Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth

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Cancer Letters 271 (2008) 314–322 www.elsevier.com/locate/canlet

Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth q Su Cheol Park a,b, Jung-Hwan Yoon c,*, Jeong-Hoon Lee c, Su Jong Yu c, Sun Jung Myung c, Won Kim c, Geum-Youn Gwak d, Sung-Hee Lee c, Soo-Mi Lee c, Ja June Jang e, Kyung-Suk Suh f, Hyo-Suk Lee c a

Department of Internal Medicine, Korea Institute of Radiological and Medical Sciences, Korea Cancer Center Hospital, Seoul, Republic of Korea b Department of Internal Medicine, Kangwon National University College of Medicine, Chuncheon, Republic of Korea c Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 28 Yungun-dong Chongno-gu, Seoul 110-744, Republic of Korea d Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea e Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea f Department of Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea Received 17 April 2008; received in revised form 17 June 2008; accepted 19 June 2008

Abstract Adrenomedullin is implicated in tumor progression and induced by hypoxia. We evaluated if adrenomedullin signaling is active in hepatocellular carcinoma (HCC), especially under hypoxic conditions, and to analyze its prognostic implication in HCC patients. HCC cells expressed adrenomedullin and its receptor, and hypoxia induced adrenomedullin expression. Adrenomedullin stimulated HCC cell growth via Akt activation, which was prevented by adrenomedullin peptide inhibitor. Clinico-pathological analysis revealed adrenomedullin extent was related to vascular invasion and N-cadherin intensity, which were reported to indicate a poor prognosis. In conclusion, adrenomedullin signaling is hypoxia-inducible and functionally active in HCCs, and its expression may be a prognostic factor. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Adrenomedullin; Hypoxia; N-cadherin; Akt; Hepatocellular carcinoma

1. Introduction q Grant support: This study was supported by the Seoul National University Hospital Research Fund (#03-2006-019) and by the Korean Foundation of Liver Research (2007) and the Korea Health 21 R&D Project (#0412-CR01-0704-0001). * Corresponding author. Tel.: +82 2 2072 2228; fax: +82 2 743 6701. E-mail address: [email protected] (J.-H. Yoon).

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies. Its major risk factors are chronic hepatitis caused by hepatitis B or C, alcohol abuse, or exposure to carcinogens, such as, aflatoxin B1 [1–5]. The major event that predisposes HCC development is liver

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.06.019

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cirrhosis, and it has been determined epidemiologically that over 80% of HCCs occur in cirrhotic livers [6,7]. Functional and morphological changes of hepatocytes, sinusoidal endothelium, Kupffer cells, and stellate cells in cirrhotic liver, impair normal liver blood supply systems and thereby cause tissue hypoxia [8–10]. Moreover, since HCCs are typically accompanied by neovascularization and hypervascularity, hypoxia is believed to participate in the genesis and progression of HCCs [11–13]. Recently, we have indeed demonstrated that hypoxia stimulates HCC cell growth by inducing hexokinase II expression [14]. Adrenomedullin is a peptide, that was first isolated from pheochromocytoma, but which was later found to be expressed by many body tissues and to play a key role as a mediator of inflammation via paracrine, autocrine, and endocrine mechanisms [15]. In addition, adrenomedullin might be involved in tumor progression by promoting tumor proliferation and inhibiting apoptosis through a number of signaling pathways [16–18], and also by acting as an angiogenic factor [16–20]. Adrenomedullin is also implicated in hepatic fibrogenesis since its expression in hepatic stellate cells is up-regulated in liver cirrhosis [21,22]. However, adrenomedullin signaling has not yet been extensively explored during HCC progression, particularly with respect to hypoxia inducibility and its biological impact on cellular proliferation. In this study, we attempted to determine if adrenomedullin signaling is hypoxia-inducible and functionally active in human HCCs, and if so, which intracellular signal is activated and how this affects tumor cell proliferation. Finally, we sought to determine whether adrenomedullin signaling has prognostic implication in HCC patients. Overall, the results of the present study demonstrate that hypoxia-inducible adrenomedullin signaling accelerates HCC cell proliferation by activating phosphatikinase (PI3K) and that dylinositol 30 adrenomedullin expression may be a useful prognostic marker in HCC patients. Thus, the selective interruption of adrenomedullin-induced signaling may be therapeutically efficacious in HCC patients. 2. Materials and methods 2.1. Cell culture and reagent Huh-BAT cells (Huh-7 cells stably transfected with a bile acid transporter [23]) which were origi-

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nally derived from a well-differentiated HCC [24], and HepG2 cells which were derived from a poorly differentiated HCC [25] were used in this study. In all experiments performed in this study, cells were serum starved overnight in order to avoid the effects of serum-induced signaling. Cells were then incubated under standard culture condition (20% O2 and 5% CO2, at 37 °C) or under hypoxic culture condition (1% O2, 5% CO2, and 94% N2, at 37 °C). Adrenomedullin (human) and adrenomedullin (22-52) (human) were obtained from Bachem AG (Bubendorf, Switzerland). LY294002 (a PI3K inhibitor) was obtained from Calbiochem (San Diego, CA). 2.2. Real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted and cDNA templates were prepared. Adrenomedullin mRNA was quantitated by real-time RT-PCR using the following primers: forward, 50 -acttggcagatcactctcttagca-30 ; reverse, 50 -atcagggcgacggaaacc-30 . Universal 18S primers (Ambion, Inc., Austin, TX) were used as controls to ensure RNA integrity. Quantification was performed by real-time PCR (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany) using Sybr green as the fluorophore (Molecular Probes, Eugene, OR). 2.3. RT-PCR Total RNA was extracted from cells, and cDNA templates were prepared using oligo-dT random primers and MoMLV (Moloney Murine Leukemia Virus) reverse transcriptase. PCR was performed using primers specific for the adrenomedullin receptor, calcitonin receptor-like receptor (CL) gene (forreverse, 50 ward, 50 -ctcctctacattatccatgg-30 ; 0 cctcctctgcaatctttcc-3 ), receptor activity modifying protein (RAMP) type 2 gene (forward, 50 -attgcctggagcactttgc-30 ; reverse, 50 -gcctcactgtctttactcc30 ), and RAMP type 3 gene (forward, 50 tcgtgggctgctactgg-30 ; reverse, 50 -ctcacagcagcgtgtcg30 ). To exclude the possible presence of genomic DNA in reaction mixture, we performed control reactions without the RT step. RT-PCR products were subcloned using TOPO TA cloning kits (Invitrogen), and positive clones were sequenced using an ABI PRISMÒ 377 Genetic Analyzer (Applied Biosystems, San Francisco). Genes were identified by BLAST searching.

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2.4. Cell proliferation

Table 1 The baseline characteristics of the HCC patients

Cell proliferation was measured using CellTiter 96 Aqueous One Solution cell proliferation assays (Promega, Madison, WI). This test is based on the cellular conversion of the colorimetric reagent MTS [3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumsalt] into soluble formazan by dehydrogenase enzymes, which are only found in metabolically active, proliferating cells. Following each treatment, 20 lL of dye solution was added to each well in a 96-well plate and incubated for 2 h. Subsequently, absorbance was recorded at 490 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, CA).

Variable

n(%)

Age (year) <50 P50

26(38) 42(62)

Sex Male Female

61(90) 7(10)

Child class A B

66(97) 2(3)

Viral hepatitis B C B+C NBNC

52(77) 7(10) 2(3) 7(10)

NBNC indicates non-B and non-C.

2.5. Immunoblot analysis Cells were lysed for 20 min on ice with lysis buffer (50 mM Tris–HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 lg/mL aprotinin, leupetin, pepstatin; 1 mM Na3VO4; 1 mM NaF) and centrifuged at 14,000 g for 10 min at 4 °C. Samples were resolved by SDS–PAGE, transferred to nitrocellulose membranes, blotted with appropriate primary antibodies (anti-phospho-Akt and anti-actin antibody at a dilution of 1:500 and 1:1000, respectively), and treated with peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA). Bound antibodies were visualized using chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) and exposed to Kodak X-OMAT film. Primary antibodies: antiphospho-Akt antibody was from Cell Signaling Technology (Beverly, MA). Goat anti-actin antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2.6. Patients Sixty-eight HCC patients (61 men, 7 women; mean age: 53 ± 10 years) who had undergone surgical resection at Seoul National University Hospital between June 1994 and December 1998 were retrospectively included in this study. Baseline characteristics of the study population are detailed in Table 1. The study protocol was approved by the Institutional Review Board of Seoul National University Hospital.

2.7. Tissue array and immunohistochemistry HCC tissue specimens from each patient were processed into 10% neutral formalin-fixed, paraffin-embedded blocks. All tumors were histologically diagnosed, graded using Edmonson’s scale, and classified as low (I–II) or high grade (III–IV). Tumors were also classified according to multiplicity and the presence or absence of microscopic vascular invasion. All specimens were evaluated immunohistochemically using a tissue-array method. Core tissue biopsies (2 mm in diameter) were taken from individual paraffin-embedded tissues (donor blocks) and arranged in a new recipient paraffin block (tissuearray block) using a trephine apparatus (Superbiochips Laboratories, Seoul). Immunohistochemical staining was performed using adrenomedullin antibody (Peninsula Laboratories, Inc., Belmont, CA) and N-cadherin antibody (Zymed Laboratories Inc., San Francisco, CA) using a streptavidin peroxidase based procedure. The paraffin sections were dewaxed and rehydrated followed by the microwave antigen retrieval procedures. For antigen retrieval, slides were soaked in 10 mmol/L citric acid (Sigma Chemical, St. Louis, MO) buffer, pH 6, and heated with microwave oven at 98 °C for 20 min. Sections were incubated with polyclonal antibody raised in rabbits against purified human adrenomedullin at a dilution of 1:500. Cells were considered positively stained when a brown granulation of the cytoplasm was revealed at low-power magnification. The adrenomedullin and N-cadherin immunostaining inten-

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sities were scored for each sample as none to weak, moderate, or strong. Extent of N-cadherin immunoreactivity was scored as less than one-third, onethird to two-thirds, or more than two-thirds, whereas adrenomedullin immunostaining extent was scored as none, <10%, 10% to <50%, or 50– 100%. These pathological and immunohistochemical findings are summarized in Supplementary Table 1. Histologic examinations were carried out by one pathologist (Jang J.J.) who was unaware of clinical information. 2.8. Data analysis All in vitro experimental data presented represent at least three independent experiments using samples from a minimum of three separate isolations and are expressed as means ± SD or SE as indicated. Differences between groups were compared using the Mann–Whitney U-test or one-way ANOVA test with post hoc Bonferroni test as indicated, and clinico-pathological data was analyzed using the v2 and Cochran and Mantel-Haenszel’s tests. P values of <0.05 were considered statistically significant. 3. Results 3.1. Hypoxia-mediated induction of adrenomedullin expression in HCC cells Initially, we investigated whether adrenomedullin expression is hypoxia-inducible by comparing adrenomedullin mRNA levels in HCC cells cultured under hypoxic or normoxic condition by using quantitative RTPCR. As shown in Fig. 1A, the copy number of adrenomedullin mRNA was significantly higher in hypoxic than normoxic cells, indicating that hypoxia induces adrenomedullin expression in human HCC cells.

Fig. 1. Hypoxia-mediated adrenomedullin induction and its receptor (calcitonin receptor-like receptor (CL), receptor activity modifying protein (RAMP) types 2 and 3) expression in human HCC cells. (A) Huh-BAT cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Quantitative RT-PCR was performed for adrenomedullin. Data are expressed as means ± SD of three individual experiments. *P < 0.05, versus normoxia. (B) RNAs were extracted from Huh-BAT and HepG2 cells, and RT-PCR was performed for CL, RAMP2, and RAMP3. RT () denotes negative controls, which were processed without reverse transcriptase.

cell growth was significantly accelerated by adrenomedullin in a dose-dependent manner, but this growth enhancement did not occur when adrenomedullin (22-52) (an adrenomedullin peptide inhibitor) was co-administered with adrenomedullin (Fig. 2B). These observations indicate that adrenomedullin signaling is functionally active and that it accelerates HCC cell growth in an autocrine and/or paracrine manner.

3.2. Functionally active adrenomedullin signaling in HCC cells

3.3. Intracellular signaling activated by adrenomedullin in HCC cells

We next evaluated whether adrenomedullin receptors (CL, RAMP2, and RAMP3) are expressed in HCC cells by using RT-PCR. As shown in Fig. 1B, the expressions of CL, RAMP2, and RAMP3 were identified in all HCC cell lines examined (Huh-BAT and HepG2 cells). Since adrenomedullin receptors were found to be expressed in these HCC cells, we assessed whether adrenomedullin affects HCC cell proliferation in an autocrine or paracrine manner. For this purpose, we monitored HCC cell growth following exogenous adrenomedullin administration by using MTS assays. As shown in Fig. 2A, HCC

Of the many kinase signals that are related to cell growth, we found that Akt (a downstream kinase of PI3K) was promptly activated following adrenomedullin treatment in HCC cells (Fig. 2C). Moreover, adrenomedullin-induced Akt phosphorylation was prevented when adrenomedullin (22-52) was co-administered with adrenomedullin (Fig. 2D). We then assessed if the PI3K/Akt signal is responsible for adrenomedullin-induced HCC cell growth acceleration. As shown in Fig. 2E, adrenomedullin-induced cell growth acceleration was effectively prevented when LY294002 (a PI3K inhibitor) was co-

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Fig. 2. Adrenomedullin-induced signaling in HCC cells. (A and B) Huh-BAT cells were grown in the presence of adrenomedullin (0, 0.1, and 1 lM) (A), or Huh-BAT cells were treated with adrenomedullin (0 or 0.1 lM) in the presence or in the absence of adrenomedullin peptide inhibitor (adrenomedullin (22-52), 5 nM) (B). At each indicated time, an MTS assay was performed according to the manufacturer’s instruction. Data are expressed as means ± SE of six individual experiments. *P < 0.05. AM indicates adrenomedullin and AM 22-52 indicates adrenomedullin (22*52). (C and D) Huh-BAT cells were cultured with or without adrenomedullin (1 lM) for the indicated times (C), or Huh-BAT cells were cultured with adrenomedullin (0.1 lM) either in the presence or in the absence of adrenomedullin peptide inhibitor (adrenomedullin (22-52), 5 nM) for the indicated times (D). Immunoblot analysis was performed using cell lysates and antisera specific for phosphorylated Akt (p0 -Akt). AM indicates adrenomedullin and AM 22-52 indicates adrenomedullin (22-52). (E) Huh-BAT cells were treated with adrenomedullin (AM, 0.1 lM) either in the presence or in the absence of a PI3K inhibitor, LY294002 (20 lM). An MTS assay was performed at each of the indicated times. Data are expressed as means ± SE of six individual experiments. *P < 0.05.

administered with adrenomedullin. These observations collectively imply that adrenomedullin stimulates cell growth through the activation of PI3K/Akt signal in HCC cells.

3.4. Prognostic implication of adrenomedullin signaling in HCC patients Univariate analysis of clinico-pathological and biological variables with respect to adrenomedullin intensity identified no significant variables associated with adreno-

medullin intensity (Supplementary Table 2). When adrenomedullin extent was categorized as none, <10%, 10% to <50%, or 50–100% in HCC tissues, it was found to be significantly correlated with vascular invasion of HCCs (P = 0.044, Supplementary Table 3, Fig. 3A). Furthermore, when adrenomedullin extent was dichotomized by 10%, a significant correlation was found between adrenomedullin extent and the intensity of N-cadherin in HCC tissues (P = 0.019, Supplementary Table 4, Fig. 3B). A stratified analysis of factors related with adrenomedullin extent grouped by 10% using the Cochran and MantelHaenszel’s test revealed that UICC T stage acts as a con-

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Fig. 3. Prognostic implications of adrenomedullin expression in HCC tissues. (A) The graph is representative of the proportions of vascular invasion in HCC tissues according to the extent of adrenomedullin (none, <10%, 10% to <50%, or 50–100%). The statistical significances were analyzed using v2 tests. P = 0.044. (B) The graph is representative of the proportions of adrenomedullin extent (<10% or 10–100%) according to N-cadherin intensity in HCC tissues (weak, moderate, or strong). The statistical significances were analyzed using v2 tests. P = 0.019.

founding factor (Table 2), which is the reason why the relation between adrenomedullin extent and vascular invasion appeared to be insignificant when adrenomedulTable 2 Stratified analysis of factors related to adrenomedullin extent grouped by 10% Adrenomedullin extent P* n(% within adrenomedullin extent) <10% T1

P10%

Vascular invasion Yes 0(0) No 12(100)

2(5) 35(95)

1.000

T2,3 Vascular invasion Yes 2(50) No 2(50)

14(93) 1(7)

0.097

N-cadherin intensity None to weak 7(58) Moderate 5(42) Strong 0(0)

9(24) 14(38) 14(38)

0.006

T2,3 N-cadherin intensity None to weak 2(50) Moderate 1(25) Strong 1(25)

6(40) 7(47) 2(13)

0.968

T1

T indicates UICC T stage. * Cochran and Mantel-Haenszel’s test for stratified analysis.

lin extent was grouped by 10%. Vascular invasion and Ncadherin expression profiles are known to predict HCC patient survival after surgical resection [26–29], and similarly, the above observations suggest that adrenomedullin extent in HCC tissues has prognostic implications in HCC patients.

4. Discussion The principal findings of this study relate to the hypoxia-induced acceleration of HCC cell growth via adrenomedullin signaling. Our results collectively demonstrate that adrenomedullin and its receptor are expressed in human HCC cells, and that hypoxia induces adrenomedullin expression, which accelerates cell proliferation via the activation of PI3K signaling. In addition, our clinico-pathological analysis demonstrates that adrenomedullin up-regulation is related to the vascular invasion of HCCs and that the extent of adrenomedullin is correlated with N-cadherin intensity, which suggests that adrenomedullin has prognostic implications in HCC patients. Adrenomedullin expression has been demonstrated in several cancer cell lines, e.g., brain tumor

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[30], neuroblastoma [31], and in a number of lung cancer cell lines [32]. In addition, the expressions of adrenomedullin and its receptor have been demonstrated in many human epithelial cancer cell lines of diverse origins (e.g., lung, colon, ovary, breast, bone marrow, prostate, and cartilage cell lines [33]. The present study demonstrates that adrenomedullin and its receptor are expressed in HCC cells. It was recently reported that adrenomedullin is expressed in pancreatic cancer, and that it plays a vital role as an autocrine factor that increases the aggressiveness of these cancers [34]. Thus, it is also likely that adrenomedullin expression in HCC cells acts to promote HCC cell growth in an autocrine manner, and in fact, the present study demonstrates that adrenomedullin dose-dependently accelerated HCC cell growth. In terms of tumor progression, adrenomedullin may affect vascular smooth muscle cells, endothelial cells, and tumor cells via various signaling pathways. Adrenomedullin has been demonstrated to enhance vascular smooth muscle cell maturation and thereby to promote tumor vessel growth via a cAMP-dependant pathway and/or via mitogen-activated protein kinase (MAPK) or PI3K/Akt signaling [15,35]. Adrenomedullin may also induce angiogenesis by activating PI3K/Akt, MAPK, and focal adhesion kinase (p125FAK) in endothelial cells [36]. In addition, adrenomedullin is implicated in the development of lymphatic vascular system, which is known to mediate tumor metastasis, although it has also been reported that the expression of lymphatic vessel endothelial hyaluronan receptor was down-regulated in HCC [37,38]. In tumor cells, adrenomedullin was found to inhibit apoptosis by up-regulating Bcl-2, and to increase cell growth and survival by activating oncogenic proteins like Ras, Raf, and PKC [17]. In the present study, adrenomedullin activated Akt in HCC cells, and this was found to be responsible for HCC cell growth enhancement by adrenomedullin. In contrast, we did not detect the activation of any other MAPK or anti-apoptotic signal in HCC cells following adrenomedullin treatment (data not shown). Thus, these findings collectively imply adrenomedullin acts via autocrine signaling in HCC cell growth by activating PI3K/Akt. Hypoxia is a common characteristic of many solid tumors and is associated with malignant progression, distant metastasis, and resistance to treatment [39]. Cancer cells adapt to hypoxic environments via the transcriptional activation of

hypoxia-inducible factor-1 (HIF-1), which activates the expressions of genes involved in the glycolytic system, angiogenesis and cell survival [40–42]. HCCs are characteristically hypervascular tumors, and therefore, hypoxia is more likely to activate survival signals in HCC than in other hypovascular solid tumors. Indeed, we previously demonstrated that hypoxia potently stimulates HCC cell growth via HIF-1-dependent pathways, in particular, via hexokinase II induction [14]. The present study also demonstrates the hypoxia-mediated induction of adrenomedullin expression in HCC cells. It has been reported that adrenomedullin is up-regulated under hypoxic condition in the rat cortex [43] and in neural cells [44–46], and that this induction is under the regulation of HIF-1 [47]. In human cancer cells, adrenomedullin expression was first reported to be hypoxia-inducible in human colorectal carcinoma cells [48], and more recently, it was reported that adrenomedullin is hypoxia-inducible and overexpressed in pancreatic adenocarcinomas [20]. In combination with our observation that adrenomedullin accelerates HCC cell growth, we consider that adrenomedullin probably promotes HCC progression, especially in those HCCs that exhibit unexpectedly aggressive growth following a hypoxic insult, such as, transarterial chemoembolization. During our clinico-pathological analysis, adrenomedullin extent in HCC tissue was found to be significantly correlated with vascular invasion. Vascular invasion is one of the most powerful predictors of tumor recurrence after surgical resection in HCC patients [27–29]. In this study, adrenomedullin extent was also found to be significantly correlated with N-cadherin intensity. N-cadherin is an adhesion molecule that is involved in the cell migration, invasion, and metastasis of cancer cells [49,50]. More recently, we also found that N-cadherin signaling exerts an anti-apoptotic effect in human HCCs and that intense N-cadherin expression is a predictor of poor recurrence free survival and overall survival in HCC patients [26]. Therefore, these findings collectively suggest that adrenomedullin up-regulation in HCC tissues is likely to be a useful prognostic factor in HCC patients. In conclusion, this study demonstrates that adrenomedullin signaling is hypoxia-inducible and functionally active in HCCs. Furthermore, our findings suggest that adrenomedullin may be a useful prognostic marker in HCC patients. Thus, the selective interruption of adrenomedullin signaling may be therapeutically efficacious in HCC patients.

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