Deregulated expression of Notch receptors in human hepatocellular carcinoma

Deregulated expression of Notch receptors in human hepatocellular carcinoma

Available online at www.sciencedirect.com Digestive and Liver Disease 40 (2008) 114–121 Liver, Pancreas and Biliary Tract Deregulated expression of...

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Available online at www.sciencedirect.com

Digestive and Liver Disease 40 (2008) 114–121

Liver, Pancreas and Biliary Tract

Deregulated expression of Notch receptors in human hepatocellular carcinoma J. Gao a,b , Z. Song c , Y. Chen b , L. Xia b , J. Wang b , R. Fan b , R. Du b , F. Zhang b , L. Hong b , J. Song b , X. Zou b , H. Xu a , G. Zheng a , J. Liu b,∗ , D. Fan b,∗

c

a Department of Digestive Diseases, Wuhan General Hospital, Guangzhou Command PLA, Wuhan, Hubei Province, PR China b Institute of Digestive Diseases, and State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, PR China Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, PR China

Received 15 February 2007; accepted 3 August 2007 Available online 4 October 2007

Abstract Background and aim. Notch signaling controls cellular differentiation and proliferation. Deregulated expression of Notch receptors is observed in a growing number of malignant tumours, however, the role of Notch signaling in hepatocellular carcinoma is still unknown. To address this, the expression of Notch receptors in human hepatocellular carcinoma was examined in both protein and ribonucleic acid levels. Patients and methods. Fifty-three hepatocellular carcinoma tissue sections were detected by immunohistochemistry. Three paired fresh surgical hepatocellular carcinoma and adjacent nontumour liver samples were analyzed by Western blot and reverse transcriptase polymerase chain reaction. Immunohistochemistry, Western blot and reverse transcriptase polymerase chain reaction are reliable methods to examine the expression of protein and RNA. Results. All of the four Notch receptors were expressed in the neoplastic cells of hepatocellular carcinoma tissues with different intensity and extensity. Notch1 and Notch4 were expressed in both cytoplasm and nucleus, and all of the nuclear staining showed up in the cytoplasm-positive cases. Cytoplasmic and nuclear Notch1 was detected in 88.7% (47/53) and 9.4% (5/53) of hepatocellular carcinoma tissues, respectively; positive rates of Notch4 were 67.9% (36/53) in cytoplasm and 52.8% (31/53) in nucleus. Notch2 and Notch3 were only in cytoplasm, with positive rates of 26.4% (14/53) and 52.8% (28/53), respectively. Compared with adjacent nontumour liver, Notch1 (cytoplasmic) and Notch4 (nuclear) were up-regulated (P < 0.05, P < 0.05), Notch2 was down-regulated (P < 0.05), while Notch1 (nuclear), Notch3 and Notch4 (cytoplasmic) showed no difference between hepatocellular carcinoma and adjacent nontumour liver. Western blot and reverse transcriptase polymerase chain reaction analysis showed a consistent result. Conclusion. Our findings indicate that the expression of Notch receptors was deregulated and Notch signaling might be involved in the development of hepatocellular carcinoma. © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: HCC; Notch1; Notch2; Notch3; Notch4

1. Introduction

∗ Corresponding authors at: Institute of Digestive Diseases, and State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, PR China. Tel.: +86 29 84773974; fax: +86 29 82539041. E-mail addresses: [email protected] (J. Liu), [email protected] (D. Fan).

Notch signaling pathway is an evolutionarily highly conserved mechanism for cell-cell communication, which is important for many types of cell fate determination in a wide range of organisms [1]. Four Notch homologs, Notch1 through Notch4, have been identified in mammals [2–4]. Activation of the Notch pathway is thought to be mediated by interactions of bordering cells via

1590-8658/$30 © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2007.08.001

J. Gao et al. / Digestive and Liver Disease 40 (2008) 114–121

cell-to-cell contact of the Notch receptors and its membraneassociated ligands [5]. During maturation, Notch polypeptide precursors are cleaved just outside their transmembrane domain by a furin-like protease to produce an extracellular subunit (NEC) and a transmembrane subunit (NTM). These two subunits associate to form the heterodimeric, mature receptor at the cellular surface [6]. Binding of Notch ligands to NEC leads to a metalloprotease-dependent cleavage in the extracellular portion of NTM, which is rapidly followed by additional cleavage in the transmembrane domain that requires a presenilin-dependent protease activity. This cleavage releases the intracellular region of NTM (ICN), which translocates to the nucleus and forms a ternary complex with a transcription factor CSL (also known as CBF1/RBP-J, Suppressor of Hairless or Lag1) and transcriptional co-activators of the mastermind-like to activate transcription of a group of downstream genes [7]. Notch receptors are widely expressed in mammals and control various biological events. Recent studies have revealed that Notch signaling is not only important in embryonic development and cell fate determination, but also plays important roles in cancer. Notch1 is identified as an oncogene responsible for acute T cell lymphoblastic leukaemia [2]. Notch2 plays a role in B-cell chronic lymphoid leukaemia [8]. Notch3 contributes to the growth of human lung cancers by its effect on mitogen-activated protein kinase pathway [9]. Notch4, which is found to be a common integration site for the mouse mammary tumour virus in mouse mammary tumours, has profound effects on mammary gland development and tumourigenesis [10]. Until now, there is no report about the overall association between Notch receptors and hepatocellular carcinoma (HCC). In this study, we examined the expression of four mammalian Notch homologs, Notch1-4, in human HCC tissues and adjacent nontumour liver, to clarify the possible roles of Notch receptors in the carcinogenesis and progress of HCC.

2. Methods 2.1. Patients and specimens Formalin-fixed, paraffin-embedded sections of HCC tissues were obtained from 53 primary HCC patients who received surgical resection at Xijing Hospital of the Fourth Military Medical University from September 2002 to December 2004 with the approval of the human research committee of university and with the patients’ consents. Among them, 32 had adjacent nontumour liver tissues. The patient’s age, gender, tumour size, histological type of the neoplasm, and tumour, node, metastasis (TNM) stage were obtained from surgical and pathological records. Three paired fresh surgical samples of HCC tissues and adja-

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cent nontumour liver were collected at the time of surgery for tumour resections in Xijing Hospital. The tissue samples were named sample 1, 2, 3 respectively and were snap-frozen and stored at −80 ◦ C for Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. 2.2. Immunohistochemical staining Horseradish peroxidase staining was used to visualize antigens on paraffin-embedded 5-micron sections. After treated with 10% normal animal serum for 30 min, sections were incubated with anti-Notch1 primary goat polyclonal antibodies (pAb) or anti-Notch2, 3, 4 primary rabbit pAb (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:100) overnight at 4 ◦ C, respectively. Sections were then washed and primary antibodies were detected with HistostainTM -SP (Zymed Lab Inc., San Francisco, CA, USA). Diaminobenzidine was used as the chromogen for the immunoperoxidase reaction. The slides were counterstained with haematoxylin, then dehydrated and mounted. In negative controls non-immune goat IgG or rabbit IgG were used to substitute for the primary antibodies. AntiNotch1 pAb is raised against a peptide mapping at the C-terminus of Notch1 of human origin (sc-6014), antiNotch2 pAb is against amino acids 25–255 mapping within an extracellular domain of Notch2 of human origin (sc-5545), anti-Notch3 pAb is against amino acids 2107–2240 mapping at the C-terminus of Notch3 of mouse origin (sc-5593), and anti-Notch4 pAb is against amino acids 1779–2003 mapping at the C-terminus of Notch4 of mouse origin (sc-5594). 2.3. Evaluation of immunostaining and statistical analysis All stained sections were evaluated by two independent investigators in a blind manner. The scoring was based on intensity and extensity according to previous report [11]. Briefly, the percentage of positive tumour cells was determined semi-quantitatively by assessing the whole tumour section, and each sample was scored on a scale of 0–4, in which 0 = negative, 1 = positive staining in 1–25% of cells, 2 in 26–50%, 3 in 51–75%, and 4 in 76–100%. The intensity of immunostaining was determined as 0 (negative staining), 1 (weakly positive staining), 2 (moderately positive staining), and 3 (strongly positive staining). The immunoreactive score of each section was calculated by sum of these two parameters. A total score was finally calculated and grade as negative (I), weak (II), moderate (III), strong (IV). Statistical analysis was performed using SPSS software (version 10.0, SPSS Inc., Chicago, USA). Mann–Whitney U-test for two groups and Kruskal–Wallis H-test for multi-groups were used to compare the differences of groups for immunohistochemistry staining. P < 0.05 was considered statistically significant.

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2.4. Protein extraction and Western blot analysis

3. Results

The three paired samples of HCC tissues and adjacent nontumour liver tissues were solubilized in a lysis buffer (150 mM NaCl; 20 mM Tris-HCl (pH 7.4); 0.1% SDS; 1% sodium deoxycholate; 1% Triton X-100, 5 ␮g/ml aprotinin, 1 ␮g/ml leupeptin) on ice. All lysates were centrifuged at 4 ◦ C at 10,000 × g for 10 min. The protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratory, Richmond, CA, USA) with bovine serum albumin as the standard protein. Immunoblotting of proteins was performed using the anti-Notch1–4 antibodies (1:300), followed by incubation with peroxidase coupled anti-goat or anti-rabbit IgG, respectively. Proteins were then visualized by enhanced chemiluminescence. Western blot for ␤-actin was used as an internal sample. The bidimensional optical densities of Notch receptors and ␤-actin proteins on the films were quantified and analyzed with Molecular Analyst software (Bio-Rad). Every experiment was repeated three times. The ratios of Notch receptors against ␤-actin were calculated. Statistical analysis was done using SPSS software. Statistical significance was assumed if P < 0.05.

3.1. Expression of Notch receptors in HCC tissues by immunohistochemical staining and its relationship with clinicopathological parameters

2.5. RNA isolation and RT-PCR Total cellular RNA of three paired samples of HCC tissues and adjacent nontumour liver tissues was extracted using the one-step RT-PCR kit (MBI Fermentas, Lithuania) according to the manufacturer’s instructions. Semi-qualitative PCR was performed using human primer sequences for Notch1–4 (Table 1). ␤-actin was used as a housekeeping gene. The conditions of PCR were as follows: after initial denaturation at 94 ◦ C for 4 min, 30 cycles of denaturation at 94 ◦ C for 45 s, annealing at each appropriate temperature as described for 30 s and extension at 72 ◦ C for 45 s. PCR products were separated by electrophoresis on a 1% agarose gel and visualized with ethidium bromide staining. Gene expression was presented by the relative yield of the PCR product from target sequences to that from ␤-actin gene. Mean values from three independent experiments were taken as results.

The expression of Notch receptors in HCC tissues were examined by immunohistochemical staining. The result showed that Notch1–4 were all expressed in the neoplastic cells of HCC tissues with different intensity and extensity. Notch1 and Notch4 were expressed in both cytoplasm and nucleus, and all of the nuclear staining showed up in the cytoplasm-positive cases. Notch2 and Notch3 were expressed only in cytoplasm. Cytoplasmic Notch1 was strong and could be detected in 88.7% (47/53) of HCC, which was significantly higher than in adjacent nontumour liver (P < 0.05); nuclear Notch1 was weak and the positive rate was 9.4% (5/53), and the level of nuclear Notch1 showed no statistical difference between HCC and adjacent nontumour liver (Fig. 1A and B). Notch2 was expressed in 26.4% (14/53) of HCC, and the signal was relatively weaker compared with adjacent nontumour liver (P < 0.05) (Fig. 1C and D). 52.8% (28/53) of HCC had Notch3 expression, however, there is no statistical difference between HCC and adjacent nontumour liver (Fig. 1E and F). Cytoplasmic and nuclear expression of Notch4 was detected in 67.9% (36/53) and 58.5% (31/53) of HCC, respectively. Although the cytoplasmic expression of Notch4 showed no statistical difference, the nuclear staining of it was much higher in HCC than in adjacent nontumour liver (P < 0.05) (Fig. 1G and H) (Table 2). According to statistical analysis of the association of the expression of Notch receptors with clinicopathological parameters, none of gender, tumour size, TNM stage was correlated with the expression of those antigens, while the levels of Notch2, Notch3 were higher significantly in old (at the age of more than 50 years old) than in younger patients (less than 50 years old) (P < 0.01, P < 0.01). About histological grade, the data showed that the expression of Notch1 (cytoplasmic), Notch2 and Notch3 had significant differences among differentiation grade, with P < 0.05, P < 0.05, P < 0.05, respectively

Table 1 Primer sequences for Notch1-4 and ␤-actin together with annealing temperatures and product sizes Gene

Primer sequence

Annealing temperature (◦ C)

Product amplified (base pairs)

Notch1

Fw 5 -CCGTCATCTCCGACTTCATCT-3 Rev 5 -GTGTCTCCTCCCTGTTGTTCTG-3

52

468

Notch2

Fw 5 -GCTGATGCTGCCAAGCGT-3 Rev 5 -CCGGGGAAGACGATCCAT-3

55

474

Notch3

Fw 5 - CTGGCGAGACTGCTTTGC-3 Rev 5 -CGACTGTGCCGCTTTGAG-3

52

661

Notch4

Fw 5 - CACTGAGCCAAGGCATAGAC-3 Rev 5 -ATCTCCACCTCACACCACTG-3

53

472

␤-actin

Fw 5 -AGCGGGAAATCGTGCGTG-3 Rev 5 -CAGGGTACATGGTGGTGCC-3

54

301

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Fig. 1. Immunohistochemical staining of Notch receptors in HCC tissues and adjacent nontumour liver. (A, C, E, G) in order showed Notch1-4 expression in HCC tissue. The arrows in (A) and (G) showed nuclear staining of Notch1 and Notch4, respectively. (B, D, F, H) showed Notch1-4 expression in adjacent nontumour liver; (I) was the negative control. Compared with adjacent nontumour liver, cytoplasmic Notch1 and nuclear Notch4 were significantly up-regulated, while Notch2 was significantly down-regulated in HCC. Nuclear Notch1, Notch3 and cytoplasmic Notch4 showed no difference between HCC and adjacent nontumour liver (magnification, ×200).

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Table 2 Expression of Notch receptors in HCC tissues and adjacent nontumour liver Cases tested

Positive cases

Score I

P value II

III

IV

Notch1 (cytoplasmic) Adjacent nontumour liver HCC

32 53

30 47

2 6

14 12

16 30

0 5

<0.05

Notch1 (nuclear) Adjacent nontumour liver HCC

32 53

0 5

32 48

0 4

0 1

0 0

NS

Notch2 Adjacent nontumour liver HCC

32 53

15 14

17 39

9 12

6 2

0 0

<0.05

Notch3 Adjacent nontumour liver HCC

32 53

17 28

15 25

8 19

9 9

0 0

NS

Notch4 (cytoplasmic) Adjacent nontumour liver HCC

32 53

24 36

8 17

10 19

14 16

0 1

NS

Notch4 (nuclear) Adjacent nontumour liver HCC

32 53

13 31

19 22

7 13

6 17

0 1

<0.05

(Table 3). We did not analyse the relationship of Notch1 (nuclear) with the clinicopathological parameters since only five HCC samples were positive for it. 3.2. Protein levels of Notch receptors in HCC tissues by Western blot analysis The protein levels of Notch1–4 in the three paired samples of HCC and adjacent nontumour liver was detected by Western blot. Fig. 2 showed that the proteins of Notch1–4 were all expressed in HCC and adjacent nontumour liver, with specific bands at ∼70–120 kDa. Among them, Notch1, Notch2 and Notch3 showed only one band, which was the NTM form [12]. Notch1 was higher in HCC than in adjacent nontumour liver in all the three samples. Notch2 was lower in HCC than in adjacent nontumour liver in sample 1 and 3 but not 2. Notch3 was equally expressed between HCC and adjacent nontumour liver in all the three samples. Notch4 showed two bands; the band with a higher molecular mass was its NTM form, the lower one was its ICN form [13]. Although Notch4 NTM was equally expressed in all the three samples, the ICN form of it was higher in HCC than in adjacent nontumour liver in sample 1 and 2 but not 3. The expression of Notch precursors (full length) were also detected with a band above 200 kDa, but it was weak and showed no difference between HCC than adjacent nontumour liver (data not shown). The results illustrated that the expression of Notch1 (NTM form) and Notch4 (ICN form) was up-regulated while Notch2 was down-regulated in HCC, and Notch3 showed no change between HCC and adjacent nontumour liver, which was consistent with our immunohischemistry result.

3.3. mRNA levels of Notch receptors in HCC tissues by RT-PCR To evaluate the mRNA expression of Notch1–4, RTPCR analysis was performed in the three paired samples of HCC and adjacent nontumour liver. The results showed that mRNA of all the four Notch receptors was found to be expressed in the three pairs of samples. Notch1 mRNA was expressed higher in all the three HCC tissues than in adjacent nontumour liver. For Notch2, samples 1 and 3 had lower mRNA levels in HCC than in adjacent nontumour liver, while sample 2 showed no difference. Notch3 mRNA showed no difference between HCC and adjacent nontumour liver in all the three samples. Notch4 mRNA was higher in HCC compared with adjacent nontumour liver in samples 1 and 2 but not 3 (Fig. 3). This result demonstrated that the alteration of mRNA levels of Notch receptors in HCC was consistent with the change of their protein levels described above.

4. Discussion HCC is one of the most common malignant tumours in the world. The molecular mechanisms of HCC have been studied for many years, and many molecules and signaling pathways are proved to be involved in the processes of HCC. Notch signaling has been showed to be involved in the carcinogenesis, progress, invasion and neurovascular formation of many malignant tumours [14–18]. Until now, the expression and localization of all of the Notch receptors in HCC

6 7 4 NS 3 6 4 4 10 8 NS 0 0 0 4 5 0 <0.05

0 1 0 NS 6 7 3 6 8 5

10 9

1 8 8

11 6

6 8 5 5 5 2 7 18 14

<0.05 3 2 0 8 13 9 <0.05 0 2 4 Differentiation Well (n = 13) Moderately (n = 24) Poorly (n = 16) P value

2 7 3

4 2 Age (years) ≤50 (n = 27) >50 (n = 26) P value

8 4

13 17 NS

2 3

<0.01

1 1 0

0 2

0 0 0

0 0

3 11 11

17 8 2 10 25 14

II IV III II I

119

0 1 0

0 1 6 11 NS 7 6 14 8 0 1 6 10 0 0 1 8 <0.01 9 10

III II I III II I II I II I

IV

Notch3 Notch2 Notch1

Table 3 Relationship between Notch receptors expression and clinicopathological parameters in 53 HCC patients

III

IV

Notch4 (cytoplasmic)

IV

Notch4 (nuclear)

IV

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Fig. 2. Protein expression of Notch1-4 in HCC tissues and adjacent nontumour liver by Western blot. The protein levels of Notch1-4 in the three paired samples of HCC and adjacent nontumour liver were detected by Western blot. Notch1, Notch2 and Notch3 showed only the NTM form. The protein level of Notch1 was higher in HCC than in adjacent nontumour liver in all three tissue samples. Notch2 protein was lower in HCC than in adjacent nontumour liver in samples 1 and 3 but not 2. Notch3 protein showed no difference between HCC and adjacent nontumour liver in all the three samples. Notch4 showed both the NTM and the ICN form. Although the NTM form was equally expressed in all the three samples, the ICN form was higher in HCC than in adjacent nontumour liver in samples 1 and 2 but not 3. Data were normalized by setting the ratios of the protein levels of Notch receptors against ␤-actin at 100%. Results are mean ± S.D. of three independent experiments. * P < 0.05, Student’s t test.

have not been reported, however, the expression and function of these Notch molecules in liver of development and disease have been investigated. Reports show that Notch signaling plays an important role in hepatoblast differentiation and all four of the known Notch receptors are expressed in adult human liver [19,20]. On the growth and proliferation of hepatocytes, some reports say that Notch signaling exerts a negative effect [21], while there are also reports showing that Notch signaling can promote the growth and proliferation of hepatocytes. For example, the expression of Notch1, Notch2, Notch3, Delta1, Jagged1 mRNA exist in hepatocytes of rat models of liver injury and regeneration [22]. After partial rat liver hepatectomy, both Notch1 and Jagged1 proteins are up-regulated, Notch1 ICN increases, and Notch1/Jagged1 signaling pathway is activated during rat liver regeneration

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Fig. 3. mRNA expression of Notch1-4 in HCC tissues and adjacent nontumour liver by RT-PCR. The mRNA levels of Notch1-4 in the three paired samples of HCC and adjacent nontumour liver were detected by RT-PCR. Notch1 mRNA was higher in HCC than in adjacent nontumour liver in all three tissue samples. Notch2 mRNA was lower in HCC than in adjacent nontumour liver in samples 1 and 3 but not in sample 2. Notch3 mRNA showed no difference between HCC and adjacent nontumour liver in all the three samples. Notch4 mRNA was higher in HCC compared with adjacent nontumour liver in samples 1 and 2 but not in sample 3. Data were normalized by setting the ratios of the mRNA levels of Notch receptors against ␤-actin at 100%. Results are mean ± S.D. of 3 independent experiments. * P < 0.05, Student’s t test.

[23]. These reports hint that Notch signaling might also play important roles in HCC. In this study, we characterized the expression and localization of Notch1–4 in human HCC tissues. By immunohistochemical staining, we find that Notch1–4 were all expressed in the neoplastic cells of HCC tissues. Notch1 and Notch4 were expressed in both cytoplasm and nucleus, and Notch2 and Notch3 were expressed only in cytoplasm. Compared with adjacent nontumour liver, Notch1 (cytoplasmic) and Notch4 (nuclear) were up-regulated, whereas Notch2 was down-regulated in HCC. The results from Western blot and RT-PCR in three paired samples of HCC and adjacent nontumour liver were consistent with the result from immunohistochemical staining. These results suggest that Notch1 and Notch4 might exert tumourigenic effects. For Notch2, reports showed that Notch2 had a weaker transactivation domain, so the down-regulation of Notch2 might make Notch1 and Notch4 take advantage of more ligands and lead to a stronger activation of Notch signaling, which contributes to the development of HCC. This cooperative antagonism effect of Notch1, Notch4 and Notch2 has also

been reported in previous researches. One study says that Notch1 and Notch4 are undetectable in normal human breast samples, whereas 67% and 44% of the ductal carcinomas in situ are positive for Notch1 and Notch4, respectively [24]. Another study shows that Notch1 and Notch2 play contrasting roles in breast tumour patients’ outlook. A high level of Notch1 is associated with a poorer outlook, while a high level of Notch2 correlated to a higher chance of survival [25]. Three previous reports have connected Notch signaling with HCC. One paper shows that Notch1 signaling could inhibit growth of human HCC through induction of cell cycle arrest and apoptosis [26], which indicates that Notch pathway plays a negative role in the progress of HCC. However, two recent papers show that Notch pathway seems to take a positive effect on HCC. One of them finds that Notch3, Jagged1, Delta1 and HES-1 are all expressed in HepG2 cell line [27]; the other demonstrates that the levels of Notch1, Jagged1, and HES-1 gene expression are increased in HCC samples relative to the adjacent HCCfree liver tissue by real-time quantitative RT-PCR [28]. Our results are consistent with the two recent papers to some extent. The up-regulated Notch1 might function in HCC by its own signal or by its effects on other signal pathways, such as TGF-␤ signaling [29]. For Notch4, the up-regulated nuclear Notch4 shows that this molecule is activated in HCC, which provides a basis for the activation of Notch signaling in HCC. In conclusion, our study indicates that Notch1, Notch2 and Notch4 are involved in the development of HCC. This result connects Notch signaling with HCC and thus provides new possible molecular mechanisms in the development of HCC.

Practice points • This paper is the first one to investigate the expression and localization of all of the four Notch receptors in HCC and shows that the expression of Notch receptors is deregulated in HCC. • Notch1 (cytoplasmic) and Notch4 (nuclear) are up-regulated in HCC compared with adjacent nontumour liver. • Notch2 is down-regulated in HCC compared with adjacent nontumour liver. • Notch1 (nuclear), Notch3 and Notch4 (cytoplasmic) showed no difference between HCC and adjacent nontumour liver. • Our findings indicate that Notch signaling might be involved in the development of HCC.

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Research agenda • The expression of Notch receptors in HCC cell lines. • The expression of Notch ligands in HCC. • The expression of downstream effector molecules of Notch signaling in HCC. • The activity of Notch signaling in HCC. • The functional role of Notch signaling in HCC.

Conflict of interest statement None declared.

Acknowledgements This work was supported by grants from National Hi-Tech Research and Development Program of China (863) (Grant No. 2006AA02Z468) and National Natural Science Foundation of China (Grants No. 30371585 and No. 30570835).

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