Med Oncol (2011) 28:94–104 DOI 10.1007/s12032-009-9408-4
ORIGINAL PAPER
Phosphorylated p27Kip1 on Thr157 is an important prognosis in human hepatocellular carcinoma in vivo and in vitro Song He • Mudan Lu • Wenqun Xue • You Wang • Yueming Zhao • Shangfeng Gao • Qing Ke • Yonghua Liu • Peng Li • Xiaopeng Cui • Chun Cheng • Aiguo Shen
Received: 25 March 2008 / Accepted: 23 October 2008 / Published online: 28 January 2010 Ó Springer Science+Business Media, LLC 2010
Abstract The p27Kip1 cyclin-dependent kinase inhibitor is a negative regulator of cell cycle progression in G1 phase; recent studies suggested that oncogenically activated kinase Akt/PKB can also phosphorylate p27kip1 at T157 inducing its relocalization to the cytoplasm. To evaluate the significance of p-p27 Thr157 and PI3K pathway in hepatocellular carcinoma (HCC), we studied 51 hepatocellular carcinomas along with corresponding nontumoral tissue and the HCC cell lines. Immunohistochemistry and western blot analysis suggested that p-p27 Thr157 was overexpressed in HCC, which was positively correlated with proliferation marker Ki-67. Correlation analysis was performed among immunohistochemistryassessed level of p-p27 Thr157, survival, and major clinical and pathological variables. Overexpressed p-p27 Thr157
was correlated with histological differentiation (P \ 0.05). Univariate analysis showed that p-p27 Thr157 and Ki-67 expression were correlated with tumor-specific survival. In a multivariate analysis, p-p27 Thr157 and Ki-67 protein expression were proved to be an independent prognostic for HCC. While in vitro, treatment of LY294002 and transduction of mutant p27 (T157A) could diminish the expression of p-p27 Thr157 protein and arrest cells growth. Our results suggested that p-p27 Thr157 protein expression may be a favorable independent poor prognostic parameter for HCC. Gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K and transduction of mutant p27 (T157A) to down-regulate p-p27 Thr157 expression could be developed for the management of HCC.
Song He, Mudan Lu, and Wenqun Xue contributed equally to this work.
Keywords Human hepatocellular carcinoma (HCC) Ki-67 P-p27 Thr157 Prognosis PI3K/Akt SMMC-7721 HepG2
S. He M. Lu Y. Wang Y. Zhao S. Gao Q. Ke Y. Liu C. Cheng (&) A. Shen Department of Pathology, Affiliated Cancer Hospital of Nantong University, Medical College of Nantong University, 226001 Nantong, China e-mail:
[email protected] A. Shen (&) The Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University, 19 Qi-xiu Road, 226001 Nantong, Jiangsu, China e-mail:
[email protected]
Abbreviations HCC Human hepatocellular carcinoma IHC Immunohistochemistry CDK2 Cyclin-dependent kinase 2 CDK4 Cyclin-dependent kinase 4 CDKI Cyclin-dependent kinase inhibitor
P. Li X. Cui Department of General Surgery, The Affiliated Hospital of Nantong University, 226001 Nantong, China
Introduction
M. Lu W. Xue Y. Wang Wuxi Maternal and Child Health Hospital affiliated Nanjing Medical University, 214002 Wuxi, China
Human hepatocellular carcinoma (HCC) is the most common form of malignancy in humans worldwide, representing 40% of all of the cancers in Southeast Asia, Japan,
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and Africa and 2–3% of all of the cancers in the United States [reviewed in 1, 2]. About 82% of cases (and deaths) are in developing countries (55% in China alone). The areas of high incidence are sub-Saharan Africa, eastern and southeastern Asia, and Melanesia. Unfortunately, most of the cases of HCC are not curable because extensive resection is not possible, there is extensive liver dysfunction caused by cirrhosis, and/or the disease is rarely identified at an early stage. Genomic amplification of oncogenes and inactivation of tumor suppressor genes are frequently associated with cancer progression. It is necessary to know the molecular mechanism of HCC. It is well known that carcinogenesis resulted from disorder of the cell cycle. A number of molecules play an important role in regulating cell cycle progression. Cell cycle progression depends on the activity of kinase complexes composed of cyclins and cyclin-dependent kinases (CDKs). The CDK activity is suppressed in part by association with CDK inhibitors, including the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/ Kip family (p21Waf1/Cip1, p27Kip1, and p57Kip2) [3]. P27Kip1, a Cip/Kip1 member, was identified as a CDK inhibitor that causes G1 arrest by inhibiting the activities of G1 cyclin/CDKs. The activity of p27Kip1 is controlled by its concentration, its distribution among different cellular complexes, its cellular localization [4, 5]. In many human cancers, reduced p27Kip1 expression is frequently observed [6]. The reduced expression of p27Kip1 is reported to correlate with tumor progression and poor patient survival [7, 8]. Thus, p27Kip1 may participate in tumor suppression by inhibiting abnormal cell cycle progression. However, recent results manifested that cytoplasmic sequestration of p27kip1 is a mechanism whereby cancer cells overcome p27kip1-imposed growth inhibition and has been reported for colon [9], esophagus [10], thyroid [11], ovarian [12], and breast carcinomas [13–15]. These studies showed that oncogenically activated kinase Akt/PKB can phosphorylate p27kip1 at T157 inducing its relocalization to the cytoplasm [13, 15]. Aktmediated cytosolic accumulation of p27kip1 is critical for Akt mitogenic signaling. Akt-mediated exclusion of wildtype p27kip1 from the nuclear compartment results in activation of nuclear CDK2 and cell cycle progression, whereas a mutation at T157 confers resistance to Aktmediated p27kip1 nuclear exclusion and impairs Aktdependent rescue of p27kip1-induced cell cycle arrest [16]. Hence, sufficient evidence has accumulated to suggest that cytoplasmic relocalization of p27kip1 might facilitate the tumor development. The presence of cytoplasmic p27kip1 (induced by phosphorylation at T157) has been shown to predict poor prognosis in breast cancer [13, 15]. It is not clear whether this also applies to other tumor types such as HCC with high Akt activity and high expression p-p27
95
Thr157 or whether this regulation is specific for breast cancer. Iorenino [17] posed that in their series of HCCs, reliable cytoplasmic p27kip1 staining was observed in 44% of cases. Biochemical studies using subcellular fractionation of protein lysates are needed to confirm the role of cytoplasmic localization in preventing p27kip1 function in HCC. We also found that p27kip1 overexpressed in both hepatocarcinoma cells and normal liver tissues. In normal liver tissues, p27kip1 localized in the nucleus, while in HCC most of p27kip1 confined to cytoplasma. Localization of p27kip1 determined its function. Moreover, many researches suggested that p27kip1 was phosphorylated by PKB/Akt on Thr157 and then detained p27kip1 in the cytoplasma. So studying the expression of p-p27 Thr157 in HCC may provide a more effective molecular target than total p27kip1. In the study, we investigated the expression of p-p27 Thr157 and Ki-67 in HCC and their clinicopathologic significance. We also examined the relationship between expression of p-p27 Thr157 and HCC survivals. More importantly, we studied the effect of PI3K inhibitor LY294002 and transfection with mutant p27 (T157A) on cell cycle in vitro to discover a new therapeutic way to HCC.
Materials and methods Patients and tissue samples HCC tissues were obtained from 51 patients. All underwent hepatic surgical resection without postoperative systemic chemotherapy at the Surgery Department, the Affiliated Hospital of Nantong University. The main clinical and pathologic variables of the patients are shown in Table 1. Forty-two patients were men and nine were women; their ages ranged from 35 to 79 (mean = 65.49 years). Thirtysix patients were positive for HBV surface antigen, 41 were positive for cirrhosis. Histological grades were classified to well differentiated (grade I; n = 6), moderately differentiated (grade II; n = 29), and poorly differentiated (grade III; n = 16). The follow-up time was 5 years for 45 patients ranging from 1 to 60 months (mean = 40.93 months). None of the patients received postoperative adjuvant therapy. Tissue samples were immediately processed after surgical removal. For histological examination, all tumorous and surrounding nontumorous tissue portions were fixed in formalin and embedded in paraffin. Protein was analyzed in eight snap-frozen tumorous and adjacent nontumorous tissue samples that were stored at -80°C. Control nontumorous tissue samples were prepared from 10 normal livers
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96 Table 1 P-p27 Thr157, Ki67 expression, and clinicopathological parameters in 51 HCC specimens
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Parameters
Total
P-p27 Thr157 expression Low B0.24
P
High [0.24
Ki-67 expression Low B0.42
P
High [0.42
Age (year) B45
15
9
6
[45
36
23
13
42
29
13
9
3
6
0.794
8
7
18
18
29
13
3
6
0.273
Gender Male Female
0.053
0.053
Histological grade Well
6
5
1
5
1
Mod
29
21
8
0.043
18
11
Poor Metastasis
16
6
10
3
13
Positive
14
7
7
Negative
37
25
12
B5
30
19
11
[5
21
13
8
0.247
4
10
22
15
0.004
0.049
Tumor size(cm) 0.917
14
16
12
9
18
18
8
7
20
21
6
4
0.461
HBsAg (?)
36
27
27
(-)
15
12
10
Positive
41
24
17
Negative
10
2
7
B50
24
16
8
[50
27
16
11
0.794
0.828
Cirrhosis
Statistical analyses were performed by the Pearson v2 test. P \ 0.05 was considered significant
0.390
AFP(ng/ml)
that were obtained at surgery for other conditions. Informed consent was obtained from all patients. Immunohistochemistry (IHC) Tissue sections (4 lm) were cut, placed on APES-pretreated slides, deparaffinized, rehydrated through graded alcohol, and quenched in 3% hydrogen peroxide. Antigen retrieval was performed by microwave heating at high power (750 W) in 10 mM sodium citrate buffer (pH 6.0) for three cycles of 5 min each. After blocking with normal serum for 1 h at room temperature, the sections were incubated overnight at 4°C with anti-human p-p27 Thr157 rabbit polyclonal antibody (diluted 1:100; R&D), anti-Ki67 mouse monoclonal antibody (diluted 1:100; clone 7B11; Zymed Laboratories, San Francisco, Calif., USA), antip27kip1 (sc-528, 1:100; Santa Cruz Biotechnology). Negative control slides were also processed in parallel using a nonspecific immunoglobulin IgG (Sigma Chemical Co., St. Louis, MO) at the same concentration as the primary antibody. The positive immunostaining of reactive lymph
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0.188
0.585
16
8
10
17
0.035
node breast carcinoma specimens represented an internalpositive control for preservation of antigenicity in the sections examined. All slides were processed using the peroxidase-antiperoxidase method (Dako, Hamburg, Germany). Diaminobenzidine was used as the final chromogen, and Gill’s hematoxylin was used for counterstaining. Human lymph node tonsil tissue was used as a positive control because this contains lymphoid tissue with variable proliferative activity. In the mantle (peripheral) zone of the follicle, the cells are mainly quiescent, whereas cells in the germinal centers are highly proliferative. All of the immunostained sections were evaluated in a blinded manner without knowledge of the clinical and pathological parameters of the patients. For assessment of p-p27 Thr157 and Ki-67, five highpower fields in each specimen were selected randomly, and nuclear (cytoplasma) staining was examined under high power magnification. More than 500 cells were counted to determine the LI, which represented the percentage of immunostained cells relative to the total number of cells [18]. In half of the samples, staining was
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repeated twice to avoid possible technical errors, but similar results were obtained in these samples. The above procedures of evaluation were performed by M.D.L. The obtained results were confirmed other investigator (Y.W) using a multihead microscope, and a consensus was achieved. Cell culture and cell treatment 7721 and HepG2, human hepatocarcinoma cell line, were obtained from the Institute of Cell Biology, Academic Sinica and cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin in 5% CO2 at 37°C. Cells were added in 6-well dishes; 24-well dishes; and 96-well dishes 3 9 103 per well. The medium was replaced 24 h later using fresh medium containing various concentrations of LY294002 (SIGMA) or for transduction. HCC cells suspended in medium contained DMSO were used as a control.
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CDK4 (1:500; Santa Cruz Biotechnology); anti-cyclin D1 (1:500; Santa Cruz Biotechnology); anti-cyclin D1 (1:500; Santa Cruz Biotechnology). Finally, horseradish peroxidaseconjugated secondary antibody was added for an additional 2 h and the blots were developed using enhanced chemiluminescence detection system (Pierce). After the chemiluminescence was exposed to X-ray films, the films were scanned using a Molecular Dynamics densitometer (Imaging Technology, Ontario, Canada). Values are responsible for at least three independent reactions. Cell cycle analysis SMMC7721 and HepG2 cells were treated with the PI3K inhibitor LY294002 at different concentration. After 24 h, cells were trypsinized, fixed with methanol, and their nuclei were labeled with propidium iodide as described [19]. A total of 2 9 104 propidium iodide-positive nuclei were gated and analyzed in a FACS/Calibur Flow Cytometer (Becton–Dickinson).
Immunoblot analysis Statistical analysis Tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M Tris–HCl pH 7.5, 1% Triton X-100, 1% NP-40 (nonidet p-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, 10 lg/ml leupeptin, 10 lg/ml aprotinin, and 1 mM PMSF, then centrifuged at 10,000 g for 30 min to collect the supernatant. Protein concentrations were determined with a Bio-Rad protein assay (BioRad, Hercules, CA, USA). The supernatant diluted in 29 SDS loading buffer and boiled. Proteins were separated with SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% dried skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20). After 2 h at room temperature, the filters were washed by TBST for three times and then incubated overnight with polyclonal antibody against using the primary antibodies described later and horseradish peroxidase-linked IgG as the secondary antibodies. Immunoreactive bands were visualized by chemiluminescence (NEN Life Science Products, Boston, MA). Antibodies used were as follows: total Akt and P-S473 Akt (Cell Signaling, Beverly MA); anti-PCNA (SC-56; 1:1000; Santa Cruz Biotechnology); A phospho-specific Thr-157 p27 antibody was from R&D (1:1000); A phospho-specific Ser10 and Thr-187 p27 antibody was from Zymed Laboratories (1:200) anti-Tubulin (1:500; Santa Cruz Biotechnology); anti-b-actin (1:4000; Sigma); anti-p21 (1:500; Santa Cruz Biotechnology); anti-p16 (1:500; Santa Cruz Biotechnology); anti-p27 (1:500; Santa Cruz Biotechnology); anti-CDK2 (1:500; Santa Cruz Biotechnology); anti-
Statistical analysis was performed using the Stat View 5.0 software package. The association between Ki-67 and p-p27 Thr157 expression and clinicopathological features was analyzed using v2 test. Ki-67 and p-p27 Thr157 expression in human hepatocellular carcinoma (HCC) was studied using the Spearman rank correlation test because the data were not normally distributed. For analysis of survival data, Kaplan–Meier curves were constructed, and the log-rank test was performed. Multivariate analysis was performed using Cox’s proportional hazards model with P \ 0.05 considered statistically significant. The results of the HCC cells are expressed as the mean ± SE. Statistical analyses of the data between LY294002-treated cells and DMSO-treated cells were done by Student’s t-test. P \ 0.05 was considered statistically significant.
Results The expression of p-p27 Thr157 and its correlation with clinicopathologic variables in HCC Making working a phosphorylated antibody on paraffinembedded clinical samples, human lymph node tonsil tissue was used as a positive control. P27Kip1 staining in human lymph node was shown primarily in the resting cells of the mantle zone, with little or no staining in the center of the follicle (Fig. 1 A a). This was an opposite pattern to the one observed with p-p27 Thr157, in which the positive staining was seen in the rapidly dividing cells in the center
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98
of the follicle, whereas cells in the mantle zone showed very little p-p27 staining (Fig. 1A b). The specific immunodetection of p27 and p-p27 Thr157 antibodies was tested against whole-tissue extract from HCC by immunoblotting. As shown in Fig. 1B, when total protein extract from HCC were separated, blotted, and then probed with p-p27 Thr157, and p27Kip1 antibodies, only a single band was recognized for either of the antibodies (27 kDa, p27) (Fig. 1B). There were no or low p-p27 Thr157 expression in normal human hepatic tissue (Fig. 1C). So our data focused on the p-p27 Thr157 expression in HCC. P-p27 Thr157 expression in HCC was scored as positive when strong cytoplasma and (or) nuclei (Fig. 1D). Its labeling index ranged from 2.02% to 79.16%. The mean percentage of positive cell was 24%. In p-p27 Thr157 high expression, 1 was Histological grade Well, 8 were Histological grade Mod and 10 were Histological grade Poor (details in Table 1). We evaluated the association of Ki-67 and p-p27 Thr157 expression with clinicopathologic variable. No differences in p27Kip1 expression were found according to gender, age, metastasis, tumor size, HBsAg, cirrhosis and AFP, but p-p27 Thr157 expression was significantly associated with Histological grade (P = 0.043) (Table 1). Furthermore, in most specimens, the proportion of p-p27 Thr157 positive tumor cells was similar to the proportion of Ki-67 positive tumor cells. A positive correlation between p-p27 Thr157 expression and Ki-67 based proliferative activity was found, with a correlation coefficient of 0.401 (P \ 0.01; Fig. 2). To explore the significance of p-p27 Thr157 in human hepatocarcinoma progression, we further examined p-p27 Thr157 protein expression using immunoblotting methods in 8 novel specimens, we also showed that strong p-p27 Thr157 expression was altered in paired normal liver and HCC biopsy samples, with a dramatically increased expression in six of eight tumors when compared with the adjacent normal tissue, which showed low p-p27 Thr157 expression (Fig. 1E). Figure 1B also showed a dramatically increased PCNA level in six of eight tumors when compared with the paired normal tissue, which displayed little PCNA expression. Prognostic value of P-p27 Thr157 Proliferation is found throughout the cell cycle (G1, S, G2, and M phases) but not in resting (G0) cells [20]. Studies suggest that the Ki-67 is considered to be the clinical value of proliferation markers for tumor malignancy [21–23]. We detected a positive correlation between p-p27 Thr157 expression and Ki-67, so we hypothesized whether p-p27 Thr157 was correlated with prognosis. Survival analysis of 45 patients with information available on clinical follow-
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up. Based on IHC positivity, patients were divided into two groups: high expressers and low expressers by p-p27 Thr157 mean value. Concerning survival, only 14 of 27 (52%) patients in the lower-expresser group died of disease versus 6 of 18 (33%) in the high-expresser group (Table 2). The Kaplan–Meier survival curves of low versus high expressers of p-p27 Thr157 showed a highly significant separation (P \ 0.05 Fig. 3a). Moreover, patients with the phenotype of p-p27 Thr157 (?)/Ki-67(?) had the worst overall survival. The 5-year overall survival rate of patients with the phenotype of p-p27 Thr157 (?)/Ki-67(?) was 28.57% less than others (Fig. 3b). When a multivariate Cox proportional hazard model was constructed (including gender, age, tumor grade, tumor size, liver metastasis, AFP, Ki-67 expression and p-p27 Thr157 expression), p-p27 Thr157 and Ki-67 were the strongest independent predictor of survival (P \ 0.05), the second predictor being liver metastasis (Table 3). Reduced p-p27 Thr157 expression and arrest cell cycle in HCC cell lines by PI3K inhibitor LY294002 The current study investigated that p-p27 Thr157-positive tumor cells was similar to the proportion of Ki-67 positive tumor cells and p-p27 Thr157 overexpression was associated with poor prognosis of HCC. P-p27 Thr157 expression may be considered as a malignancy characteristic and a strongest independent predictor of survival. Moreover, we detected that p-Akt overexpressed in HCC than normal liver tissue (Fig. 1E). Nakanishi et al. [24] proposed that the critical involvement of Akt phosphorylation in the aggressiveness of HCC and has identified Akt phosphorylation as a significant risk factor for early disease recurrence and poor prognosis. Many studies have suggested that Akt directly phosphorylates p27Kip1 on Thr157. Yet, we hypothesized that elimination of the expression of p-p27 Thr157 in HCC cells could inhibit cell proliferation. We used the PI3K inhibitor LY294002 to treat HCC cell lines HepG2 [25] and SMMC7721. Cells were treated with LY294002 or DMSO 24 h and then collected. P-p27 Thr157 expression in HepG2 treated with LY294002 was decreased significantly in comparison with controls (P \ 0.05). Levels of phosphorylated Akt (p-Akt) proteins were much lower in treated animals relative to cells (Fig. 4a left panel, b left panel). The same results were also obtained in SMMC7721 (Fig. 4a right panel, b right panel). However, the expression of p-p27 Ser10 and p-p27 Thr187 were not changed by the PI3K inhibitor (Fig. 4a). These results demonstrated PI3K inhibitor LY294002 reduced p-p27 Thr157 expression but not p-p27 Ser10 and p-p27 Thr187 expression. The proliferative activity was monitored by flow cytometric analyses of propidium iodide incorporation. At 50 lM or higher concentrations of
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Fig. 1 (A) Staining in human lymph node. Immunohistochemistry for Ki67 and p27kip1 were performed as described in the ‘‘Materials and Methods’’. The high expression of Ki-67 in the germinal center centroblasts (c) and the high expression of p27kip1 in the interfollicular (a); however, p-p27 Thr157 localized mostly in the germinal center centroblasts (b) (940). (B) Detection of p27 (lane 1), and p-p27 Thr157 (lane 2) in whole-tissue extract from a HCC (27 kDa, p27). Protein lysates were separated and blotted onto a polyvinylidene difluoride membrane; immunoblotting (IB) was performed with the indicated antibodies. MW, molecular weight. (C) The expression of p-p27 Thr157 and Ki-67 in HCC by immunochemistry. The expression of Ki-67 (a), p27 (b), and p-p27 Thr157 (c) in the normal liver tissue beside liver carcinoma (940). (D) Low expression of p-p27 Thr157 (d) is correlated with low Ki-67 (b) in the same HCC specimen while high expression of p-p27 Thr157 (c) is correlated with high Ki-67 (a) in the same HCC specimen (940). (E) Comparison of p-p27 Thr157, PCNA, p-Akt and Akt in HCC tumor cell. Details of the experiments are given in ‘‘Materials and Methods’’
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Med Oncol (2011) 28:94–104 Table 2 Survival status and clinicopathological parameters in 45 HCC specimens Total
Survival status Alive
P
Dead
Age B45
15
8
7
[45 Gender
30
12
18
Male
38
17
21
7
3
4
Female
0.396
0.629
Histological grade
Fig. 2 Relationship between Ki-67 proliferation index and p-p27 Thr157 expression in HCC. Scatterplot of Ki-67 versus p-p27 Thr157 with regression line showing a correlation of the two cell cycle regulators using Spearman’s correlation coefficient
LY294002, complete growth arrest was evident by 24 h. Similar results were observed in the flow cytometric analyses in SMMC7721 (Fig. 4b) and HepG2 (Fig. 4c). Again, C50 lM LY294002 was sufficient to cause a significant decrease in cells entering S phase. Propidium iodide staining indicated that the LY294002-treated cells were arrested predominantly at the G1 phase of the cell cycle. These dose-dependent proliferation results also showed that, under our experimental conditions, 50 lM LY294002 could induce cell cycle arrest by 24 h but did not trigger noticeable cell death.
Well
5
4
1
Mod
25
11
14
Poor/undiff.
15
5
10
Negative
31
17
14
Positive
14
3
11
B5
28
13
15
[5
17
7
10
14 31
5 15
9 16
0.428
Negative
9
5
4
0.453
Positive
36
15
21
B50
23
11
12
[50
22
9
13
Low expression
22
13
9
High expression
23
7
16
Metastasis
Negative Positive Cirrhosis
AFP (ng/ml)
Furthermore, we analyzed whether mutant p27 (T157A) arrested cell cycle than control. The article [26] progressed by our laboratory showed the successful of the transduction of mutant p27 (T157A). Flow cytometric analysis showed that the proportion of cells in G1 arrest increased and the proportion of cells in S phase decreased 36 h after induction of p27 expression [26]. P27 (T157A) clones showed more extensive G1 arrest (70.89% in p27 mutant and 50.83% in control, respectively).
p27
123
0.731
HBsAg
Ki-67
Hepatocellular carcinoma (HCC) is the most common hepatic malignancy worldwide, especially in South-east Asia. Approximately 80% of HCC patients have been associated with liver cirrhosis [27]. Even after comprehensive therapies with surgical excision, chemotherapy, ethanol injection, radiofrequency, or cryotherapy, this
0.037
Tumor size(cm)
Transduction of mutant p27 (T157A) inhibited cell growth of SMMC7721
Discussion
0.239
Low expression
22
5
17
High expression
23
15
8
Low expression
29
14
13
High expression
16
6
12
0.641
0.053
0.004
P-p27 Thr157 0.051
Statistical analyses were performed by the Pearson v2 test. P \ 0.05 was considered significant
tumor shows a high percentage of recurrence and metastasis, and the mean survival of the patients is still short, compared to other major solid tumors [28]. A deeper understanding of the molecular events associated with the HCC is necessary. Hyper-proliferation of cells conduced to carcinogenesis. The p27Kip1, as CKI, belongs to the second group of CDKI, the Cip/Kip family, is one of the most important cell cycle inhibitor, and its expression has the character of a tumor suppressor gene. Loss of the p27Kip1 protein expression may result in tumor development and/or
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101 Table 3 Contribution of various potential prognostic factors to survival by Cox regression analysis in 45 HCC specimens Hazard ratio
95% confidence interval
P
Age (year)
1.574
0.657–3.773
0.2940
Gender
0.906
0.310–2.644
0.8554
Tumor grade
1.866
0.981–3.547
0.0521
Metastasis
2.960
1.327–6.605
0.0109
Tumor size
1.109
0.497–2.470
0.8009
HBsAg
0.636
0.281–1.441
0.2908
Cirrhosis AFP
1.358 1.066
0.465–3.964 0.486–2.337
0.5623 0.8726
Ki-67
1.058
1.027–1.091
0.0001
P-p27 Thr157
1.029
1.011–1.048
0.0026 2
Statistical analyses were performed by the Pearson v test. P \ 0.05 was considered significant
Fig. 3 Kaplan–Meier survival curves for low p-p27 Thr157 expression versus high p-p27 Thr157 expression (a) in 45 patients of HCC showed a highly significant separation (P = 0.03, log-rank test). Overall survival curves according to p-p27 Thr157/Ki-67 expression (b)
progression [29]. Two different mechanisms have been implicated in p27kip1 inactivation during the process of human carcinogenesis: down-regulation of its expression and exclusion from nuclear compartment [15]. Reduction
in the level of p27kip1 protein contributes to tumor development by allowing an increase in nuclear CDK2 activity, which results in an increased rate of cell proliferation [6]. Many results demonstrated that the function of p27kip1 is determined by its localization. Cytoplasmic mislocalization of p27kip1 may ensure when its nuclear import is impaired or its export facilitated, and this is believed to affect the growth-inhibitory function of p27kip1 [11]. The nuclear/cytoplasmic distribution of p27kip1 is regulated by phosphorylation of one of three key amino acid residues: S10, T157, and T198. Phosphorylation of S10 is accomplished by hKIS and has been reported to facilitate nuclear export whereas phosphorylation of p27kip1 at T157 is accomplished by protein kinase B/Akt and is known to cause retention of p27kip1 in the cytoplasm [13, 14]. The interaction between Akt and p27kip1 and consequent phosphorylation at T157 occurs in the cytoplasmic compartment and that this modification impairs the association of p27 with importin-a, thus preventing its re-entry into the nucleus [13]. P-p27 Thr157 expression in breast carcinoma has been proved; however, it is unclear whether p-p27 Thr157 is overexpressed in HCC. This is the first report that p-p27 Thr157 is overexpressed on HCC. Moreover, we found a positive correlation between p-p27 Thr157 and Ki-67 expression (P \ 0.05) in HCC. Using immunoblotting methods, we also showed that strong p-p27 Thr157 expression was altered in paired normal liver and HCC biopsy samples, with a dramatically increased expression in six of eight tumors when compared with the adjacent normal tissue, which showed low p-p27 Thr157 expression. This is consist with Giuseppe Viglietto’s [15] investigation that p-p27 Thr157 was overexpressed in breast cancer. More importantly, our works reported that p-p27 Thr157 expression was significantly associated with Histological grade (P = 0.043). P-p27 Thr157 expression is positive with nuclear proliferation antigen Ki-67. P-p27 Thr157
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Fig. 4 a Down-regulation of p-p27 Thr157 and inhibition of Akt activation by PI3K inhibitor in hepatocarcinoma cells. SMMC7721 and HepG2 were collected after 24 h after treatment of LY294002. The protein levels of p-p27 Thr157, Akt/PKB, phospho-Akt/PKB p-p27 Ser10, p-p27 Thr157, and p-p27 Thr187 assessed by western blot analysis (a) and quantitated by densitometric analysis (b) of protein bands. Equal loading was confirmed by stripping the blot and reprobing it for b-actin. Representative experiment repeated three times with similar results. Columns mean relative density normalized
to b-actin from three experiments; bars SE. *, #, P \ 0.05. Details of the experiments are given in ‘‘Materials and Methods’’. Dosedependent effects of LY294002 on proliferation of SMMC7721 and HepG2 cells. Cell cycle analysis of the SMMC7721 (C) and HepG2 (D) cells treated with various dosages of LY294002. Exponentially growing HCC cells were incubated with various concentration of LY2940002 as indicated and harvested at 24 h for cell cycle analysis. Details of the experiments are given in ‘‘Materials and Methods’’
expression can be considered to be an independent prognosis in HCC. Furthermore, Fig. 4a showed that p-p27 Thr157 could be decreased by PI3K inhibitor LY294002. The PI3K family of enzymes is well characterized with respect to promotion of cellular growth, survival, and suppression of apoptosis in cancer cells [30–32]. PI3K inhibition by LY294002 has been shown to cause apoptosis in various human cancer cells in vitro [33–35]. Recent study suggested that Akt can also phosphorylate p27kip1 at T157 in breast carcinoma. Our results suggested that in HCC p-p27 Thr157 and Akt overexpressed in vivo and in vitro. PI3K inhibitor LY294002 can depress HCC cell lines HepG2 and SMMC7721 growth. This has certified by Caro AA [36]. The p27Kip1 protein is a member of the cdk inhibitory proteins, with an important role in both the initiation and timely exit of cells from the cell cycle in response to antimitogenic signals [37, 38]. P27Kip1 has proven to be a candidate as a cancer therapy target [39, 40]. There is considerable evidence that the inactivation of p27Kip1 is a fundamental step in the development of malignancies [41, 42]. The cytoplasmic sequestration of p27Kip1 in tumors has been identified only recently as a mechanism whereby cancer cells promote carcinogenesis in humans [37]. Because the growth-inhibitory activity of p27Kip1 depends on its nuclear localization, cytoplasmic mislocalization can effectively inactivate p27Kip1 [10, 11, 43–46], as cytoplasmic p27Kip1 is partitioned from its nuclear cyclin–cdk
target [37]. Cytoplasmic p27Kip1 appears to directly correlate with a poor prognosis and advance tumor grade of both esophagus and breast carcinomas [10, 13–15]. These findings suggest that mislocalization of p27Kip1 may also be the result of a variety of different oncogenic assaults [46]. Our results confirmed that transduction of mutant p27 (T157A) arrested cell cycle in G1 than control. In conclusion, this study suggests that p-p27 Thr157 is a useful prognosis variable for HCC. And that the inhibition of PI3K may increase HCC patients’ survival. Although this study reports a limited predictive value of p-p27 Thr157 and Ki-67 as prognostic factors in HCC, it has important distinctive features. First, Ki-67 nuclear antigen is the identification of protein markers responsible for the regulation of cell proliferation. P-p27 Thr157 expression is positively correlated with Ki-67 expression. P-p27 Thr157 expression may represent the malignancy of HCC. Secondly, function of p27Kip1 is determined by its localization, the p-p27 Thr157 expression is retained in the cytoplasma by the activated Akt. Thirdly, in vitro, we found that inhibition of the Akt signaling pathway by PI3K inhibitor LY294002 can reduce p-p27 Thr157 then arrest G1 cell cycle by regulating p27Kip1 localization in HepG2 and SMMC7721 cell lines. Fourth, transduction of mutant p27 (T157A) arrested cell cycle in G1 than control. So in clinical study, we can use chemotherapeutics such as PI3K inhibitor, PI3K siRNA, or the mutation of p27Kip1 on Thr157 to treat HCC patients who cannot accept operation.
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Using these chemotherapeutics, or the mutation of p27Kip1 on phosphorylation, patients can accept surgical treatment after tumor shrink. Gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K to downregulate p-p27 Thr157 expression could be developed for the management of HCC. Acknowledgments This work was supported by the National Natural Scientific Foundation of China Grant (No. 30770488 and No. 30872320), Natural Scientific Foundation of Jiangsu Province Grant (No. BK2003035 and No. BK2006547), College and University Natural Scientific Research Programme of Jiangsu Province (No. 03 KJB180109 and No. 04KJB320114), Technology Guidance Plan for Social Development of Jiangsu Province Grant (BS2004526), Health Project of Jiangsu Province (H200632), ‘‘Liu-Da-Ren-Cai-Gao-Feng’’ Financial Assistance of Jiangsu Province Grant (No. 2).
References 1. Akriviadis EA, Llovet JM, Efremidis SC, Shouval D, Canelo R, et al. Hepatocellular carcinoma. Br J Surg. 1998;85:1319–31. 2. Bruix J, Boix L, Sala M, Llovet JM. Focus on hepatocellular carcinoma. Cancer Cell. 2004;5:215–9. 3. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13: 1501–12. 4. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci. 2001;114: 2903–10. 5. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J. 2000;346:561–76. 6. Slingerland J, Pagano M. Regulation of the cdk inhibitor p27 and its deregulation in cancer. J Cell Physiol. 2000;183:10–7. 7. Catzavelos C, Bhattacharya N, Ung YC, Wilson JA, Roncari L, et al. Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat Med. 1997;3:227–30. 8. Mori M, Mimori K, Shiraishi T, Tanaka S, Ueo H, et al. p27 expression, gastric carcinoma. Nat Med. 1997;3:593. 9. Ciaparrone M, Yamamoto H, Yao Y, Sgambato A, Cattoretti G, et al. Localization and expression of p27KIP1 in multistage colorectal carcinogenesis. Cancer Res. 1998;58:114–22. 10. Singh SP, Lipman J, Goldman H, Ellis FH Jr, Aizenman L, et al. Loss or altered subcellular localization of p27 in Barrett’s associated adenocarcinoma. Cancer Res. 1998;58:1730–5. 11. Baldassarre G, Belletti B, Bruni P, Boccia A, Trapasso F, et al. Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells. J Clin Invest. 1999;104:865–74. 12. Masciullo V, Sgambato A, Pacilio C, Pucci B, Ferrandina G, et al. Frequent loss of expression of the cyclin-dependent kinase inhibitor p27 in epithelial ovarian cancer. Cancer Res. 1999;59: 3790–4. 13. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, et al. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med. 2002;8:1153–60. 14. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, et al. PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med. 2002;8:1145–52. 15. Viglietto G, Motti ML, Bruni P, Melillo RM, D’Alessio A, et al. Cytoplasmic relocalization and inhibition of the cyclin-dependent
103
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31. 32.
kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nat Med. 2002;8:1136–44. Fujita N, Sato S, Katayama K, Tsuruo T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14–3-3 and cytoplasmic localization. J Biol Chem. 2002;277:28706–13. Fiorentino M, Altimari A, D’Errico A, Cukor B, Barozzi C, et al. Acquired expression of p27 is a favorable prognostic indicator in patients with hepatocellular carcinoma. Clin Cancer Res. 2000;6: 3966–72. Xu X, Yamamoto H, Sakon M, Yasui M, Ngan CY, et al. Overexpression of CDC25A phosphatase is associated with hypergrowth activity and poor prognosis of human hepatocellular carcinomas. Clin Cancer Res. 2003;9:1764–72. Chakravarthy MV, Abraha TW, Schwartz RJ, Fiorotto ML, Booth FW. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3’-kinase/Akt signaling pathway. J Biol Chem. 2000;275: 35942–52. Cattoretti G, Becker MH, Key G, Duchrow M, Galle J, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol. 1992; 168:357–63. Benjamin DR. Proliferating cell nuclear antigen (PCNA) and pediatric tumors: assessment of proliferative activity. Pediatr Pathol. 1991;11:507–19. Khine MM, Aung W, Sibbons PD, Howard CV, Clapham E, et al. Analysis of relative proliferation rates of Wilms’ tumor components using proliferating cell nuclear antigen and MIB-1 (Ki-67 equivalent antigen) immunostaining and assessment of mitotic index. Lab Invest. 1994;70:125–9. Delahunt B, Farrant GJ, Bethwaite PB, Nacey JN, Lewis ME. Assessment of proliferative activity in Wilms’ tumour. Anal Cell Pathol. 1994;7:127–38. Nakanishi K, Sakamoto M, Yamasaki S, Todo S, Hirohashi S. Akt phosphorylation is a risk factor for early disease recurrence and poor prognosis in hepatocellular carcinoma. Cancer. 2005; 103:307–12. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl4H–1-benzopyran-4-one (LY294002). J Biol Chem. 1994;269: 5241–8. Lu M, Fei M, Cheng C, Wang Y, et al. Mutant p27Kip1 and its potential effect as hepatocellular gene therapy. Arch Med Res. 2008;39:573–81. Ikai I, Yamaoka Y, Yamamoto Y, Ozaki N, Sakai Y, et al. Surgical intervention for patients with stage IV-A hepatocellular carcinoma without lymph node metastasis: proposal as a standard therapy. Ann Surg. 1998;227:433–9. El-Assal ON, Yamanoi A, Soda Y, Yamaguchi M, Igarashi M, et al. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology. 1998;27:1554–62. Lloyd RV, Erickson LA, Jin L, Kulig E, Qian X, et al. p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am J Pathol. 1999;154:313–23. Franke TF, Kaplan DR, Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell. 1997;88:435–7. Toker A, Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997;387:673–6. Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990;61:203–12.
123
104 33. Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, et al. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A. 1992;89:9267–71. 34. Izuishi K, Kato K, Ogura T, Kinoshita T, Esumi H. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy. Cancer Res. 2000;60: 6201–7. 35. Kulik G, Carson JP, Vomastek T, Overman K, Gooch BD, et al. Tumor necrosis factor alpha induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells. Cancer Res. 2001;61:2713–9. 36. Caro AA, Cederbaum AI. Role of phosphatidylinositol 3-kinase/ AKT as a survival pathway against CYP2E1-dependent toxicity. J Pharmacol Exp Ther. 2006;318:360–72. 37. Min YH, Cheong JW, Kim JY, Eom JI, Lee ST, et al. Cytoplasmic mislocalization of p27Kip1 protein is associated with constitutive phosphorylation of Akt or protein kinase B and poor prognosis in acute myelogenous leukemia. Cancer Res. 2004;64: 5225–31. 38. Sgambato A, Cittadini A, Faraglia B, Weinstein IB. Multiple functions of p27(Kip1) and its alterations in tumor cells: a review. J Cell Physiol. 2000;183:18–27. 39. Blain SW, Scher HI, Cordon-Cardo C, Koff A. p27 as a target for cancer therapeutics. Cancer Cell. 2003;3:111–5. 40. Katner AL, Hoang QB, Gootam P, Jaruga E, Ma Q, et al. Induction of cell cycle arrest and apoptosis in human prostate
123
Med Oncol (2011) 28:94–104
41.
42.
43.
44.
45.
46.
carcinoma cells by a recombinant adenovirus expressing p27(Kip1). Prostate. 2002;53:77–87. Yang G, Ayala G, De Marzo A, Tian W, Frolov A, et al. Elevated Skp2 protein expression in human prostate cancer: association with loss of the cyclin-dependent kinase inhibitor p27 and PTEN and with reduced recurrence-free survival. Clin Cancer Res. 2002;8:3419–26. Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, et al. Increased proteasome-dependent degradation of the cyclindependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med. 1997;3:231–4. Jiang Y, Zhao RC, Verfaillie CM. Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34? cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity. Proc Natl Acad Sci U S A. 2000;97:10538–43. Soucek T, Yeung RS, Hengstschla¨ger M. Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc Natl Acad Sci U S A. 1998;95: 15653–8. Orend G, Hunter T, Ruoslahti E. Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorageindependent cells. Oncogene. 1998;16:2575–783. Viglietto G, Motti ML, Fusco A. Understanding p27(kip1) deregulation in cancer: down-regulation or mislocalization. Cell Cycle. 2002;1:394–400.
