Med Oncol (2014) 31:15 DOI 10.1007/s12032-014-0015-7
ORIGINAL PAPER
Increased expression of phospho-acetyl-CoA carboxylase protein is an independent prognostic factor for human gastric cancer without lymph node metastasis Wenzhang Fang • Hongmei Cui • Danyang Yu Ying Chen • Jiejun Wang • Guanzhen Yu
•
Received: 14 November 2013 / Accepted: 28 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Upregulation of acetyl-CoA carboxylase (ACC), as a rate-limiting enzyme of fatty acid synthesis,has been recognized in multiple human cancers, implicating a critical role in cancer development and progression; yet, its role in gastric cancer still remains unclear. In the present study, we detected ACC and phosphorylated form of ACC (pACC) expression in gastric cancers and explored its clinical significance. Tissue microarray blocks containing primary gastric cancer and adjacent normal mucosa specimens obtained from 1,072 Chinese patients were used for the detection of ACC and pACC expression by immunohistochemistry. Gastric cancer cell lines were treated by metformin, and pACC was measured by Western blotting. ACC overexpression was observed in all the tumor specimens. High expression of
Wenzhang Fang, Hongmei Cui, and Danyang Yu have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12032-014-0015-7) contains supplementary material, which is available to authorized users. W. Fang J. Wang G. Yu (&) Department of Medical Oncology, Changzheng Hospital, Hetian Road 64, Shanghai 200070, People’s Republic of China e-mail:
[email protected] J. Wang e-mail:
[email protected] W. Fang Department of Oncology, Fuzhou General Hospital, Fuzhou, Fujian Province, People’s Republic of China
pACC was found in 630 (58.8 %) of the 1,072 primary tumors and in 237 (66.6 %) of the 356 primary tumors without lymph node metastasis. Absent/low expression of pACC significantly correlated with advanced T stage (P \ 0.001), tumor size (P = 0.010), lymph node metastasis (P \ 0.001), advanced disease stage (P \ 0.001), and poor histological differentiation (P = 0.014) in 1,072 primary tumors, and with advanced T stage (P = 0.015), tumor size (P = 0.017), and poor histological differentiation (P = 0.001) in 356 tumors without lymph node metastasis. Kaplan–Meier analysis showed that high expression of pACC is strongly related to better survival rates in all gastric cancer patients (P = 0.006). Cox regression analysis revealed that pACC is an independent prognostic factor only in patients without lymph node metastasis (P = 0.016). Metformin treatment leaded to increased expression of pACC, which, in turn, resulted in the reduction of cell proliferation and colony formation of gastric cancer cells (P \ 0.05). Increased activation of ACC is frequent in human gastric cancer, and downregulation of pACC is an important prognostic factor, D. Yu Department of Ophthalmology, Kunming General Hospital of Chengdu Military Command, Kunming, Yunnan Province, People’s Republic of China Y. Chen Department of Pathology, Changhai Hospital, Shanghai, People’s Republic of China
H. Cui Department of No. 3 Cadre Wards, General Hospital of Jinan Military Area Command, Jinan, Shandong Province, People’s Republic of China
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suggesting that ACC/pACC might be a potential target for cancer intervention.
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Methods Human tissue specimens and patients’ information
Keywords Gastric carcinoma ACC pACC Prognosis Metformin Immunohistochemistry
Background Gastric cancer (GC) is the second leading cause of cancer death worldwide, and it has the highest mortality rates in East Asia, including China [1]. Despite advances in early diagnosis and treatment efficacy, the 5-year survival of patients with GC is around 20–30 % [2, 3]. The most important prognostic factors established for GC continue to be the TNM stage and lymph node metastasis [4–6]. However, even patients at the same stage, the prognosis after removing the primary tumor is still diverse. Therefore, it is of great value to find new prognostic and predictive markers in order to predict patients’ outcome and personalize treatments according to the individual in GC. One of the hallmarks of aggressive cancer cells is a high rate of energy-consuming anabolic processes characterized by increased synthesis of lipids, proteins, and DNA [7]. The constitutive activation of the fatty acid biosynthetic pathway produces saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), sustaining the increasing demands of new membrane phospholipids in cancer cells, implicating the lipogenic enzymes in the pathway as potential targets for cancer intervention [8]. Among these lipogenic enzymes, acetyl-CoA carboxylase (ACC) leads to increased synthesis of SFA. Recently, increasing evidences suggested a critical role of ACCs in human cancer development and progression [7]. ACC exists as active/ dephosphorylation and inactive/phosphorylation forms. Upregulation of active ACC has been observed in various type of human cancer, including breast [9, 10], prostate [11], and liver cancer [12]. Silencing of ACC induces growth inhibition and apoptosis of prostate cancer cells [13]. Moreover, inactivate (phosphorylated) form of ACC (pACC) was absent/low in 35 % lung tumors and high pACC was an independent prognostic marker for lung adenocarcinoma patients [14]. Thus, ACC has been regarded as a potential target for cancer treatment. Numerous chemical ACC inhibitors have been developed and used in clinical trials [15]. However, the status of ACC and pACC expression and its clinical significance in GC were not fully investigated. In this study, we sought to evaluate the expression of ACC and pACC in resected GCs and to determine its clinical and prognostic value by correlating ACC and pACC expression with clinicopathological features and survival.
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Tissue microarrays containing 1,072 cases with primary GC were used for the detection of ACC and pACC expression, which were preserved in the GC Tissue Bank at the Department of Medical Oncology, Changzheng Hospital Shanghai, China [16, 17]. The patients’ medical records had been previously described and listed in Table 1. Clinical follow-up results were available for 841 of 1,072 (78.5 %) patients. All of the tissue specimens were obtained for this study with patient informed consent, and the use of the human specimens was approved by the Changzheng and Changhai Hospital Institutional Review Board.
Table 1 Association between pACC expression and clinicopathological factors in gastric cancer Clinicopathological variables
N
High pACC expression
Age (years)
P value 0.118
\60
537
303 (56.4)
C60
535
327 (61.1)
Male
757
445 (58.8)
Female
315
185 (58.7)
T1/T2
312
211 (67.6)
T3/T4
760
419 (55.1)
356 716
237 (66.6) 393 (54.9)
\0.001
0.010
Sex
0.987
\0.001
pT
pN N0 N1–3 Tumor size* \6 cm
344
222 (64.5)
C6 cm
481
279 (58.0) \0.001
Disease stage I/II
427
278 (65.1)
III/IV
645
352 (54.6)
-
481
277 (57.6)
?
591
353 (59.7)
-
115
62 (53.0)
?
957
568 (59.2)
632 440
391 (61.9) 239 (54.3)
1,072
630 (58.8)
p53
PCNA
0.202
Differentiation High/moderate Poorly Total
0.479
0.014
*Data of tumor size was available for 825 cases
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Immunohistochemistry Standard procedure was performed to investigate the level of ACC and pACC expression in the tumor. Briefly, 4-lm sections of paraffin-embedded tissue microarrays were deparaffinized and rehydrated in xylene and degradation alcohol. Antigen unmasking was performed by pretreatment of the slides in 0.01-M citrate buffer (pH 6.0) at 98 °C for 5 min using a microwave oven. The slides were then cooled to room temperature. Endogenous peroxidase was cleaned by incubating the slides in 3 % hydrogen peroxide for 10 min. After washed in 0.01 M PBS (pH 7.4), the sections were incubated for 10 min at room temperature with normal goat serum, followed by incubation with anti-ACC antibody (dilution: 1:100, C83B10, CST, Beverly, MA) and anti-pACC (dilution: 1:50, Ser79, CST, Beverly, MA) overnight at 4 °C. A S-p kit (KIT-9710; MAIXIN, Fuzhou, China) was used to visualize antibody binding on the slides. Counterstaining was performed with hematoxylin. ACC and pACC protein expression in these specimens was evaluated by two individuals under an Olympus CX31 microscope (Olympus, Center Valley, PA). Evaluation of immunostaining A mean percentage of positive tumor cells was determined in at least five areas at 9400 magnifications (50–250 cancer cells per area) and assigned from 0 to 100. The intensity of immunostaining was scored as follows: negative, 0, weak, 1?, moderate, 2?; and intense, 3?. Theoretically, the percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case: ranging from 0 (0 % of cells staining) to 300 (100 % of the cells staining at 3? intensity). For convenience in reporting and statistical analysis, a quartation (negative, weak, moderate, and strong staining) was made for each antibody. The cutoff points were based on the scores: negative, 0; weak, \75; moderate, 75–150; and intense, [150. We defined the score \75 as low expression and[75 as high expression. Cell lines and culture conditions The human gastric cell line BGC823 and MKN28 was purchased from the Cell Center of Chinese Academy of Sciences, Shanghai, China. BGC823 and MKN28 were maintained in DMEN with 10 % fetal bovine serum (FBS) (Invitrogen Corp., Grand Island, NY). The cells were cultured in a 37 °C humidified atmosphere containing 95 % air and 5 % CO2. Cell proliferation assay Cells were digested, and 5,000 cells were seeded into 96-well plates and incubated in medium with 10 % FBS.
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24 h after seeding, cells were treated with metformin in a range of concentrations (0, 10, 50 mM). At 0, 24, 48, and 72 h, CCK8 assay (Dojindo Kumamoto, Japan) was carried out to quantify cell viability. The experiment was repeated three times independently. Colony formation assay Gastric cancer (GC) cells were seeded in 6-well plates in triplicate at a density of 500 cells per well. At 24 h, cells were treated with metformin (0, 10, 50 mM) for 2 weeks at 37 °C. The colonies were fixed with methanol/acetone (1:1) and stained with crystal violet. Colonies with cell numbers over 50 cells per colony were counted. Western blot Western blot analysis was carried out according to protocols provided by vendors of antibodies. Briefly, protein samples (25 lg) were subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). The membranes were sequentially blotted with a rabbit antibody against human ACC or pACC (1:1,000) and an anti-rabbit IgG antibody, which was a horseradish peroxidase-linked F(ab’)2 fragments obtained from a donkey (Amersham). Equal protein sample loading was monitored by probing the same membrane filter with an anti-bactin antibody. The immunoreactive proteins were detected by the enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). Immunofluorescence analysis Cells were fixed with 4 % formaldehyde in PBS for 15 min and rinsed three times in PBS for 5 min. Blocking specimens in blocking buffer for 60 min and then applying diluted anti-pACC antibody overnight at 4 °C. Then, the specimens were incubated with fluorochrome-conjugated secondary antibody for 1 h. After PBS washing, nuclei were stained with DAPI (Sigma, Munich, Germany) and examined by fluorescence microscopy (Keyence, Neu-Isenburg, Germany). Data analysis All statistical analyses were conducted using the SPSS 16.0 statistical software program. Categorical data were analyzed using chi-square tests. The Kaplan–Meier method was used to estimate survival rates, and the log-rank test was used to assess survival differences between groups. The Cox proportional hazards model for multivariate survival analysis was used to assess predictors related to survival. The significance of the in vitro data was
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significant difference of ACC expression was observed between normal epithelium and tumor cells. These results were further confirmed by Western blotting that the expression of ACC protein was only slightly increased in gastric tumors compared with non-neoplastic tissues (Fig. 1c, d).
determined using two-tailed Student’s t test. A two-sided P \ 0.05 was defined as statistically significant.
Results ACC expression in patients with gastric cancer
pACC expression in patients with gastric cancers To evaluate the ACC expression pattern in GC, we assessed both the proportions and locations of the positively stained tumor area. As shown in Fig. 1a, ACC-positive staining was preferentially nuclear localized and partially cytoplasm localized in normal gastric epithelium and all of the adjacent non-cancerous epithelium showed moderateto strong-positive staining of ACC. However, in cancer cells, ACC-positive staining was preferentially nuclear localized. All the tumor cells in these specimens showed intensively strong-positive staining of ACC (Fig. 1b). No
A
A1
B
B1
C
D
N ACC -actin
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T
N
T
N
T Relative expression of ACC/ -actin
Fig. 1 Analysis of ACC expression in human gastric cancers and adjacent normal mucosa specimens. a Normal gastric mucosa showed positive nuclear- and cytoplasmlocalized staining of ACC; b ACC-positive staining was preferentially nuclear localized in gastric cancer; c Western blot analysis of three paired gastric cancer (T) and normal gastric mucosa specimens (N); d relative amounts of protein in these specimens vs. b-actin from (c) analyzed by Image J. a1, b1: enlargement of tissues from a, b, respectively. Original magnification of a, b: 940; Original magnification of A1, B1: 9200
We then detected the expression of inactive form of ACC (pACC) in normal and malignant epithelium. pACC-positive staining was preferentially cytoplasm localized determined by both immunofluorescence assay and immunohistochemistry. Immunofluorescence and Western blotting revealed that pACC was strongly expressed in gastric mucosa (Fig. 2a, c, d), while a faint cytoplasmic staining was present throughout all samples (Fig. 2b). These results were further confirmed by immunohistochemistry that pACC was upregulated in normal
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A
15
B
D
N
T
N
T
N
T
pACC -actin
Relative expression of pACC/ -actin
C
Fig. 2 Analysis of pACC expression in human gastric cancers and adjacent normal mucosa specimens by immunofluorescence assay and Western blotting. a Normal gastric mucosa showed preferentially positive cytoplasm-localized staining of pACC; b gastric cancer cells showed weak cytoplasm-localized staining of pACC; c Western blot
analysis of three paired gastric cancer (T) and normal gastric mucosa specimens (N); d relative amounts of pACC levels in the specimens vs b-actin from (c) analyzed by Image J. Original magnification of a, b: 9200
gastric epithelium, while downregulated in gastric tumors (Fig. 3). The mean values of pACC in tumor tissues were 1.67 ± 1.12, significantly lower than those in normal tissue: 2.80 ± 0.55, while significantly higher than that in lymph node metastases (1.34 ± 1.20) (Fig. 3d). Among the 1,072 tumors, 630 (58.8 %) showed high expression of pACC (Fig. 3b) and 442 (41.2 %) showed low/absent expression of pACC (Fig. 3c).
(66.6 %) than that with regional LN metastasis (54.9 %; P \ 0.001). The mean score of pACC in tumors was 2.00 ± 1.16 at N0 stage, significantly higher than that at N1–3 stage: 1.51 ± 1.06 (Fig. 5a). In addition, a significant relationship was observed between high pACC expression and histological differentiation. Specifically, we observed pACC overexpression in 391 well differentiated to moderately differentiated tumors (61.9 %) and 239 poorly differentiated tumors (54.3 %) (P = 0.014). There were no statistically significant associations with advanced age, sex, p53, and proliferating cell nuclear antigen (PCNA). In tumors without lymph node metastasis, pACC expression significantly correlated with T stage, tumor size, and differentiation (Table S1).
pACC expression correlates with tumor grade and the disease stage in gastric cancer Table 1 summarized the correlation of high expression of pACC with clinicopathological variables. We observed that high pACC expression significantly inversely correlated with advanced T, N, and TNM stage. High pACC expression was more often observed in small gastric tumors (67.6 % at T1/T2) than in large ones (55.1 % at T3/T4; P \ 0.001). With regard to TNM stage, high pACC expression was significantly inversely associated with advanced disease stage: 65.1 % at stage I/II and 54.6 % at stage III/IV (P \ 0.001) and the mean value of pACC in tumors was 1.93 ± 1.13 at stage I/II, significantly higher than that at stage III/IV: 1.50 ± 1.08 (Figure S1A). As for nodal metastasis, high pACC expression occurred more frequently in gastric tumors without lymph node metastasis
Relationship of pACC expression with better outcome in patients with gastric cancer Survival analysis showed that high expression of pACC was associated with increased median survival durations. Specifically, patients with pACC high expression tumors had median survival duration of 66.6 months, whereas patients with pACC absent/low expression tumors had median survival duration of 57.3 months (P = 0.006; Fig. 4). Furthermore, multivariate analysis using the Cox proportional hazards model showed that age at diagnosis,
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A
A1
B
B1
C
C1
D
***
***
Fig. 3 Analysis of pACC expression in human gastric cancers and adjacent normal mucosa specimens by immunohistochemistry. a Normal gastric mucosa showed preferentially positive cytoplasm-localized staining of pACC; b pACC-positive staining was preferentially cytoplasm localized in gastric cancer; c low expression of pACC in
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N
T
M
Average
2.80
1.67
1.34
Std
0.55
1.12
1.20
gastric cancer; d graphical representation of the differences of pACC staining in non-neoplastic (N), cancer tissues (T), and nodal metastases (M). ***P \ 0.001. a1, b1, c1: enlargement of tissues from a, b, c. Original magnification of a, b, c: 940; original magnification of a1, b1, c1: 9200
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tumor cell differentiation, gastric wall invasion, LN metastasis, and TNM stage were independent prognostic factors. However, pACC expression was not an independent prognostic factor (Table 2). Subgroup analysis according to TNM revealed that the survival durations were significantly worse in patients of stages I and II with low/absent pACC expression (81.7 months) than those of stages I and II with pACC overexpression (92.2 months; P = 0.016) (Figure S1B). However, there was no significant difference in survival durations in patients of stages III and IV with or without pACC overexpression (Figure S1C). Therefore, the pACC overexpression status in early-stage disease significantly influences patients’ survival. Furthermore, subgroup
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analysis according to nodal metastasis also revealed that patients without regional lymph node metastasis with high pACC expression had a better survival durations (93.1 months) than those without nodal metastasis with low pACC expression (74.5 months; P = 0.001) (Fig. 5b). No significant differences in survival durations were found in patients with nodal metastasis with or without pACC overexpression (Fig. 5c). This result further supported the important role of pACC overexpression in early stage of GC. Furthermore, subgroup multivariate analysis using the Cox proportional hazards model showed that age at diagnosis, tumor cell differentiation, gastric wall invasion, and pACC were independent prognostic factors for GC patients without lymph node metastasis (Table 3). Inhibition of gastric cancer cell proliferation and colony formation by inducing expression of pACC Given the fact that pACC expression was prominently lost in GCs, we further assessed the functional significance after restoring pACC expression. As shown in Fig. 6a, d, metformin treatment could lead to increased expression of pACC protein. The restoration of pACC significantly inhibited cell viability (Fig. 6b, e) and cell colony formation (Fig. 6c, f) of BGC823 and MKN28 cells.
High pACC expression
Low pACC expression P =0.006
Discussion
pACC
OS
Std.
95% CI
Low
57.3
2.5
52.4
62.2
High
66.6
2.1
62.5
70.8
2
7.56
P
0.006
Fig. 4 Kaplan–Meier curves of survival durations in patients with gastric cancer according to the expression of pACC. Survival durations were significantly better in patients with high expression of pACC (median survival, 66.6 months) than in those with low expression of pACC (median survival, 57.3 months; P = 0.006) Table 2 Cox proportional hazards model analysis of prognostic factors
In the present study, we systemically detected ACC and pACC expression in a larger cohort of GC patients and we found that low/absent expression of pACC was significantly associated with (1) advanced tumor stage and lymph node metastasis; (2) poorly differentiation status of GC cells; and (3) shorter survival duration than those with high expression of pACC, especially in patients at early stage. Moreover, inactivation of ACC, characterized by ACC phosphorylation, by metformin treatment significantly inhibits cell proliferation and cell growth. Therefore, we provide the strong evidence that loss of pACC might play a critical role in the development of GC.
Covariate (observed value)
b
SE
Wald
Exp (B)
95 % CI
P value
Age, years (B60 vs. [60)
-.485
.100
23.392
.616
.506–.750
\.001
Gastric wall invasion (T1/T2 vs. T3/T4)
-.592
.195
9.213
.553
.377–.811
.002
Nodal involvement (negative vs. positive)
-.592
.202
8.598
.553
.372–.822
.003
Tumor size (B6 cm vs. [6 cm)
-.203
.109
3.504
.816
.660–1.010
Differentiation (well/moderate vs. poorly)
-.417
.098
17.969
.659
.544–.799
\.001
TNM stage (I/II vs. III/IV)
-.637
.236
7.301
.529
.333–.840
.007
pACC (negative vs. positive)
-.049
.098
.247
.952
.786–1.154
.619
.061
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Fig. 5 Kaplan–Meier plot of overall survival durations in the patients with different status of lymph node metastasis according to pACC expression. a Graphical representation of the differences of pACC staining between N0 stage and N1–3 stage; b survival durations were significantly worse in patients without lymph node metastasis with low expression of pACC (median survival, 74.5 months) than in those with high expression of pACC (median survival, 93.1 months; P = 0.001); c no statistical significance of the survival durations was observed between patients with regional lymph node metastasis with high expression of pACC and those with low expression of pACC
Med Oncol (2014) 31:15
A ***
B
N0
N1-3
Average
2.00
1.51
Std
1.16
1.06
C High pACC expression
Low pACC expression
Low pACC expression High pACC expression
P =0.001
pACC
OS
Low
74.5
P =0.344
Std.
95% CI
P
4.4 65.8 83.2
pACC
OS
Std.
95% CI
Low
50.2
2.8 44.6 55.86
High
47.0
2.7 41.8 52.3
0.001 High
Table 3 Cox proportional hazards model analysis of prognostic factors for patients without lymph node metastasis
93.1
0.344
2.4 88.4 97.7
Covariate (observed value)
b
SE
Age, years (B60 vs. [60)
-.813
.269
9.137
.444
.262–.571
.003
Gastric wall invasion (T1/T2 vs. T3/T4)
-.915
.270
11.480
.401
.236–.680
.001
Wald
Exp (B)
95 % CI
P value
p53 (negative vs. positive)
-.376
.238
2.507
.686
.431–1.091
.113
Tumor size (B6 cm vs. [6 cm)
-.219
.276
.630
.803
.468–1.380
.427
PCNA (negative vs. positive) TNM stage (I/II vs. III/IV)
-.783 -.178
.593 .366
1.742 .238
.457 .837
.143–1.462 .408–1.715
.457 .626
pACC (negative vs. positive)
.560
.233
5.751
.1.750
1.108–2.764
.016
Cancer is characterized with uncontrolled cell growth, along with increased synthesis of DNA, protein, and fatty acid. ACCs are rate-limiting enzymes in de novo fatty acid synthesis. Upregulation of ACC in human cancer made it a potent target for caner intervention [18]. In the present study, we observed a strong expression of ACC in the nuclear of GC cells. This phenomenon is in line with previous studies showing overexpression of ACCA in breast, prostate, and liver carcinoma [10–12]. In normal
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P
gastric mucosa, however, we found that ACC protein was expressed both in nuclear and cytoplasm of the epithelium cells. This data suggested that nuclear translocation of ACC increased fatty acid synthesis to meet the demand of gastric cells growth and proliferation. Inconsistent to scattered staining in the cytoplasm of epithelial cells in breast duct [19], we observed intensively expression of ACC in these non-neoplastic tissues. Specifically, we only found a slight increase, not significant, of ACC expression
Med Oncol (2014) 31:15 Fig. 6 Increased expression of pACC by metformin treatment inhibits gastric cancer cell proliferation and colony formation. Gastric cells (MGC823 and MKN28) were treated with metformin (0, 10, 50 mM) for 48 h, and Western blotting (a, d) was used to detect the expression of pACC; (B, E) MGC823 and MKN28 cells were incubated with different concentration of metformin, and CCK8 assay was performed at 24, 48, 72 h; *P \ 0.05, compared with untreated group or 10 mM metformin-treated group. (c, f) Gastric cancer cells were grown in six-well plates and incubated with metformin (0, 10, 50 mM) for 2 weeks. The numbers of the cell colonies ([50 cells) were obtained and calculated as: colonies/ 500 9 100. *P \ 0.05; **P \ 0.05, compared with untreated group
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BGC823
A
15
MKN28
D pACC -actin
0mM
10mM
0mM
50mM
B
10mM
50mM
Metformin
E
*
C
*
F
*
** ** ***
in GC cells compared with that in normal cells. Interestingly, ACCA silencing results in apoptotic cell death in breast, colon, and prostate cancer cells by decreasing fatty acid synthesis, but has no effect on the growth and survival of non-malignant cells [13]. Similarly, ACC inhibitors have no significant cytotoxicity to premalignant BPH-1 prostate cells [20]. Therefore, although ACC expression can be observed in gastric mucosa, the overexpression of ACC in GC cells leads to a notion that ACC may be an attractive and potential target for cancer therapy. Acetyl-CoA carboxylase (ACC) activity is balanced via feedforward allosteric activation by citrate and feedback inhibition by reversible phosphorylation and inactivation. Since upregulation of ACC leads to cell growth and survival, inactivation of ACC by adenosine monophosphatedependent protein kinase (AMPK) results in inhibition of cell proliferation and tumor growth. In the present study, pACC protein expression was lost in 41.2 % of GC specimens, significantly lower than that in normal mucosa (100 %). Cancer cells are characterized by dysregulated
epithelial differentiation. Poorly differentiated tumors have a higher frequency of recurrence and metastasis than welldifferentiated tumors do [16]. Our data revealed a significantly higher expression of inactive form of ACC (pACC) in well-differentiated tumors than that in poorly differentiated tumors, suggesting increased activity of ACC in dedifferentiated tumors. The status of disease stage and lymph node metastasis remains the most important determinants in designing treatment strategies and predicting the outcome of patients with GC [3, 21–24]. Here, we presented that pACC expression decreased with disease stage increased and lymph node metastasized, further supporting the critical role of ACC in the development and progression of GC. Although several clinicopathological factors (TNM stage and LN metastasis) have been found to be independent prognostic factors for GC patients, diverse clinical outcome of these patients has been observed in the same stage. Fortunately, additional molecular markers help to distinguish this difference [17, 25–28]. In addition
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to previously described markers, our data showed that high pACC expression is a potential molecular marker for predicting outcome in patients with resected primary GC. Our univariate analysis showed that high expression of pACC is strongly correlated with better survival rates. This notion was supported by other study that median overall survival was longer in patients with pACC positive than those with pACC negative in lung adenocarcinoma [14]. However, inconsistent with that study, pACC is not an independent prognostic factor. To further determine the prognostic role of pACC in GC, we next analyzed the survival difference of pACC high and low expression according to disease stage and lymph node metastasis. Interestingly, patients with high pACC expression had a better outcome at stage I/II, not stage III/IV, than those patients with absent/low pACC expression. Moreover, high pACC expression is an independent prognostic factor for GC patients without regional lymph node metastasis, not for those with nodal metastasis. This notion is also supported by previous study that ACC was expressed at higher levels in situ duct or lobular breast carcinoma [19]. These findings suggested that pACC downregulation is an important event in initiation of gastric tumorigenesis and is a useful biomarker for predicting outcome of patients at early stage. Mechanistically, ACC activity in mammalian cells is regulated by several genes, including AMP-activated kinase (AMPK), protein kinase A (PKA), and BRCA1 [7]. Among these regulators, AMPK appears a key regulator of ACC activity, inhibiting fatty acid synthesis and inducing apoptosis by phosphorylating ACC [7, 29]. In the present study, pACC upregulation induced by metformin, an activator of AMPK, leads to significant inhibition of cell proliferation and colony formation. This notion is clearly supported by a recent study that pACC expression was significantly increased in tumors after treated by metformin, along with decreased tumor growth and metastatic spread of ovarian cancer [30]. Thus, better understanding and targeting ACC/pACC is very essential for GC prevention and treatment. In summary, our results showed that ACC was frequently activated, while pACC was frequently lost in human GC. Moreover, low/absent expression of pACC was associated with advanced tumor stage, the presence of nodal metastasis, and poor outcome for GC patients, especially for those without lymph node metastasis. These findings further suggest that ACC/pACC could be a promising molecular target for the prevention and treatment of GC. Acknowledgments This work was partly sponsored by Natural Science Foundation of China (30901794) and Shanghai Pujiang Program (13PJD002).
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Med Oncol (2014) 31:15 Conflict of interest We declare no conflicts of interest with any other person or units.
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