P = 0.024, HR:
16
2.720, 95% CI: 1.141-6.486) in lung adenocarcinoma patients.
17
Conclusion: ERα36 had a high expression mainly in adenocarcinoma and the high
18
expression of ERα36 was strongly correlated with more advanced regional lymph
19
node metastasis and poor survival in lung adenocarcinoma.
20
Keywords
21
Estrogen receptor alpha36; Estrogen receptor alpha66; Non-small cell lung cancer;
22
Prognosis 2
1
1. Introduction
2
Lung cancer has long been thought of as a cancer that mainly affects men, but
3
over the past several decades, with the incidence rate of lung cancer in male
4
decreasing, there is a slow but steady increase in female1. Women were more likely to
5
develop lung cancer when consuming the same amount of cigarettes compared to
6
men2, 3, in addition, women non-smokers are at a 2.5-fold greater risk than male
7
non-smokers for developing lung adenocarcinoma (AC)4.
8
Estrogen has been proposed to be responsible for such gender difference1. A
9
number of epidemiological studies reported that women undertaking estrogen
10
replacement therapy had a higher risk of lung cancer, especially in lung
11
adenocarcinoma (AC).5, 6 Aromatase, which is responsible for a key step in estrogen
12
biosynthesis, proved to be a strong predictor of survival with NSCLC.
13
estrogen treatment has been demonstrated to result in enhanced growth of lung cancer,
14
8-10
15
tumor growth both in vivo and in vitro.11-14
7
Besides,
inhibition of aromatase and ER or anti-estrogen treatment down-regulated lung
16
Estrogens exert their molecular action by interaction with two subtypes of
17
estrogen receptor (ER), ERα and ERβ. Traditionally, ERs (ERα and ERβ) were a
18
group of nuclear receptors which controlled the expression of target genes through
19
estrogen-dependent genomic pathway15. There have been several studies examining
20
ER expression in lung cancers, but their results remain inconclusive.13, 16 and the
21
relationship between ER expression and prognosis remains unclear.14,
22
expression rate of ERs in lung cancer varied drastically from 0% to 100%.18-20 3
17
The
1
Recently, a novel ERα variant was discovered with a molecular mass of ∼36 kDa
2
(ERα36), It differs from ERα66 by lacking both transcriptional activation domains
3
(AF1 and AF2) (exons 7 and 8) but retains the DNA-binding domain and partial
4
dimerization and ligand-binding domains 21. Instead of binding to target gene, ERα36
5
mainly exists in the cytoplasm or on the cell membrane and exerts its effect through
6
MAPK/ERK and/or PKC pathway.
7
breast23, colorectal24, gastric25 and endometrial cancer26. In this study, we detected the
8
expression of ERα36 in 126 NSCLC patients by immunohistochemistry to see if this
9
novel receptor for estrogen is to any extent related to the clinical features of NSCLC
10
22
The expression of ERα36 was detected in
and compared it with that of ERα66.
11 12
2. Materials and methods
13
2.1. Patients and Tumor Samples
14
Tumor samples were randomly obtained from 126 patients who underwent
15
complete surgical resection for NSCLC in Provincial Hospital Affiliated to Shandong
16
University in 2008. Clinical and pathological features (including sex, smoking history,
17
family history, histological type, pathologic stage, lymph node metastasis, and tumor
18
stage) were abstracted from the patients’ charts. Pathological staging was based on the
19
current seventh edition of the TNM classification27. A total of 126 patients were
20
followed up until death or the last day of follow-up (Dec 15, 2013). The overall
21
survival (OS) was defined as the interval between the date of the definitive resection
22
and the date of the last follow-up or death, and disease-free survival (DFS) was 4
1
defined as the time interval between the date of the definitive resection and detection
2
of first disease recurrence, metastasis, or the date of the last follow-up. The study was
3
approved by the Ethic Committee of Shandong Provincial Hospital.
4 5
2.2. Immunohistochemistry
6
Immunostaining was done on paraffin embedded sections (4µm). After
7
deparaffinization and rehydration, endogenous peroxidase was quenched with 3%
8
hydrogen peroxide in methanol at room temperature (25 °C) for 30 min. Then the
9
slides were placed in a 95 °C solution of 0.01 M sodium citrate buffer (pH=6.0) for 15
10
min, and in 10% fetal bovine serum for 30 min at room temperature to block
11
non-specific protein-binding sites. Then the slides were incubated overnight at 4°C
12
with either rabbit-anti-human ERα36 polyclonal antibody22 (a polyclonal anti–ERα36
13
antibody raised against the last 20 amino acids as a custom service by Alpha
14
Diagnostic International, San Antonio, TX; dilution 1:200, kindly provided by Dr.
15
Zhaoyi Wang) or mouse-anti-human ERα66 (1/100, 1D5, Santa Cruz Biotechnology,
16
USA). Washed with PBS, the slides were incubated with biotin-labeled secondary
17
antibody for 30 min and streptavidin-horseradish peroxidase for 30 min.
18
Diaminobenzidine was used as a chromagen and slides were counterstained with
19
hematoxylin. Positive controls were from breast cancer cases and negative controls,
20
missing out the primary antibodies, were performed to discriminate background
21
staining.
22 5
1
2.3 Scoring
2
Immunohistochemistry was scored assessing separately the extent of positivity,
3
graded by determining the percentage of positive tumor cells (score 0, none; score 1,
4
<1/100; score 2, 1/100 to 1/10; score 3, 1/10 to 1/3; score 4, 1/3 to 2/3; and score
5
5,>2/3) and their staining intensity (score 0, none; score 1, weak; score 2, intermediate;
6
and score 3, strong), using previously described scoring system28, a total final score,
7
which ranged from 0 to 8, was obtained from the sum of the two scores and divided
8
into two groups: 0 to 4 was defined as low to negative expression and 5 to 8 as high
9
expression. The slides were scored by two pathologists who were blind to all clinical
10
features or survival states; disagreements were discussed with a third pathologist.
11 12
2.4. Statistical analysis
13
Associations between the expression of the receptors and patients’ clinical and
14
pathological features were assessed by chi-square or Fisher’s exact test (when the
15
number was <5) for categorical variables. Survival curves were obtained using the
16
Kaplan–Meier method and compared using the log-rank test. A multivariate model
17
using the Cox stepwise regression analysis was used to evaluate the statistical strength
18
of independent association between selected covariates and patient survival. P <0 .05
19
were considered significant. All statistical analyses were performed using SPSS
20
version 17.0.
21 22
3. Results 6
1
3.1 Clinical background
2
The study was comprised of 77 males and 49 females, the mean age of the
3
patients was 60.7 years (range, 38-84 years); there were 65 adenocarcinoma and 55
4
squamous cell carcinoma (SCC). (As shown in Table.1) The stage of the tumor was
5
determined based on the 2009 TNM system27, The lymph node ratio (LNR), defined
6
as the ratio of metastasis lymph nodes (MLNs) divided by the total number of
7
retrieved LNs, also introduced to defined the lymph node status. None received
8
pre-operative chemotherapy, radiation or both. The median follow-up time was 48
9
months (range, 5–69 months). The work has been approved by the appropriate ethical
10
committees of Shandong Provincial Hospital.
11 12
3.2 Identification of ERα36 and ERα66 in lung cancer tissues.
13
By immunohistochemistry, ERα36 and ERα66 expression were examined in 126
14
NSCLC patients and graded to two groups (intensely expressed group and lower to
15
negative group) according to the percentage of positive cells and density of staining.
16
Both receptors were restricted to tumoral tissues, no positivity observed in
17
peritumoral tissues. Staining of the ERα36 protein was identified in the cytoplasm and
18
cell membrane of cancer cells, ERα66, however, existed mostly in cytoplasma of
19
tumor cells and no membrane distribution was observed (Fig.1).
20 21 22
3.3 ERα36/ ERα66 expression and clinicopathological parameters. To evaluate the association between ERα36/ ERα66 expression and 7
1
clinicopathological parameters, patients were divided into low ERα36/ERα66
2
expression (score 0–4) and high ERα36/ERα66 expression (score 5–8) group. Table1
3
summarizes
4
clinicopathological features of the 126 NSCLC patients. There was no significant
5
association between ERα36 expressions and the age, family History, menopause,
6
tumor size, or lymph node status. However, a significant difference was noted
7
regarding histologic type, smoking status and chemotherapy. There was a tendency for
8
high ERa36 to be associated with females (p= 0.059), but this tendency was likely due
9
to the association with adenocarcinoma, which occurred at a higher proportion in
10
women. Only 16 of 56 (28.6%) squamous cell carcinomas were highly positive for
11
ERα36 versus 53 of 70 (75.7%) of the adenocarcinoma (P<0.000). For ERα66, no
12
correlation was found between its expression and any biological or clinical
13
characteristics. Co-expression of ERα36 and ERα66 was observed in 14 out of 126
14
cases (7 in female adenocarcinoma，4 in male adenocarcinoma，2 in female squamous
15
carcinoma and 1 in male squamous carcinoma).
the
relationship
between
the
ERα36/ERα66
expression
and
16
Given the imbalance expression on histology subtypes, further analysis of ERα36
17
correlation with clinicopathological variables subdivided according to histology type
18
performed. In adenocarcinoma patients, high expression of ERα36 was found in 92.3%
19
of cases with lymphatic metastasis, and in 65.9% of cases without metastasis, a strong
20
correlation was found between ERα36 expression and lymph node metastasis
21
(P=0.014, Table 2). No such difference was seen in the squamous carcinoma group.
22 8
1
3.4 Correlation of ERα36 Tissue Status and Prognosis.
2
Fig.2 shows the survival curves of all patients according to the ERα36 expression.
3
Patients with high expression of ERα36 seems to have a poorer prognosis than did
4
those in low ERα36 expression group, and has a correlation with disease-free survival
5
(P= 0.035). In this analysis, high expressing patients tended (P = 0.061) a lower
6
overall survival. Analysis was repeated after stratifying by histological subtype
7
because there was a significant interaction between ERα36 expression and lung
8
adenocarcinoma. This histological differential in survival associated with ERα36
9
expression is illustrated in Fig.3. In adenocarcinoma, ERα36 high expression patients
10
were associated with poorer OS (P=0.020, Fig. 3A) and DFS (P=0.024, Fig. 3B) than
11
ERα36 low expression patients (Fig. 3A, B), In contrast, there was no such difference
12
in patients with squamous carcinoma (P=0.809, P=0.777, Fig. 3C, D).
13 14
3.5 Multivariate Analysis
15
Multivariate survival analysis was performed to evaluate the independence of
16
ERα36 expression as a prognostic factor, the analysis revealed that gender, smoking
17
status, LNR, pT stage and ERα36 status were recognized as independent prognostic
18
factors for overall survival, and the LNR, pT stage and ERα36 status also emerged as
19
the independent prognostic parameters for disease-free survival (Table 3).
20
Multivariate analysis, which has been done in the adenocarcinoma subset, has also
21
shown that the ERα36 expression is an independent prognostic factor (Table 4).
22 9
1
4. Discussion
2
Although lung was not the main target organ of estrogen, there were increasing
3
reports suggesting that estrogen is involved in the development of normal lung tissue
4
as well as lung cancer.29-33 In the present study, we evaluated the expression of ERα36,
5
a novel variant of ERα, in the tumor tissues of NSCLC patients and its correlation
6
with clinicopathological parameters. As shown in Fig.4, ERα36 lacks the two
7
transactivational domains, AF-1 and AF-2, which were essential for the function of
8
ERα66 as a transcriptional factor. However, it retains the functions of ligand-binding,
9
dimerizing and binding to specific DNA sequences. The 1D5 antibody for ERα66 we
10
employed has a high affinity for the AF-1 domain which is absent in ERα36; and the
11
primary antibody against ERα36 targets at the unique 20-amino acid sequence in its
12
C-terminal.21,
13
theoretically prevented.
22
Hence, the crosstalk between the two primary antibodies was
14
Our study demonstrated a significant greater prevalence of ERα36 in NSCLC
15
patients compared to ERα66. ERα66 were identified in merely 16.7% of the NSCLC
16
patients and the distribution has no correlation with any clinical or pathological
17
feature analyzed. By contrast, ERα36 was detected in 54.8% of the patients and was
18
in close relation to the histological subtype, smoking status, and receiving
19
chemotherapy. Considering its distinct distribution in tumor cells, the greater
20
prevalence of ERα36 suggested a closer relationship to NSCLC compared to ERα66.
21
Estrogen receptors are members of the nuclear steroid receptor superfamily, the
22
classical model for the mechanism of action of estrogen receptors is that they undergo 10
1
translocation to the nucleus when bound to estrogen34. Our present study reveals an
2
aberrant cytoplasmic localization that is inconsistent with its role as a nuclear
3
transcription factor. It has been reported that lung tumors express mostly splice
4
variants of ERα.16, 35 The anti-ERα antibody we used was mouse monoclonal 1D5
5
which recognizing the amino-terminal domain, therefore, false negative findings may
6
have resulted if the antibodies targeted epitopes that are not present in splice variants
7
found in lung tumors.
8
ERα36 mainly existed on the plasma membrane or in the cytoplasm of NSCLC
9
cancer cells, these results were in accordance with other studies reporting the same
10
distribution pattern in breast cancer22 and gastric cancer36. It has been reported that
11
ERα36 exerts its effects through the membrane (non-genomic) pathway by rapid
12
phosphorylation of p42/p44 MAPK and/or Akt36. As early as 1977, the membrane
13
pathway of estrogen has been considered of greater importance in a series of
14
malignancies including lung cancer
15
ER has long been open to question. As some researchers claimed that the
16
non-genomic ER pathway may interact with other membrane growth factor pathways,
17
such as the epidermal growth factor receptor (EGFR), others reported that estrogen
18
activates non-genomic signaling pathways in other systems, leading to increases in
19
second messengers such as cAMP, calcium, and activation of PI3K and MAPK.29, 30, 38
20
Since ERα36 was shown to activate the MAPK and/or PI3K pathways, the
21
identification of ERα36 in NSCLC patients provides strong evidence for the role of
22
these membrane pathway of estrogen in lung cancer.
37
. However, the identity of the membrane based
11
1
There are well-known differences in the etiology, location, lymph node spread,
2
and prognosis of patients with lung adenocarcinoma and squamous cell carcinoma.
3
We found that ERα36 expression was significantly associated with histological type.
4
Strong cytoplasmic and membrane staining of the protein was noted in 75.7% of
5
adenocarcinoma, whereas its expression in squamous carcinoma was not frequent
6
(28.6%, P<0.000). When the entire set of patients was analysed, smoking status, and
7
receiving chemotherapy were associated with ERα36 status (P=0.036, P=0.045),
8
however, in the histological subset analysis, neither smoking status nor chemotherapy
9
was found related to the expression of ERα36.It’s well known that smoking is a
10
contributory factor for SCC and considering the imbalance histological distribution of
11
ERα36, we concluded that ERα36 was closely related to adenocarcinoma regardless
12
of smoking status or whether receiving chemotherapy.
13
In our present study, we also found an association between ERα36 expression
14
and lymph node metastasis in lung adenocarcinoma, there are several lines of
15
experimental evidence supporting such involvement in the process of tumor
16
progression. ERα36 has been shown to have a high association with lymph node
17
metastasis in patients with gastric cancer36; Reyhaan et al39 reported that ERα36 can
18
enhance the metastasis of breast cancer cells by promoting a number of metastatic
19
factors. In pulmonary adenocarcinoma, ERα36 may also play a part in the acquisition
20
of an invasive and metastatic phenotype. Further study is needed to confirm this role
21
and the possible mechanism.
22
In this study, high ERα36 expression among lung adenocarcinoma patients was 12
1
associated with significantly worse survival. In squamous carcinoma, survival was not
2
statistically significantly different by ERα36 status. It may be speculated that
3
association between high ERα36 and increased lymphatic spread indicates a role for
4
the receptor isoform in tumor progression. Further studies on the effects of ERα36 on
5
cellular proliferation and mobility are required to examine this possibility.
6
Several inherent limitations to the study must be considered. First, only NSCLC
7
patients who underwent radical resection in our institution were enrolled in the
8
present study, which restricted the total study population to a relatively low number.
9
Second, the retrospective design lacks some detailed clinical information which could
10
have influenced on survival. To address this limitation, multicenter, randomized
11
studies should be conducted to confirm whether ERα36 can be used as an
12
accurateprognostic maker for adenocarcinoma. Third, our study using the
13
immunohistochemical staining to assess the expression of ERα36. Clearly it would
14
have been helpful to measure both protein and mRNA in the same samples, but the
15
amounts of tissue were limiting. Additionally, the current lack of commercial antibody
16
for human ERα36 precludes this type of study at the present time.
17 18
5. Conclusion
19
In conclusion, our study suggested a close relation between ERα36 expression
20
and NSCLC, and the ERα36 expression level significantly correlate lymph node
21
metastases and poor survival in patients with lung adenocarcinoma. Furthermore, Cox
22
regression model analysis showed that ERα36 was a meaningful independent 13
1
prognostic factor for both overall and disease-free survival. Although previous studies
2
provided evidence that antiestrogens may be a useful strategy for lung cancer
3
therapy35, a recent study in breast cancer demonstrated that antiestrogens (tamoxifen
4
and fulvestrant) act as agonist to promote cell proliferation through ERα36 induced
5
membrane pathways, which partly explained antiestrogen therapy resistance22. Given
6
the distinct distribution and biological response of ERs between the lung and breast
7
tumors, continued effort is required to confirm the underlying biology of ERα36 in
8
the development and/or metastasis of lung cancer and identify specific antagonists for
9
this form of ER.
10 11 12
6. Acknowledgements
13
This study was supported by Provincial science and technology foundation of
14
Shandong (2011GGH21819). The authors thank Professor Zhaoyi Wang for kindly
15
providing the antibody of ERα36.
16 17
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and regeneration in mice. Am J Physiol Lung Cell Mol Physiol
16
2004;287:L1154-9.
17
[33]Morani A, Barros RP, Imamov O, et al. Lung dysfunction causes systemic hypoxia
18
in estrogen receptor beta knockout (ERbeta-/-) mice. Proc Natl Acad Sci U S A
19
2006;103:7165-9.
20 21 22
[34]Ribeiro RC, Kushner PJ, Baxter JD. The nuclear hormone receptor gene superfamily. Annu Rev Med 1995;46:443-53. [35]Stabile LP, Davis ALG, Gubish CT, et al. Human non-small cell lung tumors and 18
1
cells derived from normal lung express both estrogen receptor α and β and
2
show
3
2002;62:2141-2150 %@ 0008-5472.
biological
responses
to
estrogen.
Cancer
research
4
[36]Deng H, Huang X, Fan J, et al. A variant of estrogen receptor-alpha, ER-alpha36 is
5
expressed in human gastric cancer and is highly correlated with lymph node
6
metastasis. Oncol Rep 2010;24:171-6.
7
[37]Acconcia F, Marino M. The Effects of 17beta-estradiol in Cancer are Mediated by
8
Estrogen Receptor Signaling at the Plasma Membrane. Front Physiol
9
2011;2:30.
10
[38]Hershberger PA, Stabile LP, Kanterewicz B, et al. Estrogen receptor beta (ERbeta)
11
subtype-specific ligands increase transcription, p44/p42 mitogen activated
12
protein kinase (MAPK) activation and growth in human non-small cell lung
13
cancer cells. J Steroid Biochem Mol Biol 2009;116:102-9.
14
[39]Chaudhri RA, Olivares-Navarrete R, Cuenca N, et al. Membrane estrogen signaling
15
enhances tumorigenesis and metastatic potential of breast cancer cells via
16
estrogen receptor-alpha36 (ERalpha36). J Biol Chem 2012;287:7169-81.
17 18 19
19
1 2
Table1. Clinicopathologic Characteristics and ERα36 / ERα66 status Characteristics Gender Male Female Age <60 ≥60 Histology Subtype AC SCC Smoking* Yes No Family History Yes No Mensopause Yes No Location Left Right pT stage T1 T2 T3-4 Lymphatic metastasis Positive Negative MLN 0 1-3 ≥4 LNR 0 0-35% ≥35% Chemotherapy Yes No
3 4 5 6
ERα36 low high 40 17
37 32
24 33
30 39
17 40
53 16
33 24
27 42
7 50
16 53
14 43
21 48
29 28
33 36
15 38 4
33 30 6
P value 0.059
ERα66 low high 65 40
12 9
48 57
6 15
58 47
12 9
50 55
10 11
18 87
5 16
30 75
5 16
54 51
8 13
38 60 7
10 8 3
0.878
0.150
.000
0.874
0.036
1.000
0.116
0.474
0.468
0.660
0.736
0.268
0.086
0.717
0.190 22 35
34 35
35 12 10
37 15 17
35 18 4
37 22 10
22 35
39 30
P value 0.686
0.818 46 59
10 11
61 21 23
11 6 4
61 32 12
11 8 2
51 54
10 11
0.310
0.884
0.268
0.732
0.045
0.937
*Smoking: yes: former smokers or current smokers; no: never smokers. AC: adenocarcinoma; SCC: squamous cell carcinoma; MLN: metastasis lymph nodes; LNR: lymph node ratio. 20
1 2 3
Table2 Clinicopathologic Characteristics and ERα36 status in AC and SCC ERα36(AC) low high
4 5
ERα36(SCC) low high
Characteristics P value P value Gender 0.900 0.813 Male 6 23 34 14 Female 11 30 6 2 Age 0.771 0.737 <60 7 21 17 6 ≥60 10 29 23 10 Smoking 0.952 0.928 Yes 5 16 28 11 No 12 37 12 5 Family History 0.134 0.143 Yes 2 16 5 0 No 15 37 35 16 Mensopause 0.733 0.763 Yes 5 18 9 3 No 12 35 31 13 Location 0.094 0.869 Left 8 25 21 8 Right 9 28 19 8 pT stage 0.768 0.064 T1 7 24 8 9 T2 10 26 28 5 T3-4 0 3 4 2 Lymphatic metastasis 0.014 0.534 Positive 2 24 20 6 Negative 15 29 20 10 MLN 0.034 0.389 0 15 31 20 6 1-3 1 10 11 5 ≥4 1 12 9 5 LNR 0.034 0.372 0 15 31 20 6 0-35% 1 14 17 8 ≥35% 1 8 3 2 Chemotherapy 0.338 0.092 Yes 7 29 15 10 No 10 24 25 6 *Smoking: yes: former smokers or current smokers; no: never smokers. AC: adenocarcinoma; SCC: squamous cell carcinoma; MLN: metastasis lymph nodes; LNR: lymph node ratio.
6 7 8 9 10 21
1 2 Table 3 Multivariate analysis of factors contributing to overall survival and disease-free survival in 3 NSCLC patients. 4 OS 5 Variables HR 95%CI P value 6 2.075 1.029-4.185 0.041 Gender 7 1.975 0.989-3.943 0.054 Smoking status 8 1.348 1.091-1.666 0.006 LNR 9 pT stage 1.825 1.200-2.776 0.005 10 1.737 1.011-2.984 0.045 11 ERα36 12 13 Variables HR 95%CI P value14 1.318 1.083-1.605 0.006 15 LNR 1.571 1.066-2.316 0.022 16 pT stage 1.785 1.074-2.968 0.025 17 ERα36 18 OS, Overall survival; DFS, Disease-free survival; CI, confidence interval; HR, hazard ratio; LNR, lymph node ratio. DFS
19 20 21 22 23
24 Table 4 Multivariate analysis of factors contributing to overall survival and disease-free survival in 25 lung adenocarcinoma patients. Variables
26
OS
DFS
pT stage
HR (95%CI) 2.192(1.276-3.767)
P-values 0.004
HR (95%CI) 1.718(0.993-2.971)
P value 0.053
ERα36
3.142(1.215-8.128)
0.018
2.720(1.141-6.486)
0.024
OS, Overall survival; DFS, Disease-free survival; CI, confidence interval; HR, hazard ratio.
27
22
1 2
Figure legends
3
Figure1. Immunohistochemical staining of ERα36/ ERα66 in NSCLC tissues. (A).
4
ERα66 negative staining in the lung tumor tissue; (B). ERα66 positive staining in the
5
cytoplasm of lung tumor tissue; (C). ERα36 positive staining in the cytoplasm of lung
6
tumor tissue; (D) ERα36 positive staining in the membrane of lung tumor tissue.
7 8
Figure2 Kaplan-Meier survival curves according to ERα36 expression in NSCLC
9
patients. (A) Overall survival (OS) for ERα36 expression in NSCLC (log-rank
10
P=0.061). (B) Disease-free survival (DFS) for ERα36 expression in NSCLC (log-rank
11
P=0.035).
12 13
Figure3 Kaplan-Meier survival curves according to ERα36 expression in NSCLC
14
patients. (A) Overall survival (OS) for ERα36 expression in lung adenocarcinoma
15
(log-rank P=0.020). (B) Disease-free survival (DFS) for ERα36 expression in lung
16
adenocarcinoma (log-rank P=0.024). (C) OS for ERα36 expression in SCC (log-rank
17
P=0.809). (D) DFS for ERα36 expression in SCC (log-rank P=0.777).
18 19
Figure4 Schematic illustration of domain structure of ERα66 and ERα36. Domains of
20
ERα66 were designated from A to F. It contains two transactivation domains (AF-1 in
21
A/B, AF-2 in E/F), a DNA binding domain(C), a variable hinge region (D), and a
22
ligand-binding domain (E). The two transactivation domains, AF-1 and AF-2 and part 23
1
of the ligand binding domain were missed out in ERα36. Instead, it contains an extra,
2
unique 27-animo acid sequence in its C-terminal. The binding sites of the primary
3
antibodies employed were also indicated.
4
24
1 2
3 4
25
1
2 3
26
1
2 3
27
1
2 3
28
1 2 3
Highlights
4 5 6 7 8 9
ERα36 was more highly expressed in NSCLC patients compared to ERα66. ERα36 mainly existed on the plasma membrane or in the cytoplasm of NSCLC cells. ERα36 was strongly correlated with more advanced regional lymph node metastasis. ERα36 status was a significant independent prognostic factor of OS and DFS in lung adenocarcinoma patients.
10
29
S0039-128X(14)00134-2 http://dx.doi.org/10.1016/j.steroids.2014.05.021 STE 7583
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
11 March 2014 18 May 2014 23 May 2014
Please cite this article as: Zhang, S., Qiu, C., Wang, L., Liu, Q., Du, J., The Elevated Level of ERα36 is correlated with Nodal Metastasis and Poor Prognosis in Lung Adenocarcinoma, Steroids (2014), doi: http://dx.doi.org/10.1016/ j.steroids.2014.05.021
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1
The Elevated Level of ERα36 is correlated with Nodal
2
Metastasis and Poor Prognosis in Lung Adenocarcinoma
3
*1 2 Shuming Zhang, *1Chen Qiu, 3Lei Wang ↑1 Qi Liu, ↑ 3Jiajun Du
4 5
1. Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong
6
University, Shandong University. 324 Jingwu Road, Jinan 250021, P.R. China.
7
2. State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital,
8
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
9
100871, P.R. China
10
3. Department of Thoracic surgery, Shandong Provincial Hospital Affiliated to
11
Shandong University, Shandong University. 324 Jingwu Road, Jinan 250021, P.R.
12
China.
13
* These authors equally contributed to this work.
14
↑
15
Email: [email protected], [email protected]
16
Tel: +86-15964524862
17
Fax: +86-0531-68777100
Corresponding author: Jiajun Du, Qi Liu
18 19 20 21 22 23 1
1
Abstract
2
Introduction: ERα36 is a recently cloned variant of estrogen receptor-alpha which
3
has been proved to play an active role in a series of malignant diseases.
4
Method: ERα36 expression was examined using immunohistochemical methods with
5
sections from 126 resected NSCLC specimens. The immunoreactivity of ERα66 was
6
also studied as a comparison. Kaplan-Meier method and multivariable Cox
7
proportional hazards regression analyses were used to examine the relationship
8
between ERα36 and survival.
9
Result: ERα36 was more highly expressed in NSCLC patients compared to ERα66.
10
ERα36 expression has a strong correlation with histology (AC: 53/70, SCC: 16/56,
11
P<0.000) and had a significantly positive correlation with lymphatic metastasis
12
(P=0.014) in adenocarcinoma. High ERα36 expression was correlated with poorer
13
overall survival (OS) (P = 0.020) and disease-free survival (DFS) (P= 0.024) in
14
adenocarcinoma. Furthermore, ERα36 status was a significant independent prognostic
15
factor of OS (P = 0.018, HR: 3.142, 95% CI: 1.215-8.128) and DFS (P = 0.024, HR:
16
2.720, 95% CI: 1.141-6.486) in lung adenocarcinoma patients.
17
Conclusion: ERα36 had a high expression mainly in adenocarcinoma and the high
18
expression of ERα36 was strongly correlated with more advanced regional lymph
19
node metastasis and poor survival in lung adenocarcinoma.
20
Keywords
21
Estrogen receptor alpha36; Estrogen receptor alpha66; Non-small cell lung cancer;
22
Prognosis 2
1
1. Introduction
2
Lung cancer has long been thought of as a cancer that mainly affects men, but
3
over the past several decades, with the incidence rate of lung cancer in male
4
decreasing, there is a slow but steady increase in female1. Women were more likely to
5
develop lung cancer when consuming the same amount of cigarettes compared to
6
men2, 3, in addition, women non-smokers are at a 2.5-fold greater risk than male
7
non-smokers for developing lung adenocarcinoma (AC)4.
8
Estrogen has been proposed to be responsible for such gender difference1. A
9
number of epidemiological studies reported that women undertaking estrogen
10
replacement therapy had a higher risk of lung cancer, especially in lung
11
adenocarcinoma (AC).5, 6 Aromatase, which is responsible for a key step in estrogen
12
biosynthesis, proved to be a strong predictor of survival with NSCLC.
13
estrogen treatment has been demonstrated to result in enhanced growth of lung cancer,
14
8-10
15
tumor growth both in vivo and in vitro.11-14
7
Besides,
inhibition of aromatase and ER or anti-estrogen treatment down-regulated lung
16
Estrogens exert their molecular action by interaction with two subtypes of
17
estrogen receptor (ER), ERα and ERβ. Traditionally, ERs (ERα and ERβ) were a
18
group of nuclear receptors which controlled the expression of target genes through
19
estrogen-dependent genomic pathway15. There have been several studies examining
20
ER expression in lung cancers, but their results remain inconclusive.13, 16 and the
21
relationship between ER expression and prognosis remains unclear.14,
22
expression rate of ERs in lung cancer varied drastically from 0% to 100%.18-20 3
17
The
1
Recently, a novel ERα variant was discovered with a molecular mass of ∼36 kDa
2
(ERα36), It differs from ERα66 by lacking both transcriptional activation domains
3
(AF1 and AF2) (exons 7 and 8) but retains the DNA-binding domain and partial
4
dimerization and ligand-binding domains 21. Instead of binding to target gene, ERα36
5
mainly exists in the cytoplasm or on the cell membrane and exerts its effect through
6
MAPK/ERK and/or PKC pathway.
7
breast23, colorectal24, gastric25 and endometrial cancer26. In this study, we detected the
8
expression of ERα36 in 126 NSCLC patients by immunohistochemistry to see if this
9
novel receptor for estrogen is to any extent related to the clinical features of NSCLC
10
22
The expression of ERα36 was detected in
and compared it with that of ERα66.
11 12
2. Materials and methods
13
2.1. Patients and Tumor Samples
14
Tumor samples were randomly obtained from 126 patients who underwent
15
complete surgical resection for NSCLC in Provincial Hospital Affiliated to Shandong
16
University in 2008. Clinical and pathological features (including sex, smoking history,
17
family history, histological type, pathologic stage, lymph node metastasis, and tumor
18
stage) were abstracted from the patients’ charts. Pathological staging was based on the
19
current seventh edition of the TNM classification27. A total of 126 patients were
20
followed up until death or the last day of follow-up (Dec 15, 2013). The overall
21
survival (OS) was defined as the interval between the date of the definitive resection
22
and the date of the last follow-up or death, and disease-free survival (DFS) was 4
1
defined as the time interval between the date of the definitive resection and detection
2
of first disease recurrence, metastasis, or the date of the last follow-up. The study was
3
approved by the Ethic Committee of Shandong Provincial Hospital.
4 5
2.2. Immunohistochemistry
6
Immunostaining was done on paraffin embedded sections (4µm). After
7
deparaffinization and rehydration, endogenous peroxidase was quenched with 3%
8
hydrogen peroxide in methanol at room temperature (25 °C) for 30 min. Then the
9
slides were placed in a 95 °C solution of 0.01 M sodium citrate buffer (pH=6.0) for 15
10
min, and in 10% fetal bovine serum for 30 min at room temperature to block
11
non-specific protein-binding sites. Then the slides were incubated overnight at 4°C
12
with either rabbit-anti-human ERα36 polyclonal antibody22 (a polyclonal anti–ERα36
13
antibody raised against the last 20 amino acids as a custom service by Alpha
14
Diagnostic International, San Antonio, TX; dilution 1:200, kindly provided by Dr.
15
Zhaoyi Wang) or mouse-anti-human ERα66 (1/100, 1D5, Santa Cruz Biotechnology,
16
USA). Washed with PBS, the slides were incubated with biotin-labeled secondary
17
antibody for 30 min and streptavidin-horseradish peroxidase for 30 min.
18
Diaminobenzidine was used as a chromagen and slides were counterstained with
19
hematoxylin. Positive controls were from breast cancer cases and negative controls,
20
missing out the primary antibodies, were performed to discriminate background
21
staining.
22 5
1
2.3 Scoring
2
Immunohistochemistry was scored assessing separately the extent of positivity,
3
graded by determining the percentage of positive tumor cells (score 0, none; score 1,
4
<1/100; score 2, 1/100 to 1/10; score 3, 1/10 to 1/3; score 4, 1/3 to 2/3; and score
5
5,>2/3) and their staining intensity (score 0, none; score 1, weak; score 2, intermediate;
6
and score 3, strong), using previously described scoring system28, a total final score,
7
which ranged from 0 to 8, was obtained from the sum of the two scores and divided
8
into two groups: 0 to 4 was defined as low to negative expression and 5 to 8 as high
9
expression. The slides were scored by two pathologists who were blind to all clinical
10
features or survival states; disagreements were discussed with a third pathologist.
11 12
2.4. Statistical analysis
13
Associations between the expression of the receptors and patients’ clinical and
14
pathological features were assessed by chi-square or Fisher’s exact test (when the
15
number was <5) for categorical variables. Survival curves were obtained using the
16
Kaplan–Meier method and compared using the log-rank test. A multivariate model
17
using the Cox stepwise regression analysis was used to evaluate the statistical strength
18
of independent association between selected covariates and patient survival. P <0 .05
19
were considered significant. All statistical analyses were performed using SPSS
20
version 17.0.
21 22
3. Results 6
1
3.1 Clinical background
2
The study was comprised of 77 males and 49 females, the mean age of the
3
patients was 60.7 years (range, 38-84 years); there were 65 adenocarcinoma and 55
4
squamous cell carcinoma (SCC). (As shown in Table.1) The stage of the tumor was
5
determined based on the 2009 TNM system27, The lymph node ratio (LNR), defined
6
as the ratio of metastasis lymph nodes (MLNs) divided by the total number of
7
retrieved LNs, also introduced to defined the lymph node status. None received
8
pre-operative chemotherapy, radiation or both. The median follow-up time was 48
9
months (range, 5–69 months). The work has been approved by the appropriate ethical
10
committees of Shandong Provincial Hospital.
11 12
3.2 Identification of ERα36 and ERα66 in lung cancer tissues.
13
By immunohistochemistry, ERα36 and ERα66 expression were examined in 126
14
NSCLC patients and graded to two groups (intensely expressed group and lower to
15
negative group) according to the percentage of positive cells and density of staining.
16
Both receptors were restricted to tumoral tissues, no positivity observed in
17
peritumoral tissues. Staining of the ERα36 protein was identified in the cytoplasm and
18
cell membrane of cancer cells, ERα66, however, existed mostly in cytoplasma of
19
tumor cells and no membrane distribution was observed (Fig.1).
20 21 22
3.3 ERα36/ ERα66 expression and clinicopathological parameters. To evaluate the association between ERα36/ ERα66 expression and 7
1
clinicopathological parameters, patients were divided into low ERα36/ERα66
2
expression (score 0–4) and high ERα36/ERα66 expression (score 5–8) group. Table1
3
summarizes
4
clinicopathological features of the 126 NSCLC patients. There was no significant
5
association between ERα36 expressions and the age, family History, menopause,
6
tumor size, or lymph node status. However, a significant difference was noted
7
regarding histologic type, smoking status and chemotherapy. There was a tendency for
8
high ERa36 to be associated with females (p= 0.059), but this tendency was likely due
9
to the association with adenocarcinoma, which occurred at a higher proportion in
10
women. Only 16 of 56 (28.6%) squamous cell carcinomas were highly positive for
11
ERα36 versus 53 of 70 (75.7%) of the adenocarcinoma (P<0.000). For ERα66, no
12
correlation was found between its expression and any biological or clinical
13
characteristics. Co-expression of ERα36 and ERα66 was observed in 14 out of 126
14
cases (7 in female adenocarcinoma，4 in male adenocarcinoma，2 in female squamous
15
carcinoma and 1 in male squamous carcinoma).
the
relationship
between
the
ERα36/ERα66
expression
and
16
Given the imbalance expression on histology subtypes, further analysis of ERα36
17
correlation with clinicopathological variables subdivided according to histology type
18
performed. In adenocarcinoma patients, high expression of ERα36 was found in 92.3%
19
of cases with lymphatic metastasis, and in 65.9% of cases without metastasis, a strong
20
correlation was found between ERα36 expression and lymph node metastasis
21
(P=0.014, Table 2). No such difference was seen in the squamous carcinoma group.
22 8
1
3.4 Correlation of ERα36 Tissue Status and Prognosis.
2
Fig.2 shows the survival curves of all patients according to the ERα36 expression.
3
Patients with high expression of ERα36 seems to have a poorer prognosis than did
4
those in low ERα36 expression group, and has a correlation with disease-free survival
5
(P= 0.035). In this analysis, high expressing patients tended (P = 0.061) a lower
6
overall survival. Analysis was repeated after stratifying by histological subtype
7
because there was a significant interaction between ERα36 expression and lung
8
adenocarcinoma. This histological differential in survival associated with ERα36
9
expression is illustrated in Fig.3. In adenocarcinoma, ERα36 high expression patients
10
were associated with poorer OS (P=0.020, Fig. 3A) and DFS (P=0.024, Fig. 3B) than
11
ERα36 low expression patients (Fig. 3A, B), In contrast, there was no such difference
12
in patients with squamous carcinoma (P=0.809, P=0.777, Fig. 3C, D).
13 14
3.5 Multivariate Analysis
15
Multivariate survival analysis was performed to evaluate the independence of
16
ERα36 expression as a prognostic factor, the analysis revealed that gender, smoking
17
status, LNR, pT stage and ERα36 status were recognized as independent prognostic
18
factors for overall survival, and the LNR, pT stage and ERα36 status also emerged as
19
the independent prognostic parameters for disease-free survival (Table 3).
20
Multivariate analysis, which has been done in the adenocarcinoma subset, has also
21
shown that the ERα36 expression is an independent prognostic factor (Table 4).
22 9
1
4. Discussion
2
Although lung was not the main target organ of estrogen, there were increasing
3
reports suggesting that estrogen is involved in the development of normal lung tissue
4
as well as lung cancer.29-33 In the present study, we evaluated the expression of ERα36,
5
a novel variant of ERα, in the tumor tissues of NSCLC patients and its correlation
6
with clinicopathological parameters. As shown in Fig.4, ERα36 lacks the two
7
transactivational domains, AF-1 and AF-2, which were essential for the function of
8
ERα66 as a transcriptional factor. However, it retains the functions of ligand-binding,
9
dimerizing and binding to specific DNA sequences. The 1D5 antibody for ERα66 we
10
employed has a high affinity for the AF-1 domain which is absent in ERα36; and the
11
primary antibody against ERα36 targets at the unique 20-amino acid sequence in its
12
C-terminal.21,
13
theoretically prevented.
22
Hence, the crosstalk between the two primary antibodies was
14
Our study demonstrated a significant greater prevalence of ERα36 in NSCLC
15
patients compared to ERα66. ERα66 were identified in merely 16.7% of the NSCLC
16
patients and the distribution has no correlation with any clinical or pathological
17
feature analyzed. By contrast, ERα36 was detected in 54.8% of the patients and was
18
in close relation to the histological subtype, smoking status, and receiving
19
chemotherapy. Considering its distinct distribution in tumor cells, the greater
20
prevalence of ERα36 suggested a closer relationship to NSCLC compared to ERα66.
21
Estrogen receptors are members of the nuclear steroid receptor superfamily, the
22
classical model for the mechanism of action of estrogen receptors is that they undergo 10
1
translocation to the nucleus when bound to estrogen34. Our present study reveals an
2
aberrant cytoplasmic localization that is inconsistent with its role as a nuclear
3
transcription factor. It has been reported that lung tumors express mostly splice
4
variants of ERα.16, 35 The anti-ERα antibody we used was mouse monoclonal 1D5
5
which recognizing the amino-terminal domain, therefore, false negative findings may
6
have resulted if the antibodies targeted epitopes that are not present in splice variants
7
found in lung tumors.
8
ERα36 mainly existed on the plasma membrane or in the cytoplasm of NSCLC
9
cancer cells, these results were in accordance with other studies reporting the same
10
distribution pattern in breast cancer22 and gastric cancer36. It has been reported that
11
ERα36 exerts its effects through the membrane (non-genomic) pathway by rapid
12
phosphorylation of p42/p44 MAPK and/or Akt36. As early as 1977, the membrane
13
pathway of estrogen has been considered of greater importance in a series of
14
malignancies including lung cancer
15
ER has long been open to question. As some researchers claimed that the
16
non-genomic ER pathway may interact with other membrane growth factor pathways,
17
such as the epidermal growth factor receptor (EGFR), others reported that estrogen
18
activates non-genomic signaling pathways in other systems, leading to increases in
19
second messengers such as cAMP, calcium, and activation of PI3K and MAPK.29, 30, 38
20
Since ERα36 was shown to activate the MAPK and/or PI3K pathways, the
21
identification of ERα36 in NSCLC patients provides strong evidence for the role of
22
these membrane pathway of estrogen in lung cancer.
37
. However, the identity of the membrane based
11
1
There are well-known differences in the etiology, location, lymph node spread,
2
and prognosis of patients with lung adenocarcinoma and squamous cell carcinoma.
3
We found that ERα36 expression was significantly associated with histological type.
4
Strong cytoplasmic and membrane staining of the protein was noted in 75.7% of
5
adenocarcinoma, whereas its expression in squamous carcinoma was not frequent
6
(28.6%, P<0.000). When the entire set of patients was analysed, smoking status, and
7
receiving chemotherapy were associated with ERα36 status (P=0.036, P=0.045),
8
however, in the histological subset analysis, neither smoking status nor chemotherapy
9
was found related to the expression of ERα36.It’s well known that smoking is a
10
contributory factor for SCC and considering the imbalance histological distribution of
11
ERα36, we concluded that ERα36 was closely related to adenocarcinoma regardless
12
of smoking status or whether receiving chemotherapy.
13
In our present study, we also found an association between ERα36 expression
14
and lymph node metastasis in lung adenocarcinoma, there are several lines of
15
experimental evidence supporting such involvement in the process of tumor
16
progression. ERα36 has been shown to have a high association with lymph node
17
metastasis in patients with gastric cancer36; Reyhaan et al39 reported that ERα36 can
18
enhance the metastasis of breast cancer cells by promoting a number of metastatic
19
factors. In pulmonary adenocarcinoma, ERα36 may also play a part in the acquisition
20
of an invasive and metastatic phenotype. Further study is needed to confirm this role
21
and the possible mechanism.
22
In this study, high ERα36 expression among lung adenocarcinoma patients was 12
1
associated with significantly worse survival. In squamous carcinoma, survival was not
2
statistically significantly different by ERα36 status. It may be speculated that
3
association between high ERα36 and increased lymphatic spread indicates a role for
4
the receptor isoform in tumor progression. Further studies on the effects of ERα36 on
5
cellular proliferation and mobility are required to examine this possibility.
6
Several inherent limitations to the study must be considered. First, only NSCLC
7
patients who underwent radical resection in our institution were enrolled in the
8
present study, which restricted the total study population to a relatively low number.
9
Second, the retrospective design lacks some detailed clinical information which could
10
have influenced on survival. To address this limitation, multicenter, randomized
11
studies should be conducted to confirm whether ERα36 can be used as an
12
accurateprognostic maker for adenocarcinoma. Third, our study using the
13
immunohistochemical staining to assess the expression of ERα36. Clearly it would
14
have been helpful to measure both protein and mRNA in the same samples, but the
15
amounts of tissue were limiting. Additionally, the current lack of commercial antibody
16
for human ERα36 precludes this type of study at the present time.
17 18
5. Conclusion
19
In conclusion, our study suggested a close relation between ERα36 expression
20
and NSCLC, and the ERα36 expression level significantly correlate lymph node
21
metastases and poor survival in patients with lung adenocarcinoma. Furthermore, Cox
22
regression model analysis showed that ERα36 was a meaningful independent 13
1
prognostic factor for both overall and disease-free survival. Although previous studies
2
provided evidence that antiestrogens may be a useful strategy for lung cancer
3
therapy35, a recent study in breast cancer demonstrated that antiestrogens (tamoxifen
4
and fulvestrant) act as agonist to promote cell proliferation through ERα36 induced
5
membrane pathways, which partly explained antiestrogen therapy resistance22. Given
6
the distinct distribution and biological response of ERs between the lung and breast
7
tumors, continued effort is required to confirm the underlying biology of ERα36 in
8
the development and/or metastasis of lung cancer and identify specific antagonists for
9
this form of ER.
10 11 12
6. Acknowledgements
13
This study was supported by Provincial science and technology foundation of
14
Shandong (2011GGH21819). The authors thank Professor Zhaoyi Wang for kindly
15
providing the antibody of ERα36.
16 17
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19
1 2
Table1. Clinicopathologic Characteristics and ERα36 / ERα66 status Characteristics Gender Male Female Age <60 ≥60 Histology Subtype AC SCC Smoking* Yes No Family History Yes No Mensopause Yes No Location Left Right pT stage T1 T2 T3-4 Lymphatic metastasis Positive Negative MLN 0 1-3 ≥4 LNR 0 0-35% ≥35% Chemotherapy Yes No
3 4 5 6
ERα36 low high 40 17
37 32
24 33
30 39
17 40
53 16
33 24
27 42
7 50
16 53
14 43
21 48
29 28
33 36
15 38 4
33 30 6
P value 0.059
ERα66 low high 65 40
12 9
48 57
6 15
58 47
12 9
50 55
10 11
18 87
5 16
30 75
5 16
54 51
8 13
38 60 7
10 8 3
0.878
0.150
.000
0.874
0.036
1.000
0.116
0.474
0.468
0.660
0.736
0.268
0.086
0.717
0.190 22 35
34 35
35 12 10
37 15 17
35 18 4
37 22 10
22 35
39 30
P value 0.686
0.818 46 59
10 11
61 21 23
11 6 4
61 32 12
11 8 2
51 54
10 11
0.310
0.884
0.268
0.732
0.045
0.937
*Smoking: yes: former smokers or current smokers; no: never smokers. AC: adenocarcinoma; SCC: squamous cell carcinoma; MLN: metastasis lymph nodes; LNR: lymph node ratio. 20
1 2 3
Table2 Clinicopathologic Characteristics and ERα36 status in AC and SCC ERα36(AC) low high
4 5
ERα36(SCC) low high
Characteristics P value P value Gender 0.900 0.813 Male 6 23 34 14 Female 11 30 6 2 Age 0.771 0.737 <60 7 21 17 6 ≥60 10 29 23 10 Smoking 0.952 0.928 Yes 5 16 28 11 No 12 37 12 5 Family History 0.134 0.143 Yes 2 16 5 0 No 15 37 35 16 Mensopause 0.733 0.763 Yes 5 18 9 3 No 12 35 31 13 Location 0.094 0.869 Left 8 25 21 8 Right 9 28 19 8 pT stage 0.768 0.064 T1 7 24 8 9 T2 10 26 28 5 T3-4 0 3 4 2 Lymphatic metastasis 0.014 0.534 Positive 2 24 20 6 Negative 15 29 20 10 MLN 0.034 0.389 0 15 31 20 6 1-3 1 10 11 5 ≥4 1 12 9 5 LNR 0.034 0.372 0 15 31 20 6 0-35% 1 14 17 8 ≥35% 1 8 3 2 Chemotherapy 0.338 0.092 Yes 7 29 15 10 No 10 24 25 6 *Smoking: yes: former smokers or current smokers; no: never smokers. AC: adenocarcinoma; SCC: squamous cell carcinoma; MLN: metastasis lymph nodes; LNR: lymph node ratio.
6 7 8 9 10 21
1 2 Table 3 Multivariate analysis of factors contributing to overall survival and disease-free survival in 3 NSCLC patients. 4 OS 5 Variables HR 95%CI P value 6 2.075 1.029-4.185 0.041 Gender 7 1.975 0.989-3.943 0.054 Smoking status 8 1.348 1.091-1.666 0.006 LNR 9 pT stage 1.825 1.200-2.776 0.005 10 1.737 1.011-2.984 0.045 11 ERα36 12 13 Variables HR 95%CI P value14 1.318 1.083-1.605 0.006 15 LNR 1.571 1.066-2.316 0.022 16 pT stage 1.785 1.074-2.968 0.025 17 ERα36 18 OS, Overall survival; DFS, Disease-free survival; CI, confidence interval; HR, hazard ratio; LNR, lymph node ratio. DFS
19 20 21 22 23
24 Table 4 Multivariate analysis of factors contributing to overall survival and disease-free survival in 25 lung adenocarcinoma patients. Variables
26
OS
DFS
pT stage
HR (95%CI) 2.192(1.276-3.767)
P-values 0.004
HR (95%CI) 1.718(0.993-2.971)
P value 0.053
ERα36
3.142(1.215-8.128)
0.018
2.720(1.141-6.486)
0.024
OS, Overall survival; DFS, Disease-free survival; CI, confidence interval; HR, hazard ratio.
27
22
1 2
Figure legends
3
Figure1. Immunohistochemical staining of ERα36/ ERα66 in NSCLC tissues. (A).
4
ERα66 negative staining in the lung tumor tissue; (B). ERα66 positive staining in the
5
cytoplasm of lung tumor tissue; (C). ERα36 positive staining in the cytoplasm of lung
6
tumor tissue; (D) ERα36 positive staining in the membrane of lung tumor tissue.
7 8
Figure2 Kaplan-Meier survival curves according to ERα36 expression in NSCLC
9
patients. (A) Overall survival (OS) for ERα36 expression in NSCLC (log-rank
10
P=0.061). (B) Disease-free survival (DFS) for ERα36 expression in NSCLC (log-rank
11
P=0.035).
12 13
Figure3 Kaplan-Meier survival curves according to ERα36 expression in NSCLC
14
patients. (A) Overall survival (OS) for ERα36 expression in lung adenocarcinoma
15
(log-rank P=0.020). (B) Disease-free survival (DFS) for ERα36 expression in lung
16
adenocarcinoma (log-rank P=0.024). (C) OS for ERα36 expression in SCC (log-rank
17
P=0.809). (D) DFS for ERα36 expression in SCC (log-rank P=0.777).
18 19
Figure4 Schematic illustration of domain structure of ERα66 and ERα36. Domains of
20
ERα66 were designated from A to F. It contains two transactivation domains (AF-1 in
21
A/B, AF-2 in E/F), a DNA binding domain(C), a variable hinge region (D), and a
22
ligand-binding domain (E). The two transactivation domains, AF-1 and AF-2 and part 23
1
of the ligand binding domain were missed out in ERα36. Instead, it contains an extra,
2
unique 27-animo acid sequence in its C-terminal. The binding sites of the primary
3
antibodies employed were also indicated.
4
24
1 2
3 4
25
1
2 3
26
1
2 3
27
1
2 3
28
1 2 3
Highlights
4 5 6 7 8 9
ERα36 was more highly expressed in NSCLC patients compared to ERα66. ERα36 mainly existed on the plasma membrane or in the cytoplasm of NSCLC cells. ERα36 was strongly correlated with more advanced regional lymph node metastasis. ERα36 status was a significant independent prognostic factor of OS and DFS in lung adenocarcinoma patients.
10
29