Med Oncol (2012) 29:3070–3076 DOI 10.1007/s12032-012-0268-y

ORIGINAL PAPER

Overexpression of RPS6KB1 predicts worse prognosis in primary HCC patients Pin Dong Li • Wen Jie Zhang • Mei Ying Zhang Lin Jing Yuan • Yin Lian Cha • Xiao Fang Ying Gang Wu • Hui Yun Wang

• •

Received: 12 May 2012 / Accepted: 24 May 2012 / Published online: 10 June 2012 Ó Springer Science+Business Media, LLC 2012

Abstract RPS6KB1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) plays a key role in regulating protein translation. The role of RPS6KB1 in HCC (hepatocellular carcinoma) has not been fully investigated. This study was undertaken to determine the clinicopathological features and prognostic value of RPS6KB1 in HCC. We examined RPS6KB1 expression in 30 paired liver cancer tissues and adjacent noncancerous tissues by reverse transcription real-time PCR and Western blotting. In addition, we analyzed RPS6KB1 expression in 87 HCC samples by immunohistochemistry. The expression of RPS6KB1 was significantly increased in cancer tissues. Clinicopathological analysis showed that the expression of RPS6KB1 was significantly correlated with tumor size, histopathologic classifications, and serum alpha-fetoprotein (AFP). Kaplan–Meier survival curves revealed that increased

This work was performed in Sun Yat-sen University Cancer Center. P. D. Li  M. Y. Zhang  L. J. Yuan  Y. L. Cha  X. F. Ying  H. Y. Wang (&) State Key Laboratory of Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangdong 510060, China e-mail: [email protected] P. D. Li  G. Wu Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430023, Hubei, China W. J. Zhang Cancer Genetics Program, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China

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expression of RPS6KB1 correlated with poor prognosis of HCC patients. RPS6KB1 expression was an independent prognostic marker for overall survival of HCC patients by multivariate analysis. Our data suggest that RPS6KB1 may play an important role in the progression of HCC and could serve as a potential molecular target for HCC therapy. Keywords RPS6KB1  Hepatocellular carcinoma  Prognosis  Western blotting  Immunohistochemistry

Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases in both developed and developing countries and therefore a serious disease burden for public health [1]. In recent years, the morbidity and mortality rates of HCC have been soaring, placing HCC as the second leading cause of deaths among nine most common cancers in China [2]. Due to the lack of sensitive and specific biomarkers in the clinic, the existing early-stage screening strategies have limited benefits to the prevention of HCC. Even with advanced treatments, such as surgery, chemotherapy, and radiotherapy, HCC still presents a dismal prognosis with overall survival rate as low as 25–39 % [3]. Therefore, it is desirable to find new avenues for early diagnosis and efficacious regimens for treating HCC in order to improve patients’ outcome. In the past few decades, many genes and cellular signaling pathways have been implicated in the pathogenesis of hepatocellular carcinoma, including MTOR (mechanistic target of rapamycin) signaling pathway [4, 5]. The MTORmediated pathway has been shown to promote tumorigenesis through coordinated phosphorylation of proteins that directly/indirectly regulate cell cycle progression and

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metabolism, as well as through transcription factors including RPS6KB1 that regulate the expression of genes involved in the oncogenic processes [6]. RPS6KB1 (also known as PS6K or S6K) is highly conserved evolutionarily consisting of 525 amino-acid residues with two non-identical kinase catalytic domains, which phosphorylates several residues of the S6 ribosomal protein [7]. RPS6KB1 is a major substrate of MTOR and acts as a crucial effector of MTOR signaling pathway. RPS6KB1 plays a key role in cell growth and proliferation by regulating insulin sensitivity, metabolism, protein synthesis, and cell cycle. In addition, deregulation of RPS6KB1 contributes to the progression of type 2 diabetes, obesity, aging, and cancer [6, 8, 9]. Recent studies have found that RPS6KB1 is up-regulated in a set of human cancers, including ovarian and breast carcinomas [10, 11]. However, the significance of altered expression of RPS6KB1 in HCC has not been fully elucidated.

Materials and methods Patients and tissue samples To investigate RPS6KB1 expression in hepatocellular carcinoma, thirty HCC cancerous tissues and paired adjacent non-tumor tissues, not less than 2 cm away from HCC, were collected, respectively, from HCC cancer patients undergoing hepatectomy at Sun Yat-sen University Cancer Center between March and June, 2009. The diagnosis of HCC was confirmed by histopathology. Surgically removed fresh tissues were immediately frozen in liquid nitrogen and stored at -80 °C until use. In addition, another 87 paraffin-embedded HCC tissues were collected between 2003 and 2004. These patients did not receive preoperative chemotherapy or radiotherapy. The follow-up data of the HCC patients included in this study were obtained as a routine practice. Our outpatient department performed postoperative follow-up on HCC patients at intervals of every 3 months for the first 2 years, every 6 months from the 3rd to the 5th year, and thereafter annually for additional 5 years or until patient death, whichever occurred first. Overall survival was defined as the time from operation to patient’s death or the last followup and was used as a measure of prognosis. While diseasefree survival was defined as the time from operation to disease recurrence or metastasis. The histological types of HCC were defined according to the classification criteria of WHO. This study was approved by the Institutional Ethics Review Board (IERB) of Sun Yat-sen University Cancer Center, and written informed consent was obtained from all patients studied.

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RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer’s protocol. Next, two microgram of RNA was reversely transcribed into firststrand cDNA by M-MLV Reverse Transcriptase (Promega, M1705) according to the manufacturer’s instructions. RPS6KB1 and GAPDH were then amplified by quantitative real-time PCR using the following primers: RPS6KB1: forward: 50 -CAGTGAAAGTGCCAATCAGG TCTT-30 , reverse: 50 -GGCTGACAGGTGTTCGTGG GCTGCC-30 ; GAPDH: forward: 50 -CTCCTCCTGTTCGA CAGTCAGC-30 , reverse: 50 -CCCAATACGACCAAATC CGTT-30 . Gene-specific amplification was performed in an ABI 7900HT real-time PCR system (Life Technologies, USA) with a 15-ll PCR mix containing 0.5 ll of cDNA, 7.5 ll of 2 9 SYBR Green master mix (Invitrogen, 11760500), and 200 nM of the appropriate primers. The mixture was preheated for 10 min at 95 °C and followed by 45 cycles of amplification (30 s at 95 °C and 1 min at 60 °C, respectively). The resolution curve was measured at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. The CT (threshold cycle) value of each sample was calculated, and the relative expression of RPS6KB1 mRNA was normalized to the GAPDH value (2-DCt method). Western blot analysis The frozen HCC samples including tumor or non-tumor tissue control were homogenated in a RIPA lysis buffer. After centrifugation at 12,000 rpm, 4 °C for 20 min, about 40 lg of protein samples was run on a 12 % SDS–PAGE gel and transferred to polyvinylidene difluoride membrane (PVDF, Millipore). After blocking non-specific binding sites for 60 min with 5 % non-fat milk, the membranes were incubated with rabbit monoclonal antibody against RPS6KB1 (1:1,000; Cell signaling Technology), and GAPDH (1:1,000; Sigma-Aldrich) at 4 °C over night, respectively. Then, membranes were washed with TBST for three times, 15 min each time and then incubated with HRP-conjugated anti-rabbit secondary antibody (1:10,000; Santa Cruz, CA, USA) for 45 min at room temperature. The membrane was developed by an enhanced chemiluminescence system (ECL; cell signaling) after washed with TBST for three times. The intensity of the protein bands was determined by densitometry using Image J software (Image J; National Institutes of Health). Immunohistochemistry Paraffinic-embedded tissues were sectioned for immunohistochemistry analysis. The sections were deparaffinized

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and rehydrated through a graded series of aqueous ethanol solutions. For the antigen retrieval, the slides were immersed in EDTA (1 mmol/L, pH 8.0) and boiled for 20 min in the microwave oven. After rinsing with PBS, endogenous peroxidase was blocked with 0.3 % hydrogen peroxide for 15 min at room temperature. The slides were incubated with the primary antibody (1:100; Cell signaling Technology) in a humidified chamber at 4 °C overnight. Following additional washed with PBS for three times, the sections were sequentially incubated with horseradish peroxidase-conjugated secondary antibody (EnvisionTM Detection Kit, GK500705, Gene Tech) at 37 °C for 30 min and then washed three times with PBS. Finally, 3, 30 -diaminobenzidine tetrahydrochloride (DAB) was used for the signal development and then the sections were lightly counterstained with 20 % hematoxylin. The slides were dehydrated and mounted on cover slips. For negative controls, PBS was used in place of primary antibody. The total RPS6KB1 immunostaining scores were calculated as the sum of percent positivity of stained tumor cells and the staining intensity. The percent positivity was scored as ‘‘0’’ (\ 5 %, negative), ‘‘1’’ (5–25 %, sporadic), ‘‘2’’ (25–50 %, focal), ‘‘3’’ ([ 50 %, diffuse). The staining intensity was scored as ‘‘0’’ (no staining), ‘‘1’’ (weakly stained), ‘‘2’’ (moderately stained), and ‘‘3’’ (strongly stained). Both percent positivity of cells and staining intensity were determined under double-blind conditions. The RPS6KB1 immunostaining score was calculated with the value of percent positivity score 9 staining intensity score, ranged from 0 to 9. RPS6KB1 expression level was defined as following: ‘-’ (score 0–1), ‘?’ (score 2–3), ‘??’ (score 4–6), and‘???’ (score [ 6). Based on the RPS6KB1 expression levels, the HCC patients were divided into two groups: low RPS6KB1 expression group (RPS6KB1 - or RPS6KB1 ?) and high RPS6KB1 expression group (RPS6KB1 ?? or RPS6KB1 ???).

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Results RPS6KB1 mRNA expression in primary HCC tissues Real-time quantitative PCR was conducted on 30 paired surgical samples of HCC (cancerous tissues and the corresponding adjacent non-cancerous tissues from the same patients) to explore expression of RPS6KB1 at mRNA level. As shown in Fig. 1, the median RPS6KB1 mRNA expression was significantly higher in cancer tissues than that in corresponding adjacent non-tumor tissues (P \ 0.0001), and 80 % of the subjects (24/30) displayed higher mRNA expression of RPS6KB1 in cancer tissues. All of the 30-paired tissues were independently tested twice. RPS6KB1 protein expression in primary HCC tissue samples To investigate whether the expression of RPS6KB1 was increased at the protein level, Western blotting was performed on these 30-paired HCC cancer tissues and their corresponding adjacent non-cancer tissues. Consistent with the qRT-PCR results, Western blot analyses showed that RPS6KB1 expression was increased in 66.67 % (20/30) of cancerous tissues compared with their corresponding noncancerous tissues (P \ 0.001, Fig. 2). RPS6KB1 expression in HCC and its relationship with clinicopathological features RPS6KB1 expression in 87 HCC specimens was investigated by immunohistochemical staining. Among them, 43.7 % of cases (38/87) showed low expression of RPS6KB1 (RPS6KB1 - or RPS6KB1 ?), while 49 of 87 (56.3 %) cases exhibited high RPS6KB1 expression (RPS6KB1 ?? or RPS6KB1 ???; Table 1). In positive cases, RPS6KB1 was localized in the cytoplasm of the

Statistical analysis Statistical analyses were performed using programs included the Statistical Package for the Social Sciences, version 17.0 (SPSS Inc, Chicago, IL, USA). Paired-samples t-test was used to compare protein expression of RPS6KB1 in HCC with paired adjacent non-tumor tissue samples. Comparisons of RPS6KB1 tumor expression with clinical and pathologic features were evaluated by v2 tests. Follow-up time was censored if the patient was lost to follow-up. Survival curves were calculated using the Kaplan–Meier method and compared by the log rank test. Cox proportional-hazard analysis was used for univariate and multivariate analysis to explore the effect of RPS6KB1 expression in HCC clinicopathological variables on survival. Differences were considered significant when P was less than 0.05.

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Fig. 1 Up-regulated mRNA expression of RPS6KB1 in hepatocellular carcinoma tissues as assessed by real-time quantitative RT-PCR

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Fig. 2 Increased protein expression of RPS6KB1 in hepatocelluar cancer tissues determined by Western blotting. a Representative results of RPS6KB1 protein expression levels in paired hepatocelluar

cancer tissues and the matched adjacent non-cancerous tissues from four patients. b All of the paired hepatocelluar cancer tissues and the matched adjacent noncancerous tissues from 30 patients

Table 1 Correlation of RPS6KB1 expression and clinicopathological data’s in 87 hepatocarcinoma cases

cancer cells (Fig. 3). However, RPS6KB1 expression was found negative in distant normal liver tissues (Fig. 3b, g). In cases with adjacent hyperplastic tissues, we observed a sharp contrast between infiltrative tumor areas of positive staining and the adjacent non-tumor tissues of negative staining (Fig. 3a, f). The relationship between expression of RPS6KB1 and various clinicopathological features was presented in Table 1. As shown, the expression of RPS6KB1 was significantly correlated with tumor size (P = 0.027), histological differentiation (P = 0.020), and serum AFP (P = 0.001). Poorly differentiated cases showed strongly positive expression of RPS6KB1 (Fig. 3e, j), moderately differentiated cases showed positive expression of RPS6KB1 (Fig. 3d, i), and in well-differentiated cases, we only detected weak expression of RPS6KB1 (Fig. 3c, h). There were no statistically significant differences between RPS6KB1 expression and age, gender, liver cirrhosis, HBV or metastasis (P [ 0.05).

Clinicopathologic features

Number of each group

RPS6KB1 expression Low

High

87

38

49

\50

57

22

25

350

30

16

24

All cases Age (years)

0.523

Gender

0.375

Male

72

5

10

Female

15

33

39 0.001a

Serum AFP \400 lg/l

40

24

16

3400 lg/l

47

14

33 0.020a

Histological differentiation Well

6

6

0

48

22

24

33

10

25

No

26

11

15

Yes

61

27

34

Negative

13

6

7

Positive

74

32

42

Moderate Poor Liver cirrhosis

0.845

0.027a

Tumor size (cm) \5

31

18

13

35

56

20

39

No

42

20

22

Yes

45

18

27

No

73

32

41

Yes

14

6

8

Recurrence

0.474

Metastasis

P value \ 0.05

Relationship between RPS6KB1 expression and patients’ survival 0.866

Serum HBsAg

a

P value

0.946

The prognostic value of RPS6KB1 on overall and diseasefree survival of HCC patients was evaluated between patients with high and low RPS6KB1 expression. High RPS6KB1 expression was significantly associated with poorer prognosis of HCC patients using Kaplan–Meier curve analysis. HCC patients with high RPS6KB1 protein levels had obviously lower overall and disease-free survival compared with those with low RPS6KB1 protein levels (Fig. 4, P \ 0.001, log rank test). Univariate and multivariate analyses of prognostic variables in HCC patients Univariate and multivariate analyses were carried out using Cox proportional-hazard model to compare the impacts of RPS6KB1 expression on other clinical pathological features of HCC patients. RPS6KB1 expression, histological

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Fig. 3 Immunohistochemistry analysis of RPS6KB1 protein expression in primary hepatocellular carcinoma surgical specimens. a and f Immunostaining of HCC tumor area and the adjacent non-tumor area. b and g Distant normal liver tissue scored as RPS6KB1 (-). c and h Well-differentiated HCC scored as RPS6KB1 (?). d and

i Moderately differentiated HCC scored as RPS6KB1 (??). e and j poorly differentiated HCC scored as RPS6KB1 (???). N nontumor tissue; T tumor tissue (a–e with 9 200 magnification; f– j with 9 400 magnification)

Fig. 4 Kaplan–Meier survival analysis of primary HCC patients (n = 87) after surgical resection with high RPS6KB1 expression (n = 49) and low RPS6KB1 expression (n = 38). a The overall survival for patients in the RPS6KB1 high group was significantly

lower than that for patients in the RPS6KB1 low group. b Patients with RPS6KB1 high expression showed shorter disease-free survival than RPS6KB1 low expression patients

grading, serum AFP, tumor size, and recurrence were significant prognostic factors based on univariate analysis (Table 2). Multivariate Cox regression analysis showed that RPS6KB1 was an independent prognostic parameter. Thus, RPS6KB1 expression may play a role in predicting the overall survival in HCC patients (P = 0.042, Table 2).

HCC samples using qRT-PCR and Western blotting. The results demonstrated that RPS6KB1 was up-regulated in most of primary HCC cancer tissues at both transcriptional and translational levels. Consistently, immunohistochemical analyses also showed that RPS6KB1 expression was increased in most HCC cancer tissues compared with their corresponding non-cancer tissues. These results suggested that up-regulated expression of RPS6KB1 may play a role in HCC development. In the immunohistochemical analysis, overexpression of RPS6KB1 in HCC was significantly associated with tumor size, tumor histological differentiation, and serum AFP.

Discussion In the present study, we examined the expression of RPS6KB1 at mRNA and protein levels in paired primary

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Table 2 Univariate and multivariate analyses of overall survival in 87 hepatocarcinoma cases Variables

Univariate analysis

Multivariate analysis

HR

95 % CI

P value

HR

95 % CI

P value

RPS6KB1

3.061

1.409–6.651

0.005a

2.608

1.399–4.926

0.042a

Age

0.886

0.574–1.366

0.583

Gender

0.763

0.331–1.762

0.527

Tumor size

3.976

1.643–9.621

0.002a

2.837

1.107–7.269

0.030a

a

1.866

0.872–3.994

0.108

2.101

0.967–4.565

0.061

1.626

1.004–2.632

0.048a

Histologic grade

2.460

1.349–4.487

0.003

Liver cirrhosis

1.024

0.501–2.091

0.948

HBsAg status Serum AFP

0.859 2.748

0.356–2.074 1.318–5.730

0.736 0.007a a

Recurrence

1.616

1.008–2.591

0.046

Metastasis

1.493

0.613–3.638

0.378

HR hazard ratio, CI confidence interval a

P value \ 0.05

The relationship between high RPS6KB1 expression and large tumor size suggested that excessive RPS6KB1 may facilitate rapid expansion of the tumor. Additionally, most well-differentiated HCC tissues showed low RPS6KB1 expression, contrasting to profoundly higher RPS6KB1 expression in moderately and poorly differentiated tumors samples. These findings indicate that gain of RPS6KB1 expression may suppress the differentiation of HCC cells and further promote the progress of HCC. The high expression of RPS6KB1 is associated with high serum AFP level. It has been demonstrated that p53 repress hepaticspecific AFP gene expression by direct interaction with a composite HNF-3/p53 DNA binding element [12]. Lai et al. [13] have shown that deactivation of RPS6KB1 signaling leads to p53 ubiquitination and promotes p53 nuclear export and degradation by MDM2, by which decreases transcriptional activity p53. Thus, it is plausible that RPS6KB1 regulates AFP expression through modifying p53 function. Surace et al. [14] have found RPS6KB1 overexpression in anaplastic meningiomas, resulting in increased tumor size in vivo. These observations are consistent with our clinicopathologic findings that high expression of RPS6KB1 in HCC is associated with larger tumor size. Kaplan–Meier survival analysis revealed that the high RPS6KB1 expression was significantly correlated with a poor prognosis of HCC patients after surgical resection. Furthermore, RPS6KB1 expression is an independent prognostic factor relative to overall and disease-free survival using multivariate analysis. Consistent with our observations, overexpression of RPS6KB1 in anaplastic oligoastrocytoma is significantly associated with a poor survival status of HCC patients [15]. These findings suggest that RPS6KB1 may serve as a new and independent

predictor of prognosis for HCC patients after surgical resection. In conclusion, this study demonstrates that RPS6KB1 expression is up-regulated in the majority of HCC tissues at both mRNA and protein levels, and the high RPS6KB1 expression may indicate dismal prognosis of HCC patients. Our findings provide the evidence that RPS6KB1 may be a novel prognostic biomarker for HCC and serve as a potential therapeutic candidate for HCC. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81171891). Conflict of interest interest exists.

The authors have declared that no conflict of

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