Ann Surg Oncol (2012) 19:3556–3565 DOI 10.1245/s10434-012-2396-1
ORIGINAL ARTICLE – HEPATOBILIARY TUMORS
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker for Human Hepatocellular Carcinoma After Curative Resection Dongtai Chen, MD1,2, Wei Xing, MD1,2, Jian Hong, MD, PhD1,3, Meibing Wang, MD1,2, Yang Huang, MD1,2, Cuizhen Zhu, MD1,2, Yunfei Yuan, MD1,3, and Weian Zeng, MD, PhD1,2 1
State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China; 2Department of Anesthesiology, Sun Yat-Sen University Cancer Center, Guangzhou, Guangdong, China; 3 Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China
ABSTRACT Background. The beta2-adrenergic receptor (Beta2-AR) is overexpressed and highly associated with poor prognosis in many malignancies. Nevertheless, the role of Beta2-AR in hepatocellular carcinoma (HCC) has not been thoroughly elucidated. The aim of this study is to investigate the expression of Beta2-AR and its clinicopathological/ prognostic value in HCC patients after curative resection. Materials and Methods. Semiquantitative reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR) were used to measure Beta2-AR RNA expression in 60 pairs of HCC tumors and matched nontumorous tissues. Beta2-AR expression was detected in HCC cell lines by Western blot analysis. Furthermore, we investigated Beta2-AR expression in correlation with the clinicopathological features and analyzed the potential prognostic significance of Beta2-AR in 192 HCC patients by immunohistochemistry (IHC). Results. Upregulation of Beta2-AR mRNA was significantly higher in HCC tumor tissues than in their paired nontumorous liver specimens. The expression of Beta2-AR protein was detected in five HCC cell lines. Positive Beta2-
Dongtai Chen and Wei Xing contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-012-2396-1) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2012 First Received: 21 October 2011; Published Online: 17 May 2012 W. Zeng, MD, PhD e-mail:
[email protected]
AR protein expression was significantly associated with a high a-fetoprotein (AFP) level (P = 0.001), large tumor size (P \ 0.001), tumor encapsulation (P = 0.002), vascular invasion (P = 0.004), microsatellite formation (P = 0.002), and poor differentiation (P \ 0.001). In univariate and multivariate analyses, Beta2-AR was an excellent predictive factor for both recurrence-free survival and overall survival (OS). Beta2-AR expression status was associated with poor prognosis independent of AFP, tumornode-metastasis stage and Edmondson stage. Conclusions. The Beta2-AR is a potential prognostic biomarker for survival and tumor recurrence in HCC patients after curative resection.
In males, hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed human malignancy worldwide and has a poor prognosis.1 HCC is ranked as the second most frequent cause of cancer-related death in the world, especially in southeast Asia and China.2 Surgical resection is the preferred curative method; however, a high incidence of postoperative metastasis and recurrence (HCC has a 5year recurrence rate of 50–70 %) is a considerable challenge, as this cancer is notably resistant to conventional systemic radiotherapy and chemotherapy.3 Therefore, investigating biomarkers for better evaluation of the diagnosis and prognosis of HCC can guide molecular targeting therapy for the improvement of more effective treatments and the inhibition of metastasis. The beta2-adrenergic receptor (Beta2-AR), a member of the family of transmembrane G protein-coupled receptors (GPCRs), initiates multiple signaling cascades and regulates cell proliferation through a classical cyclic-adenosine-monophosphate (cAMP)/protein kinase A (PKA) pathway.4,5
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker
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Tumor recurrence is a multifactorial process that includes tumor cell migration, invasion, and metastasis. Neurotransmitters and chemokines are ligands that bind to GPCRs and play important roles as the prominent regulatory factors of tumor recurrence.6,7 Beta2-AR overexpression has been reported in diverse human cancers, including breast cancer, oral cancer, prostate cancer, and melanoma. More importantly, tumorassociated Beta2-AR expression was found to be highly correlated with poor clinicopathological features, tumor recurrence, metastasis, and reduced survival.8–11 These findings suggest that Beta2-AR may be a cancer-relevant biomarker in carcinogenic processes. In cancer cell experiments performed in vitro, Beta2-AR offers a potential target for cancer therapy.12 However, the role of Beta2-AR in primary hepatocellular carcinoma development, growth, and progression, including recurrence and metastasis, has not yet been thoroughly elucidated. In this study, we aim to explore the clinical significance of the expression of Beta2-AR in HCC patients using immunohistochemical analysis. Furthermore, we investigate the potential prognostic significance of Beta2-AR expression status by comparing the survival rates of Beta2-AR-negative and Beta2-AR-positive HCC patients after surgical resection.
selected samples from 192 HCC tissues) and ten cases of normal liver tissues without cirrhosis from nontumor tissues that were also snap-frozen in liquid nitrogen and later used for semiquantitative reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR). A total of 192 pairs of primary HCC samples and adjacent nontumor liver tissues were used in the immunohistochemistry (IHC) analysis.
MATERIALS AND METHODS Cell Culture Five human HCC cell lines (Huh7, MHCC-97H, MHCC-97L, BEL7402, and HepG2) and one normal hepatic cell line (LO2) were obtained from the Chinese Type Culture Collection (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C in a humidified atmosphere of 5 % carbon dioxide and 95 % air. All culture media contained 100 units/ml penicillin and 100 lg/ml streptomycin (Invitrogen).
HCC Patients and Tissue Specimens All HCC samples and adjacent nontumor liver tissues were collected from 192 patients who had undergone surgical resections for primary HCC between 2003 and 2006 at the Department of Hepatobiliary Oncology, Sun Yat-sen University (Guangzhou, China). All nontumor and tumor tissues were confirmed histologically. The whole group of patients did not receive previous radiotherapy and chemotherapy before hepatectomy. There were 60 pairs of HCC tumor and matched nontumorous tissues (randomly
Follow-Up In all patients (170 males and 22 females), the mean age was 48.2 years (range: 20–80 years). The median followup period was 51 months (range: 2–133 months). Recurrences were confirmed by serum a-fetoprotein (AFP) levels, abdomen ultrasound every 2 months, and computed tomography (CT) or magnetic resonance imaging (MRI) every 6 months after hepatectomy. The main causes of death were HCC recurrence or complicated cirrhosis of the liver. Tumor stages were classified according to the sixth edition of the tumor-node-metastasis (TNM) system of the International Union Against Cancer by the 2002 American Joint Committee. Tumor differentiation was defined using the Edmondson-Steiner grading system. The studies using human materials were obtained and approved by the Committee for Ethical Review of Research at Sun Yat-Sen University Cancer Center. Overall survival (OS) was calculated from the date of surgery to the date of death or last follow-up. Relapse-free survival (RFS) was defined as from the date of surgery to the date of relapse or from the period of resection to the date of the last observation taken. Total RNA Extraction, Semiquantitative RT-PCR, and Real-Time qPCR Total RNA was extracted from HCC cell lines or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The total RNA (2 lg) from each sample was reverse transcribed using a PrimeScript RT Kit (Takara, Dalian) for first-strand cDNA synthesis. The primer sequences were: Beta2-AR, forward, 50 -TTC CTCTTTGCATGGAATTTG-30 , reverse, 50 -AGAGGAG TGGGGGAAGAGTC-30 ; GAPDH, forward, 50 -AGAAGG CTGGGGCTCATTTG-3, reverse, 50 -AGGGGCCATCC ACAG TCTTC-3. The RT-PCR conditions for the detection of Beta2-AR mRNA expression were: the template cDNA was first denatured at 94 °C for 5 min. The amplification step included 35 cycles of denaturation for 30 s at 95 °C, 30 s of annealing at 60 °C, and elongation at 72 °C for 30 s. The final extension step was at 72 °C for 7 min. The PCR products were electrophoresed on a 1.5 % (w/v) agarose gel in 1 9 TAE buffer. The human GAPDH gene was used
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as the endogenous control. Also, qPCR was performed with SYBR Green qPCR SuperMix and the ABI 7900HT Sequence Detector System (Applied Biosystems). All cDNA samples were normalized to the housekeeping gene GAPDH as the endogenous control; 2DDCT normalization was used to calculate the mRNA copy number ratios. Western Blot Proteins were extracted from HCC cell lines using RIPA buffer containing protease inhibitor cocktail (Roche), and protein was quantified using a BCA Protein Assay Kit (Thermo) according to the manufacturer’s protocol. Equal amounts of proteins (40 lg per lane) were separated on 10 % SDS-PAGE and transferred to polyvinylidene fluoride membrane (PVDF; Millipore). The membranes were probed with a Beta2-AR rabbit polyclonal antibody (1:100 dilution, Epitomics) and a GAPDH mouse monoclonal antibody (1:1,000 dilution, Santa Cruz Biotechnology) at 4 °C overnight and were subsequently incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution, Santa Cruz Biotechnology). Immunohistochemistry HCC tissues were fixed in 10 % neutral buffered formalin, embedded in paraffin and used for pathological evaluation. A standard indirect peroxidase labeling method was used for immunohistochemical staining. Tissue sections were deparaffinized and hydrated and were subsequently incubated with 0.3 % hydrogen peroxide. The sections were microwaved for antigen retrieval in 0.01 M citrate buffer (pH 6.0) for 20 min and were incubated with a Beta2-AR rabbit polyclonal antibody (1:100 dilution, Epitomics) overnight at 4utes°C. Subsequently, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody (DAKO, Carpinteria, CA) for 30 minutes at room temperature. To evaluate IHC staining of Beta2-AR, the expression of Beta2AR was scored as absent staining (0), weak staining (1), moderate staining (2), and strong staining (3). In this study, we characterize a low (0–1) score of Beta2-AR in HCC tumor tissue as ‘‘Beta2-AR-negative’’ and a high (2–3) score of Beta2-AR as ‘‘Beta2-AR-positive,’’ respectively.13 Compared with HCC matched nontumorous tissues, the high (2–3) IHC score of Beta2-AR in the tumor was defined as ‘‘overexpression of beta2-AR in HCC.’’ Assessments of the staining were scored in a double-blinded manner by two experienced pathologists. Statistical Analysis Statistical analysis was performed with SPSS software, version 16.0 (Chicago, IL). The mRNA level of Beta2-AR
D. Chen et al.
was ascertained by the paired t test. The chi-square test was used to analyze the association of Beta2-AR overexpression with the clinicopathological features of HCC patients. Multivariate analysis was performed on all prognostic factors that were found to be significant on the univariate level using the Cox proportional hazards regression model. The survival analyses were assessed using Kaplan–Meier plots and log-rank tests. Differences were considered statistically significant when P \ 0.05. RESULTS Beta2-AR is Overexpressed in HCC Semiquantitative RT-PCR and real-time qPCR were performed to compare mRNA expression levels of Beta2AR between 60 pairs of HCC tumor and matched nontumorous tissues. Overexpression of Beta2-AR mRNA was detected in 40 of 60 primary HCC samples (66.7 %; Supplementary Fig. 1) and was significantly higher than in their matched liver nontumorous tissues or normal liver tissues (both P \ 0.001); no significant difference was identified between matched nontumorous tissues and normal liver tissues (P [ 0.05; Fig. 1a, b). Western blot results indicated that protein expression of Beta2-AR was detectable in five HCC cell lines (Huh7, MHCC-97H, MHCC-97L, BEL7402, and HepG2), and expression of Beta2-AR was not detected in a normal hepatic cell line (LO2) (Fig. 1c). Overexpression of Beta2-AR was observed in 118 of 192 of the primary HCC specimens (56 %). The immunohistochemical analysis showed that Beta2-AR expression in HCC tumor tissues was significantly higher than in their matched noncancerous liver tissues (P \ 0.05; paired t test; Supplementary Fig. 2). Positive staining was detected in the nuclear and cytoplasmic regions. The expression of Beta2-AR was classified into negative or weak positive (- and ?; Fig. 1d, e) and moderate positive or strong positive (?? and ???; Fig. 1f, g) staining (Supplementary Table 1). Clinicopathological Correlation of Beta2-AR Protein Expression in HCC To better understand the clinical significance of Beta2AR expression in HCC, we investigated the clinicopathological features of Beta2-AR in positive-expression or negative-expression HCC samples. As shown in Table 1, a positive Beta2-AR protein level was significantly associated with a high a-fetoprotein (AFP) level (P = 0.001), large tumor size (P \ 0.001), tumor encapsulation (P = 0.002), vascular invasion (P = 0.004), microsatellite formation (P = 0.002), and poor differentiation (P \ 0.001). In contrast, Beta2-AR expression was not correlated with gender,
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker
HCC 1
10
GAPDH
8
c Huh 7
6 4
TN
TN
LO2
Beta2-AR
N
Hep G2
12
HCC 3
BEL7402
T
HCC 2
MHCC-97L
b
Relative Beta2-AR mRNA level
MHCC-97H
a
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Beta2-AR
2
GAPDH N (n = 10)
NT (n = 60)
T (n = 60)
d
Beta2-Ar (−)
e
Beta2-Ar (+)
f
Beta2-Ar (++)
g
Beta2-Ar (+++)
FIG. 1 Beta2-AR is overexpressed in HCC. a, b Relative expression levels of Beta2-AR RNA in 60 pairs of HCC tissues (T) and matched nontumorous tissues (NT) or ten normal liver tissues (N) were measured by RT-PCR and qPCR. The differences between HCC tissues, matched nontumorous tissues and normal liver tissues are significant (both P \ 0.001), and no significant difference was found between matched nontumorous tissues and normal liver tissues (P [ 0.05). c Expression levels of Beta2-AR protein in 5 HCC cell
lines (Huh7, MHCC-97H, MHCC-97L, BEL7402, and HepG2) and a normal hepatic cell line (LO2) were assayed by Western blot. GAPDH was used as a loading control. d–g Immunohistochemical staining of Beta2-AR protein expression in 192 HCC tissues. Original magnification, 9200. d, e Negative (-) or weak (?) staining were defined as negative expression; f, g Moderate (??) or strong (???) staining were defined as positive expression
age, HBsAg (hepatitis B surface antigen), Child-Pugh classification, GGT (gamma-glutamyltransferase), PVTT (portal vein tumor thrombi), and cirrhosis (all P [ 0.005).
Effects of Tumor Beta2-AR Protein Levels on Prognosis Based on the complete clinicopathological and followup data from the entire study population, the recurrence-
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free survival (RFS) and overall survival (OS) rates were 37.4 and 60.3 % at 3 years and 30.7 % and 43.6 % at 5 years, respectively. In univariate analysis, overexpression of Beta2-AR (P \ 0.001), GGT level (P = 0.001), tumor size (P \ 0.001), microsatellite formation (P = 0.001), tumor encapsulation (P = 0.002), vascular invasion (P = 0.037), PVTT (P = 0.016), and TNM stage (P = 0.003) were negative prognostic factors for OS in HCC patients. Furthermore, Kaplan-Meier analysis demonstrated that Beta2-AR overexpression (P \ 0.001), HBsAg (P = 0.030), GGT level (P = 0.002), tumor size (P \ 0.001), microsatellite formation (P \ 0.001), tumor encapsulation (P = 0.005), vascular invasion (P \ 0.001), tumor differentiation (P = 0.048), and TNM stage (P = 0.007) were significantly associated with the poor RFS rates of HCC patients (Table 2). The RFS and OS rates at 5 years were 19.9 and 30.5 % for Beta2-AR-positive patients compared with 48.1 and 65.0 % for Beta2-AR-negative patients, respectively (both P \ 0.001; Fig. 2a). In addition, the means of the RFS times in Beta2-AR-positive (n = 118) and Beta2-ARnegative (n = 74) HCC patient subgroups were 31.5 months (95% confidence interval [95% CI], 24.4–38.6 months) and 56.9 months (95% CI, 47.2–66.6 months), respectively, and the means of the OS times were 55.7 months (95% CI, 47.0–64.5 months) and 93.8 months (95% CI, 83.1–104.5 months), respectively. These results indicated that Beta2-AR protein levels might portend the prognosis of hepatocellular carcinoma.
D. Chen et al. TABLE 1 Clinicopathological correlation of Beta2-AR protein expression in HCC (n = 192) Characteristics
As TNM stage is based on information derived from tumor size, tumor number, and vascular invasion, we did not enter this data into the multivariate analysis. The multivariate Cox model analysis demonstrated that Beta2-AR overexpression (P = 0.002), microsatellite formation (P = 0.003), tumor size (P = 0.002), and GGT level (P = 0.013) were independent negative predictor factors for RFS in HCC patients enrolled in this study. By multivariate Cox regression analysis, Beta2-AR overexpression (P \ 0.001), microsatellite formation (P = 0.013), tumor size (P = 0.010), and GGT level (P = 0.002) were identified as independent risk factors for OS. Beta2-AR expression-positive patients were almost two times more likely to suffer from relapse than Beta2-AR expressionnegative patients (hazard ratio, 1.807; 95% CI, 1.239–2.636). In addition, the expression of Beta2-AR was determined to be a poor independent prognostic factor for OS in HCC patients (hazard ratio, 2.492; 95% CI, 1.572–3.951; Table 3).
Beta2-AR protein Positive expression
Negative expression
P value
Gender Female Male
22
17 (77.3%)
5 (22.7%)
170
101 (59.4%)
69 (40.6%)
0.105
Age (years) B50
106
70 (66.0%)
36 (44.0%)
[50
86
48 (55.8%)
38 (44.2%)
Negative
26
15 (57.7%)
11 (42.3%)
Positive
166
103 (62.0%)
63 (38.0%)
0.148
HBsAg 0.671
Child-Pugh classificationa A
185
112 (60.5%)
73 (39.5%)
B
7
6 (85.7%)
1 (14.3%)
0.179
59 133
25 (42.4%) 93 (69.9%)
34 (57.6%) 40 (30.1%)
0.001
AFP (ng/ml) \20 [20 GGT (units/l) B50
73
41 (56.2%)
32 (43.8%)
[50
119
77 (64.7%)
42 (35.3%)
0.238
Tumor size (cm) B5
83
37 (44.6%)
46 (55.4%)
[5
109
81 (74.3%)
28 (25.7%)
\0.001
Tumor encapsulation No/incomplete
92
67 (72.8%)
25 (27.2%)
100
51 (51.0%)
49 (49.0%)
No
171
99 (57.9%)
72 (42.1%)
Yes
21
19 (90.5%)
2 (9.5%)
0.004
183 9
110 (60.1%) 8 (88.9%)
73 (39.9%) 1 (11.1%)
0.083
Yes
Strong Beta2-AR Overexpression is an Independent Prognostic Factor
Cases
0.002
Vascular invasion
PVTT No Yes
Microsatellite formation No
152
85 (55.9%)
67 (44.1%)
Yes
40
33 (82.5%)
7 (17.5%)
Absent
33
25 (75.8%)
8 (24.2%)
Present
159
93 (58.5%)
66 (41.5%)
104
47 (45.2%)
57 (54.8%)
88
71 (80.7%)
17 (19.3%)
0.002
Cirrhosis 0.064
Tumor differentiation I/II III/IV
\0.001
HCC hepatocellular carcinoma, HBsAg hepatitis B surface antigen, AFP a-fetoprotein, GGT gamma-glutamyltransferase, PVTT portal vein tumor thrombi a
No patients with Child-Pugh class C was found
Bold values indicate P \ 0.05
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker TABLE 2 Univariate analyses of RFS and OS rates for 192 HCC patients after curative resection
Variables
Cases
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RFS rate (%) 3 years
P value
5 years
OS rate (%)
P value
3 years
5 years
54.5
43.8
59.9
43.7
Gender Female
22
40.9
31.8
170
36.9
30.6
B50
106
38.5
31.6
[50
86
34.9
23.8
Male
0.947
0.996
Age (years) 59.3
47.1
0.336
60.5
39.8
0.474
HBsAg Negative
26
53.6
48.2
69.2
54.5
Positive
166
34.2
27.8
0.030
57.7
41.1
0.181
185 7
38.2 14.3
31.3 0
0.300
60.4 42.9
43.7 0
0.814
\20
59
42.4
33.6
62.7
48.4
[20
133
35.2
29.4
58.5
41.6
B50
73
50.4
43.3
73.8
59.1
[50
119
28.6
22.9
51.3
34.3
145
39.1
33.2
64.7
47.1
47
29.8
23.0
44.7
33.1
B5
83
56.4
43.6
[5
109
22.9
20.9
92 100
29.3 44.7
24.8 36.1
No
171
40.2
32.6
Yes
21
14.3
0
183
38.7
31.6
9
11.1
0
No
152
41.9
34.9
Yes
40
20.0
14.6
104
43.0
35.1
88
29.5
25.7
Child-Pugh classification A B AFP (ng/ml) 0.367
0.337
GGT (units/L) 0.002
0.001
Number of tumor nodules Single Multiple
0.099
0.064
Tumor size (cm) 75.5
57.5
\0.001
47.7
33.2
\0.001
0.005
50.0 69.9
32.7 53.9
0.002
61.3
45.6
\0.001
42.9
28.6
61.1
44.8
0.003
22.2
0
67.0
48.2
\0.001
32.5
27.3
65.2
45.9
0.048
53.4
41.2
54.5
51.5
61.5
42.4
67.3
49.9
44.4
31.0
76.8
65.0
48.3
30.5
Tumor encapsulation No/incomplete Yes Vascular invasion 0.037
PVTT No Yes
0.016
Microsatellite formation 0.001
Tumor differentiation I/II III/IV
0.233
Cirrhosis Absent
33
42.4
38.9
Present TNM stage
159
36.3
29.1
129
41.7
34.9
63
27.0
22.0
Negative
74
55.2
48.1
Positive
118
26.3
19.9
I II/III TNM tumor-node-metastasis, RFS recurrence-free survival, OS overall survival Bold values indicate P \ 0.05
0.560
0.007
0.605
0.003
Beta2-AR expression \0.001
\0.001
3562 FIG. 2 Kaplan–Meier survival analysis of Beta2AR expression in HCC patients. a Kaplan-Meier OS and RFS survival curves of 192 HCC patients in association with Beta2-AR expression. The OS and RFS rates were significantly decreased in Beta2-AR-positive HCC patients (n = 118) compared with Beta2-AR-negative HCC patients (n = 74), both P \ 0.001. b–d HCC patients in different subgroups of Beta2-AR-positive patients showed a significantly poor survival rate compared with Beta2-AR-negative patients. b AFP B 20 ng/ ml and AFP [ 20 ng/ml; c TNM stage I and TNM stage II–III; d Edmondson I–II and Edmondson III–IV
D. Chen et al.
a Overall survival
P < 0.001
1.0
Beta-2AR (−) (n = 74) Beta-2AR (+) (n = 118)
Recurrence-free survival
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
24
48
72
96
P < 0.001
1.0
120
Beta-2AR (−) (n = 74) Beta-2AR (+) (n = 118)
0
Time after surgery (months)
24
48
72
96
Time after surgery (months)
b Recurrence-free survival AFP ≤ 20 ng/ml 1.0
P = 0.015
Beta-2AR (−) (n = 34) Beta-2AR (+) (n = 25)
Recurrence-free survival AFP > 20 ng/ml 1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
24
48
72
96
P = 0.015
Beta-2AR (−) (n = 40) Beta-2AR (+) (n = 93)
0
Time after surgery (months)
24
48
72
96
Time after surgery (months)
c Recurrence-free survival TNM stage I 1.0
P = 0.003
Beta-2AR (−) (n = 64) Beta-2AR (+) (n = 65)
Recurrence-free survival TNM stage II–III 1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
24
48
72
96
P = 0.014
Beta-2AR (−) (n = 10) Beta-2AR (+) (n = 53)
0
Time after surgery (months)
24
48
72
96
Time after surgery (months)
d Recurrence-free survival Edmonson I–II 1.0
P = 0.003
Beta-2AR (−) (n = 57) Beta-2AR (+) (n = 47)
Recurrence-free survival Edmonson III–IV 1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 0
24
48
72
Time after surgery (months)
96
P = 0.011
Beta-2AR (−) (n = 17) Beta-2AR (+) (n = 71)
0
24
48
72
Time after surgery (months)
96
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker TABLE 3 Cox multivariate analysis of prognostic factors to RFS and OS in 192 HCC patients after curative resection
Variables
3563
b
SE
Hazard ratio (95% CI)
P valuea
RFS GGT level
0.460
0.185
1.584 (1.103–2.276)
0.013
Tumor size
0.582
0.185
1.790 (1.246–2.571)
0.002
-0.283
0.185
0.754 (0.525–1.082)
0.126
Vascular invasion
0.080
0.323
1.084 (0.575–2.042)
0.803
PVTT
0.662
0.468
1.939 (0.774–4.856)
0.157
Microsatellite formation
0.605
0.204
1.831 (1.229–2.728)
0.003
Beta2-AR
0.592
0.193
1.807 (1.239–2.636)
0.002
0.674
0.220
1.963 (1.276–3.018)
0.002
Tumor encapsulation
OS GGT level Tumor size
95% CI 95% confidence interval a
Cox proportional hazards regression model
0.544
0.212
1.723 (1.138–2.608)
0.010
Tumor encapsulation
-0.325
0.208
0.723 (0.481–1.086)
0.118
Vascular invasion
-0.247
0.328
0.781 (0.411–1.486)
0.452
PVTT
0.561
0.453
1.753 (0.721–4.262)
0.215
Microsatellite formation Beta2-AR
0.553 0.913
0.222 0.235
1.738 (1.125–2.686) 2.492 (1.572–3.951)
0.013 \0.001
Bold values indicate P \ 0.05
To further investigate the prognostic value of Beta2-AR, patients were divided into 3 subgroups: (1) AFP B 20 ng/ml versus AFP [ 20 ng/ml, (2) TNM stage I versus TNM stage II-III, and (3) Edmondson I–II versus Edmondson III–IV. For the AFP B 20 ng/ml group, the 5-year RFS rate was 20.0 % for Beta2-AR-positive patients compared with 44.1 % for Beta2-AR-negative patients (P = 0.015; Fig. 2b). In the TNM stage I group, the 5-year RFS rate was 22.5 % for Beta2AR-positive patients compared with 44.8 % for Beta2-ARnegative patients (P = 0.003; Fig. 2c). In the Edmondson stage I–II group, the 5-year RFS rate was 21.3 % for Beta2AR-positive patients compared with 55.8 % for Beta2-ARnegative patients (P = 0.003; Fig. 2d). DISCUSSION HCC is the second most common cause of cancer-related death worldwide and has a poor outcome, mainly due to the high rate of metastasis and tumor recurrence observed after curative resection.1,14 As a result, the 5-year recurrence rate of HCC patients ranges from 50 to 70 %.15 Therefore, dependable tumor molecular biomarkers for HCC will be helpful for designing personalized treatments and for predicting prognoses in HCC patients. The Beta2-AR is a member of the transmembrane GPCRs, which regulate tumor progression by multiple mechanisms.16 Beta2-AR expression is significantly correlated with tumor recurrence and metastasis, thereby implicating Beta2-AR as a prognostic biomarker of malignancy.8–11,17 Nevertheless, the role of Beta2-AR in HCC has not been thoroughly elucidated. In this study, we first measured Beta2-AR mRNA expression levels in 60 paired HCC tumor tissues and matched tumor-adjacent tissues and in 10 cases of normal liver-
tissue specimens. It was determined that Beta2-AR was remarkably upregulated in HCC samples. Importantly, the correlations of Beta2-AR protein expression with clinical pathological factors and its prognostic effects were determined for 192 HCC patients using immunohistochemistry analysis. The results demonstrated that positive Beta2-AR protein levels were significantly associated with high AFP levels, large tumor size, tumor encapsulation, vascular invasion, microsatellite formation, and poor differentiation. In addition, Kaplan–Meier analysis showed that in general, Beta2-AR-positive patients had worse prognoses than did Beta2-AR-negative patients by both RFS and OS. Moreover, the multivariate Cox model analysis indicated that Beta2-AR expression status was identified as an independent risk factor for both RFS and OS. These data support the hypotheses that Beta2-AR might play a pivotal role in HCC carcinogenic processes and that Beta2-AR could be a potential prognostic biomarker of HCC. AFP is a tumor-associated antigen that is useful for the diagnosis and predicted prognosis of HCC and for monitoring metastasis and tumor recurrence in HCC patients with high AFP levels after hepatectomy.18,19 However, it is hard to predict the prognosis and metastatic recurrence of normal-AFP HCC patients after curative resection. To investigate further the prognostic value of Beta2-AR, patients were divided into two groups according to AFP level. Subgroup analysis revealed significant differences in the 5-year RFS rates between patients that were positive and negative for Beta2-AR expression (20.0 and 44.1 %) in the group with normal AFP levels. The predictive function of Beta2-AR with normal AFP levels could guide clinicians in distinguishing HCC patients with high rates of recurrence after curative hepatectomy.
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Since HCC patients with the same TNM stage I often exhibit various clinical outcomes in tumor progression and prognosis.20 In particular, TNM stage I HCC patients are likely to experience recurrence because of a lack of personalized adjuvant therapy after surgery. In this study, we determined that the 5-year RFS rates for Beta2-AR-positive and Beta2-AR-negative patients were 22.5 and 44.8 %, respectively, in HCC patients with TNM stage I. These results suggest that Beta2-AR may be useful as an additional tool for distinguishing TNM stage I HCC patients with worse prognoses from those with better prognoses and for providing more information to clinicians for improving therapeutic strategies for TNM stage I HCC patients. Moreover, the identical correlation existed in the Edmondson stage I–II group. Taken together, our results demonstrate that Beta2-AR may be a useful prognostic molecular biomarker for HCC, especially in patients with normal AFP levels and those with TNM stage I and Edmondson stage I–II HCC, the consequences of which are difficult to predict using traditional clinical parameters. Several studies have demonstrated that the Beta2-AR agonist isoproterenol (ISO) promoted the growth of human cancer cells in vitro via Beta2-AR-mediated activation of cAMP/PKA, mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase 1/2 (ERK1/2), and PI3-kinase (PI3 K)/protein kinase B (AKT) signaling pathways and that ISO activated MAPK/ERK1/2 by a Beta2-AR-mediated and vascular endothelial growth factor (VEGF)-independent mechanism.21–24 Stimulation of Beta2-AR has been shown to induce cell proliferation and cell adhesion.25,26 Therefore, Beta2-AR has recently received attention as a potential therapeutic target in the treatment of cancer. Furthermore, in vitro studies have demonstrated that norepinephrine-induced stimulation of cell proliferation and migration was significantly inhibited by the Beta2-AR antagonist propranolol in human prostate, colorectal, ovarian, and breast cancer cell lines.27–30 In addition, propranolol has been shown to suppress prostate cancer cell growth in a nude mouse xenograft model.31 Additionally, a clinical report has shown that the Beta2-AR antagonist could be a therapy for breast cancer by reducing metastasis, recurrence, and mortality.32,33 In summary, Beta2-AR expression was significantly associated with high AFP levels, large tumor size, tumor encapsulation, vascular invasion, microsatellite formation, and poor differentiation. Furthermore, we also determined that Beta2-AR might not only be a potential prognostic biomarker for RFS and OS of HCC, but may also be used as an effective tool for identifying patients with worse or better prognoses and for providing personalized adjuvant therapies for HCC patients after curative resection. Taken together, we have demonstrated that overexpression of Beta2-AR might play an important role in HCC
D. Chen et al.
tumorigenesis and prognosis and might serve as a promising therapeutic target in HCC treatment. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (30972849).
REFERENCES 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90. 2. McGlynn KA, Tarone RE, El-Serag HB. A comparison of trends in the incidence of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in the United States. Cancer Epidemiol Biomarkers Prev. 2006;15:1198–203. 3. Gish RG, Porta C, Lazar L, Ruff P, Feld R, Croitoru A, et al. Phase III randomized controlled trial comparing the survival of patients with unresectable hepatocellular carcinoma treated with nolatrexed or doxorubicin. J Clin Oncol. 2007;25:3069–75. 4. Maki T, Kontula K, Harkonen M. The beta-adrenergic system in man: physiological and pathophysiological response. Regulation of receptor density and functioning. Scand J Clin Lab Invest Suppl. 1990;201:25–43. 5. Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, et al. The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multireceptor complex with the epidermal growth factor receptor. J Biol Chem. 2000;275:9572–80. 6. Entschladen F, Drell TL 4th, Lang K, Joseph J, Zaenker KS. Tumour-cell migration, invasion, and metastasis: navigation by neurotransmitters. Lancet Oncol. 2004;5:254–8. 7. Entschladen F, Drell TLt, Lang K, Joseph J, Zaenker KS. Neurotransmitters and chemokines regulate tumor cell migration: potential for a new pharmacological approach to inhibit invasion and metastasis development. Curr Pharm Des. 2005;11:403–11. 8. Ramberg H, Eide T, Krobert KA, Levy FO, Dizeyi N, Bjartell AS, et al. Hormonal regulation of beta2-adrenergic receptor level in prostate cancer. Prostate. 2008;68:1133–42. 9. Yang EV, Kim SJ, Donovan EL, Chen M, Gross AC, Webster Marketon JI, et al. Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression. Brain Behav Immun. 2009;23:267–75. 10. Shang ZJ, Liu K, Liang de F. Expression of beta2-adrenergic receptor in oral squamous cell carcinoma. J Oral Pathol Med. 2009;38:371–6. 11. Shi M, Liu D, Duan H, Qian L, Wang L, Niu L, et al. The beta2adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Breast Cancer Res Treat. 2011;125:351–62. 12. Schuller HM. Beta-adrenergic signaling, a novel target for cancer therapy? Oncotarget. 2010;1:466–9. 13. Chen L, Yuan YF, Li Y, Chan TH, Zheng BJ, Huang J, et al. Clinical significance of CHD1L in hepatocellular carcinoma and therapeutic potentials of virus-mediated CHD1L depletion. Gut. 2011;60:534–43. 14. Tung-Ping Poon R, Fan ST, Wong J. Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg. 2000;232:10–24. 15. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132 :2557–76. 16. Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nat Rev Cancer. 2007;7:79–94. 17. Takezaki T, Hamajima N, Matsuo K, Tanaka R, Hirai T, Kato T, et al. Association of polymorphisms in the beta-2 and beta-3
The Beta2-Adrenergic Receptor is a Potential Prognostic Biomarker
3565
adrenoceptor genes with risk of colorectal cancer in Japanese. Int J Clin Oncol. 2001;6:117–22. Soresi M, Magliarisi C, Campagna P, Leto G, Bonfissuto G, Riili A, et al. Usefulness of alpha-fetoprotein in the diagnosis of hepatocellular carcinoma. Anticancer Res. 2003;23:1747–53. Johnson PJ. The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma. Clin Liver Dis. 2001;5:145–59. Lee JS, Chu IS, Heo J, Calvisi DF, Sun Z, Roskams T, et al. Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling. Hepatology. 2004;40:667–76. Pullar CE, Isseroff RR. The beta 2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms. J Cell Sci. 2006;119:592–602. Yuan A, Li Z, Li X, Yi S, Wang S, Cai Y, et al. The mitogenic effectors of isoproterenol in human hepatocellular carcinoma cells. Oncol Rep. 2010;23:151–7. Liu X, Wu WK, Yu L, Li ZJ, Sung JJ, Zhang ST, et al. Epidermal growth factor-induced esophageal cancer cell proliferation requires transactivation of beta-adrenoceptors. J Pharmacol Exp Ther. 2008;326:69–75. Guo K, Ma Q, Wang L, Hu H, Li J, Zhang D, et al. Norepinephrine-induced invasion by pancreatic cancer cells is inhibited by propranolol. Oncol Rep. 2009;22:825–30. Stork PJ, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12:258–66.
26. Bos JL. Linking rap to cell adhesion. Curr Opin Cell Biol. 2005;17:123–8. 27. Drell TL 4th, Joseph J, Lang K, Niggemann B, Zaenker KS, Entschladen F. Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells. Breast Cancer Res Treat. 2003;80:63–70. 28. Sood AK, Bhatty R, Kamat AA, Landen CN, Han L, Thaker PH, et al. Stress hormone-mediated invasion of ovarian cancer cells. Clin Cancer Res. 2006;12:369–75. 29. Masur K, Niggemann B, Zanker KS, Entschladen F. Norepinephrine-induced migration of SW 480 colon carcinoma cells is inhibited by beta-blockers. Cancer Res. 2001;61:2866–9. 30. Lang K, Drell TL 4th, Lindecke A, Niggemann B, Kaltschmidt C, Zaenker KS, et al. Induction of a metastatogenic tumor cell type by neurotransmitters and its pharmacological inhibition by established drugs. Int J Cancer. 2004;112:231–8. 31. Palm D, Lang K, Niggemann B, Drell TL 4th, Masur K, Zaenker KS, et al. The norepinephrine-driven metastasis development of PC-3 human prostate cancer cells in BALB/c nude mice is inhibited by beta-blockers. Int J Cancer. 2006;118:2744–9. 32. Powe DG, Voss MJ, Zanker KS, Habashy HO, Green AR, Ellis IO, et al. Beta-blocker drug therapy reduces secondary cancer formation in breast cancer and improves cancer specific survival. Oncotarget. 2010;1:628–38. 33. Powe DG, Entschladen F. Targeted therapies: using beta-blockers to inhibit breast cancer progression. Nat Rev Clin Oncol. 2011;8:511–2.
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19.
20.
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