Enhanced tumorigenesis and lymphatic metastasis of CD133+ hepatocarcinoma ascites syngeneic cell lines mediated by JNK signaling pathway in vitro and in vivo

Biomedicine & Pharmacotherapy 67 (2013) 337–345

Available online at

www.sciencedirect.com

Original article

Enhanced tumorigenesis and lymphatic metastasis of CD133+ hepatocarcinoma ascites syngeneic cell lines mediated by JNK signaling pathway in vitro and in vivo Yanling Jin a, Jun Mao a,b, Huanxi Wang a, Zhenhuan Hou a, Wei Ma a, Jun Zhang a, Bo Wang a, Yuhong Huang a, Shizhu Zang c, Jianwu Tang a,d,**, Lianhong Li a,b,* a

Department of Pathology, Dalian Medical University, 9 West Lvshun Southern Road, Dalian 116044, PR China The Key Laboratory of Tumor Stem Cell Research of Liaoning Province, Dalian Medical University, Dalian 116044, PR China Department of Biotechnology, Dalian Medical University, 9 West Lvshun Southern Road, Dalian 116044, PR China d The Key Laboratory of Tumor Metastasis of Liaoning Province University, Dalian Medical University, Dalian 116044, PR China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 January 2013 Accepted 3 February 2013

Cancer stem cells (CSCs), stem-like cells, or tumor-initiating cells (TICs) may initiate tumorigenesis and metastasis, but neither the basic cell biology of CSCs nor the mechanisms of CSC-mediated tumor growth and lymphoid node metastasis are understood. Evidence suggests that CSC phenotype is maintained, at least in part, by altered JNK signaling. In this study, factors influencing the growth and metastatic potential of CSCs were examined by comparing CD133 surface antigen expression, proliferation, clonogenicity, invasive capacity, tumorigenicity, and expression of JNK-associated signaling molecules between the highly metastatic mouse hepatocarcinoma ascites syngeneic cell line Hca-F and the low metastasis potential line Hca-P. The Hca-F line exhibited higher clonogenic, proliferative, and invasive capacities than Hca-P cells, and a greater proportion of Hca-F cells were CD133 positive. In both cell lines, the CD133+ subpopulation showed significantly enhanced tumorigenicity and metastatic potential. An in vivo tumorigenicity assay in nude mice indicated that Hca-F cells possessed significantly higher tumorigenicity than Hca-P cells as indicated by larger tumors after inoculation. Expression levels of Ecadherin (CDH1), annexin VII, and JNK1 proteins were inversely correlated with CD133 expression in both Hca-F and Hca-P cells. These results demonstrate that CD133+ subpopulations of both Hca-F and Hca-P lines show CSC-like properties. However, Hca-F cells showed greater tumorigenicity and invasiveness, consistent with greater lymphatic metastasis capacity. We propose that tumorigenesis and lymphatic metastasis are regulated by JNK/P53/annexin VII and JNK/ATF-2/CDH1/annexin VII signal transduction pathways. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Liver cancer stem cells Lymph node metastasis (LNM) JNK signal transduction pathway

1. Introduction Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver and the fifth most frequently diagnosed cancer worldwide [1–3]. Traditional chemotherapies can shrink the tumor but fail to eradicate it fully, leading to recurrence. Lymphatic metastasis is a critical event in determining recurrence and long-term patient survival. There is growing support for the cancer stem cell (CSC) hypothesis of tumorigenesis and metastasis, which asserts that primary tumors are initiated, maintained, and spread by a small subpopulation of cancer cells with ‘‘stem cell’’ characteristics. Characterization of CSC marker

* Corresponding author. Department of Pathology, Dalian Medical University, 9 West Lvshun Southern Road, Dalian 116044, PR China. Tel.: +86 411 86110050; fax: +86 411 86110050. ** Corresponding author. Tel.: +86 411 86110002; fax: +86 411 86118866. E-mail addresses: [email protected] (J. Tang), [email protected] (L. Li). 0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.02.006

expression, proliferation, and migration, as well as the cell signaling pathways that regulate these processes may therefore contribute greatly to our understanding of tumor growth and metastasis [4]. A lymph node (LN) metastatic carcinoma cell line (UP-LN1) producing carcinoembyonic antigen (CEA) and characterized by the persistent appearance of adherent (A) and floating (F) cells in culture was isolated to investigate the tumor distribution and tumorigenic mechanisms of CSCs [5]. The cell line Hca-F and its syngeneic cell line Hca-P are mouse hepatocarcinoma cell lines with high and low potential for lymphatic metastasis, respectively, and have proven to be valuable cell models to explore the process of HCC growth and lymphatic metastasis [6–15]. The goal of the current study was to explore mechanisms of lymphatic metastasis by identifying a liver CSC subpopulation characterized by CD133 expression within the Hca-F and Hca-P lines. The surface antigen CD133 (also known as prominin-1 or AC133), originally classified as a hematopoietic stem cell marker, can be used for enrichment of

338

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

CSCs from HCC. In this study, we demonstrated that CD133 expression was higher in Hca-F cells than Hca-P cells and that CD133 expression in both cells types was associated with higher proliferative and metastatic capacities, consistent with the association between CD133 and CSC phenotype in HCC [16–20]. The current consensus definition describes CSCs as cells within a tumor that are able to self-renew and produce the heterogeneous lineages that comprise the tumor [21]. In addition to CD133, annexin VII and c-Jun NH2-terminal kinase (JNK) genes were also more highly expressed in Hca-F cells than Hca-P cells at both the mRNA and protein levels [6–9], suggesting that the JNK pathway may regulate HCC growth and lymphatic metastasis. Indeed, early work on JNK1 [22] and annexin VII genes [23,24] in our laboratory indicated that both are involved in LN metastasis of HCC. In addition, loss of E-cadherin (CDH1) expression may also be related to the invasive capacity and metastatic potential of tumor cells [25]. Furthermore, the mTOR [26], Wnt [27], and PTEN/Akt/PI3K signaling pathways [28] have been implicated in the maintenance of CSCs. Lymphatic metastasis may also depend on changes to multiple JNK signaling pathways [29,30]. In the present study, we demonstrate that the enhanced tumorigenic potential of CD133+ liver CSCs derived from Hca-F and Hca-P lines is correlated with changes in molecules within the JNK signal transduction pathway, including annexin VII, JNK1, and CDH1. Specifically, all these signaling components were expressed at lower levels in CD133+ liver CSCs than in CD133– liver cells in both Hca-F and Hca-P lines. These findings may provide insight into JNK signaling pathways that confer the high clonogenicity, proliferation, migration, invasion, and tumorigenicity characteristic of CSCs, properties that ultimately lead to HCC growth and lymphatic metastasis. Moreover, these results define possible molecular targets to suppress HCC growth and lymphatic metastasis, which could lead to more specific therapies to enhance patient survival.

10 days under these culture conditions, floating liver CSC spheres derived from Hca-F or Hca-P cells were counted, harvested by centrifugation at 800 rpm, and photographed under a microscope (Nikon, Japan). 2.4. Flow cytometry analysis Cells at a density of 106/ml were collected and labeled with Phycoerythrin (PE) anti-mouse CD133 (Biolegend, USA). Analysis was performed using a FACS Calibur (BD Biosciences, Falcon Lakes, NJ). Analysis was done using the Flow-Jo program (Tree Star, Ashland, OR). Positive and negative gates were determined using isotype-matched mouse immunoglobulin G (IgG) as control. 2.5. Isolation of CD133+ and CD133– Hca-F and Hca-P cells by magnetic-activated cell sorting (MACS) The Miltenyi MACS system was used according to the manufacturer’s protocol. Cells of both Hca-F and Hca-P lines were cultured without FBS for 10 days as described and harvested by centrifugation at 800  g for 3 minutes. The resulting CSC spheres were collected and resuspended in PBS at 106 cells/100 ml. PE antimouse CD133 (Biolegend, USA) or IgG-PE as a control (Biolegend, USA) was added at 5 ml per 100 ml cell suspension and the mixture incubated at 4 8C for 15 minutes in the dark. Labeled cells were then washed three times in PBS, harvested by centrifugation at 800  g for 3 minutes, and then resuspended in PBS at 107 cells/ 80 ml. To this cell suspension (80 ml) was added 20 ml anti-PE MicroBeads (Miltenyi, Germany) and the mixture incubated at 4 8C for 15 minutes in the dark. The cells were washed three times with PBS, centrifuged at 800  g for 3 minutes, and separated into CD133+ and CD133– fractions using a Miltenyi LS column. In this way, we obtained four distinct cell phenotypes: CD133+ Hca-F cells (Hca-F-CD133+), CD133– Hca-F cells (Hca-F-CD133–), CD133+ Hca-P cells (Hca-P-CD133+), and CD133– Hca-P cells (Hca-PCD133–).

2. Materials and methods 2.6. Cell proliferation assay in vitro 2.1. Ethics statement All animal experiments were conducted in accordance with protocols approved by the Experimental Animal Ethical Committee of Dalian Medical University (Permit Number: L2012012). 2.2. Cell lines and cell culture The mouse hepatocarcinoma cell lines Hca-F and Hca-P were established in our laboratory and maintained under conditions described previously [6–10]. The Hca-F and Hca-P cells lines were cultured in dulbecco’s modified eagle’s medium (DMEM)/F-12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 2 mmol/L glutamine, 100 U/ml penicillin, and 100 mg/ ml streptomycin (Invitrogen, Canada), and maintained in a humidified 37 8C incubator under a 5% CO2 atmosphere. 2.3. Liver CSC sphere formation assay Cells of the two lines were collected, washed to remove serum, resuspended in FBS-free DMEM/F-12 supplemented with 100 IU/ ml penicillin, 2% B27 supplement (Invitrogen, Canada), 20 ng/ml epidermal growth factor (EGF) (Sigma, USA), 20 ng/ml basic fibroblast growth factor (b-FGF) (Invitrogen, Canada), 4 g/ml heparin (Stem cell Technologies, Canada), and 5 g/ml insulin (Sigma, USA), and plated in separate wells at 5000 cells/well in ultra low attachment 6-well plates (Corning, USA). Plates were incubated in a humidified 37 8C incubator with 5% CO2. After

Cell proliferation was assessed by the Cell counting kit-8 (CCK8) (Dojindo, Japan) according to the manufacturer’s instructions. A cell suspension was plated at 3  104 cells/ml in 96-well plates and cells incubated at 37 8C under 5% CO2. At 24, 48, 72, and 96 h, 100 ml of CCK8 solution was added to each well. Cell numbers in triplicate wells were estimated by the absorbance (at 450 nm or A450). 2.7. Transwell migration assay The upper chambers were seeded with 3  105 cells in FBS-free DMEM/F-12. The lower chambers were filled with DMEM/F-12 supplemented with 10% FBS containing 10 mg/ml fibronectin (10 mg/ml). After incubation at 37 8C in a 5% CO2 incubator for 24 h, cells remaining in the upper chamber were removed completely with a cotton swab. The filters were fixed in methanol, stained with 0.1% gentian violet, and washed with 33% acetate acid. The A450 of the acetate acid elution solution was measured. 2.8. Transwell invasion assay Cell invasion was measured as described for the migration assay except that transwell units were precoated with artificial ECM by incubation in a commercial ECM mixture (Sigma, USA) for 1 h at 37 8C. The membranes were rehydrated with 100 ml FBS-free DMEM/F-12 and all subsequent steps were as performed for the cell migration assay.

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

2.9. Western blotting Protein extracts were separate by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Invitrogen, Canada). The primary antibodies used were as follows: monoclonal anti-annexin VII (A4475, Sigma USA, 1:1500), anti-CDH1 (Abcam, USA, 1:800), anti-mouse CD133 (eBioscience, USA, 1:200), antiJNK1 (BioWorld, USA, 1:400), and anti-GAPDH (Kang Chen, China, 1:7500). Membranes were blocked in 5% dried milk for 1 h and incubated with horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. After extensive washing, bands were detected by ECL (Santa Cruz, USA) and quantified as the densitometric ratio of target protein density to GAPDH density (Bio-Rad, USA). 2.10. Tumorigenicity assay in nude mice Freshly sorted cells were collected in sterile DMEM/F-12 without FBS and injected into nude mice (male, 6- to 8-weekold, SCXK(LIAO)2008-0002) provided by the Experimental Animal Center of Dalian Medical University. Mice were maintained under standard conditions and treated according to the institutional guidelines for the use of laboratory animals. Twenty nude mice were randomly divided into four groups. Group 1 was inoculated with Hca-F-CD133+ cells, group 2 with Hca-F-CD133– cells, group 3 with Hca-P-CD133+ cells, and group 4 with Hca-P-CD133– cells.

339

Cells were suspended in 100 ml of FBS-free culture medium and subcutaneously injected into the back-limb at 5  105 cells/50 ml. On days 6, 9, 12, 15, 18, and 21 after inoculation, the tumor mass was measured using calipers. Tumor volumes (V) were calculated by the formula V = L  W2  0.5. On day 21 post-inoculation, the mice were sacrificed. For histological study, tumors were isolated and fixed in 10% neutral-buffered formalin, paraffin-embedded, and cut into 4-mm sections. Sections were then processed for immunohistochemistry (below) or stained with hematoxylineosin (H&E). 2.11. Immunohistochemistry The expression levels of annexin VII, CDH1, and CD133 were detected by immunohistochemical staining of tumor sections using mouse monoclonal antibodies against annexin VII (Sigma, USA, 1:400), CDH1 (Abcam, USA, 1:50), and CD133 (eBioscience, USA, 1:100). Immunostaining intensities and the uniformity of nuclear/cytoplasmic staining were graded. The percentage of stained cells was scored as 1 ( 10%), 2 (11–50%), 3 (51–75%), or 4 (> 75%). Staining intensity was scored as 0 (no staining), 1 (stramineous color), 2 (yellow), or 3 (brown). These scores were combined to give a final score for each section from – to +++ (–: no signal; +: weak/indeterminate signal; ++: moderate signal; +++: strong signal). All sections were stained simultaneously with the appropriate positive and negative controls.

Fig. 1. The higher clonogenic capacity of Hca-F cells compared to Hca-P cells. A. Representative CD133+ liver CSC spheres derived from Hca-F and Hca-P cells after 10 days’ cultivation in FBS-free DMEM/F-12 (200). B. The number of CD133+ liver CSC spheres derived from Hca-F or Hca-P cells after 10 days. In cultures inoculated with Hca-F cells, 70 of 5000 spheres were CD133+ compared to only 46 of 5000 spheres derived from Hca-P cells. C. The fraction of CD133+ cells in Hca-F cultures was 1.3-fold higher than the fraction in Hca-P cultures (21.31% vs. 16.45%). IgG-PE was used as the control. **P < 0.05.

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

340

Table 1 Proliferation of CD133+ and CD133– Hca-F and Hca-P cells under identical culture conditions. Group

The optical density at various times in culture (h) 24

48

72

96

Hca-F-CD133+ Hca-F-CD133– Hca-P-CD133+ Hca-P-CD133–

0.2958  0.0245 0.2864  0.0182 0.2891  0.0823 0.2743  0.0302

0.4739  0.0119 0.3942  0.0298 0.3825  0.0462 0.3729  0.0710

0.6935  0.0209 0.5179  0.0581 0.5942  0.0617 0.4916  0.0507

0.9765  0.0211 0.6193  0.0391 0.7203  0.0561 0.5723  0.0719

2.12. Statistical analysis The results are expressed as the mean  SEM. Group means were compared using two-tailed Student’s t-tests. A P value < 0.05 was considered significant. 3. Results 3.1. Hca-F cells exhibited stronger clonogenic capacity than Hca-P cells We compared the clonogenic potential of Hca-F and Hca-P cell lines by counting the number of liver CSC spheres derived from these lines under the culture conditions described in

Materials and Methods. After 10 days in culture, CSCs were identified by the surface marker CD133. Floating spherical CSC colonies were generated by small numbers of Hca-F or Hca-P cells (Fig. 1A). A significantly larger number of liver CSC spheres were derived from Hca-F cells compared to Hca-P cells (P < 0.05, Fig. 1B), indicating that Hca-F cells exhibit a stronger clonogenic capacity. We then quantified the ratio of CD133+ to CD133– cells in both Hca-F- and Hca-P-derived spheres by flow cytometry analysis and found that 21.31% of Hca-F cells were CD133+ (Fig. 1C, top right) compared to only 16.45% of Hca-P cells (Fig. 1C, bottom right). Thus, a greater proportion of Hca-F cells exhibited the CSC phenotype, consistent with the higher clonogenic capacity of the Hca-F cell line.

Fig. 2. The proliferative, migration, and invasive capacities of CD133+ and CD133– cells isolated from Hca-F and Hca-P cultures. A. Cell proliferation rate was estimated by the CCK8 viable cell assay. In both lines, the CD133+ fraction showed more rapid proliferation, while Hca-F-derived CD133+ cells proliferated more rapidly than Hca-P-derived CD133+ cells. B. Cell migration and invasion capacities were determined by transwell assays and expressed by the number of cells that passed into and through the filter (identified by crystal violet staining, 200). Again, the CD133+ fractions of both lines showed greater migration and invasiveness, and CD133+ Hca-F cells exhibited higher migration and invasive capacities than CD133+ Hca-P cells, **P < 0.05, *P < 0.01.

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

341

3.2. Hca-F cells exhibited stronger proliferative capacity in vitro than Hca-P cells

lines, CD133+ Hca-F cells had a greater proliferative capacity than CD133+ Hca-P cells in vitro.

The CD133+ and CD133– cells derived from Hca-F and Hca-P lines were isolated by magnetic-activated cell sorting (MACS) and the proliferative capacity of these subpopulations compared after 24, 48, 72, and 96 h in vitro using the CCK8 viable cell assay (Table 1, Fig. 2A). Starting with the same number of CD133+ and CD133– cells, the estimated number of Hca-F-CD133– cells was 97% of the CD133+ Hca-F cell number after 24 h, 83% after 48 h, 75% after 72 h, and 63% after 96 h, indicating that Hca-F-CD133+ cells proliferated more rapidly under the same culture conditions. Similarly, the estimated number of Hca-P-CD133– cells was 95% of the Hca-P-CD133+ cell number after 24 h, 97% after 48 h, 83% after 72 h, and 79% after 96 h. Thus, CD133 expression was associated with more rapid proliferation of both Hca-F and Hca-P cells. Starting from equal population sizes, the subpopulation Hca-PCD133+ population was 98% of Hca-F-CD133+ subpopulation after 24 h, 81% after 48 h, 86% after 72 h, and 74% after 96 h, indicating that while CD133 expression enhanced proliferation in both cell

3.3. Hca-F cells exhibited greater migration and invasion capacities than Hca-P cells in vitro A transwell migration assay demonstrated that CD133+ Hca-F and Hca-P had significantly stronger migration capacities than CD133– Hca-F and Hca-P cells. The number of Hca-F-CD133+ CSCs that passed through the transwell filter was significantly higher than the number of migrating Hca-F-CD133– cells (280.03  19.75 vs. 170.4  12.87; P < 0.05). The number of Hca-P-CD133+ CSCs that passed through the filter was significantly higher than the number of Hca-P-CD133– cells (178.03  11.60 vs. 92.27  9.83; P < 0.05). The migration of CD133+ Hca-F cells was significantly higher than the migration of CD133+ Hca-P cells (280.03  19.75 vs. 178.03  11.60; P < 0.05, Fig. 2B). Similarly, invasive capacity was also stronger in CD133+ cells in vitro. The number of Hca-F-CD133+ cells that passed through the filter and adhered to the lower surface (coated with an artificial extracellular matrix, ECM) was significantly higher than the

Fig. 3. Expression of annexin VII, JNK1, and CDH1 (E-cadherin) in CD133+ and CD133– Hca-F and Hca-P cells. A. Hca-F and Hca-P cells were cultured without FBS for 10 days. Magnetic cell sorting (MACS) was used to separate CD133+ from CD133– cells. Protein expression levels of annexin VII, JNK1, CDH1, and CD133 were measured in Hca-FCD133+, Hca-F-CD133–, Hca-P-CD133+, and Hca-P-CD133– cells by western blotting. Expression of GAPDH was measured as a gel loading control, **P < 0.05, *P < 0.01. There was an inverse relationship between CD133 expression and the expression levels of annexin VII, JNKI, and E-cadherin (CDH1). B. The signaling network connecting annexin VII, JNK1, and E-cadherin. JNK1 is the key junction point connecting E-cadherin and annexin VII. Both E-cadherin and annexin VII are downstream of JNK1.

342

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

number of invading and ECM-adherent Hca-F-CD133– cells (147.13  9.38 vs. 111.03  10.06; P < 0.05). Similarly, a significantly higher number of Hca-P-CD133+ liver CSCs passed through the filter and adhered to the ECM compared to Hca-P-CD133– cells (95.47  14.10 vs. 85.00  8.93; p < 0.05). Overall, CD133+ Hca-F cells exhibited much stronger invasive capacity than Hca-P cells (147.13  9.38 vs. 95.47  14.10 invading cells; P < 0.05; Fig. 2B). In summary, the high-metastatic potential Hca-F line exhibited greater migration and invasion compared to the low-metastatic potential Hca-P line in vitro. These results are consistent with recent findings demonstrating that CD133 is a marker for CSCs and that CD133 expression is predictive of tumor invasion and metastasis in gastric cancer [31]. 3.4. The expression of annexin VII, JNK1, and CDH1 (E-cadherin) Cancer stem cells and stem-like tumor cells exhibit elevated expression of the surface marker CD133 [32]. In an attempt to

characterize the molecular mechanisms by which CD133 regulates tumor formation and growth, we examined the relationship between CD133 expression and the expression levels of annexin VII, JNK1, and CDH1, signaling molecules previously associated with tumor aggression and metastasis. Protein levels of annexin VII, CDH1, and JNK1 were lower in Hca-F-CD133+ cells than in HcaF-CD133– cells (Fig. 3A, P < 0.05). Expression of annexin VII, CDH1, and JNK1 proteins were also significantly lower in Hca-P-CD133+ cells compared to Hca-P-CD133– cells (Fig. 3A, P < 0.05). The potential interactions between annexin VII, JNK1, and E-cadherin are mapped in Fig. 3B to reveal the possible effects of these changes in expression. Annexin VII and JNK1 interact indirectly via hnRNPk and PU.1, with annexin VII downstream of JNK1. There is also an indirect relationship between CDH1 (E-cadherin) and JNK1. Again, E-cadherin acts downstream of JNK1 via a multitude of pathways. However, there is no relationship between E-cadherin and annexin VII except through JNK1. Hence, JNK1 is a key signaling molecule linking E-cadherin and annexin VII.

Fig. 4. Tumorigenicity of CD133+ and CD133– Hca-F and Hca-P cells following injection in nude mice (n = 5 per group). A. Growth curves of xenograft tumors in nude mice. The volume of the tumor was measured 6, 9, 12, 15, 18, and 21 days after inoculation. Bars represent the mean tumor size in each group (SEM).*P < 0.01 compared to the corresponding CD133+ group at the same time point after innoculation. B. After 3 weeks of tumor growth, the mice were sacrificed and the tumors excised. The size of each was measured. Tumors derived from CD133+ cells grew faster than those derived from CD133– cells for both Hca-F and Hca-P cell lines. The tumorigenic potential of CD133+ Hca-F cells was higher than that of CD133+ Hca-P cells. C. Tumors were fixed in 10% neutral-buffered formalin, paraffin-embedded, and cut into 4-mm sections for HE staining and immunohistochemistry (200). The expression levels of annexin VII and CDH1 were lower in tumors derived from CD133+ liver cells for both Hca-F and Hca-P cell lines (P < 0.05).

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

3.5. Hca-F cells showed greater tumorigenicity than Hca-P cells in nude mice in vivo To compare the tumorigenicity of CD133+ and CD133– cells isolated from Hca-F and Hca-P lines in vivo, we established a tumor model in nude mice. Tumor development was compared between nude mice injected with CD133+ Hca-F cells, CD133– Hca-F cells, CD133+ Hca-P cells, or CD133– Hca-P cells (n = 5 per group) on days 6, 9, 12, 15, 18, and 21 after inoculation. Tumors derived from CD133+ Hca-F and CD133+ Hca-P cells grew more rapidly than tumors derived from CD133– Hca-F and Hca-P cells (Fig. 4A, P < 0.05), while tumors derived from CD133+ Hca-F cells grew significantly faster than those derived from CD133+ Hca-P cells. On the 21st day after inoculation, the mice were sacrificed and the tumor sizes measured. Again, tumors derived from CD133+ Hca-F cells were larger than those derived from CD133– Hca-F cells or from CD133+/– Hca-P cells (Fig. 4B), consistent with a report indicating that CD133+ cells isolated by MACS from a Hep-2 cell line had greater tumorigenicity [33]. The xenografts were stained with hematoxylin and eosin (HE) and examined under a light microscope (Fig. 4C). No obvious morphological differences were noted between tumor cells derived from the two cell lines. We also examined the expression of CDH1 and annexin VII in these tumor sections by immunohistochemistry. The subcellular localization of annexin VII

343

protein in xenograft tumors was confined mainly to the cytoplasm, while CDH1 was localized to the membrane (Fig. 4C). Expression levels of annexin VII and CDH1 were lower in tumors derived from Hca-F-CD133+ cells than tumors derived from Hca-F-CD133– cells (P < 0.05) and lower in tumors derived from Hca-P-CD133+ cells than tumor derived from Hca-PCD133– cells (P < 0.05), consistent with the negative correlation between CD133 expression and both CDH1 and annexin VII expression levels observed in vitro. 3.6. The greater tumor lymphatic metastasis potential of CD133+ cells is mediated by changes in the JNK signal transduction pathway In this study, we used in vitro proliferation, migration, and invasion assays as well as an in vivo tumorigenicity assay to investigate the differences in tumorigenesis between CD133+ and CD133– liver CSCs. Based on our findings, we proposed a novel mechanism: CD133+ CSCs have a high tumor lymphatic metastasis capacity due to downregulation of the JNK1 signal transduction pathway (Fig. 5). These findings provide potential mechanistic insights into the roles of the JNK/P53/annexin VII and JNK/ATF-2/ CDH1/annexin VII pathways in HCC progression and lymphatic metastasis. Our findings identify CDH1 and annexin VII as novel downstream targets of the JNK1 signal transduction pathway that may regulate tumor lymphatic metastasis in HCC.

Fig. 5. The JNK signal transduction pathway. JNK1/P53/annexin VII and JNK1/ATF-2/CDH1/annexin VII signaling pathways may be involved in tumor lymphatic metastasis. Annexin VII is the final and indirect substrate of p53 and ATF-2, which in turn are activated by the JNK1 cascade.

344

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345

4. Discussion Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver. Although the mechanisms of cancer metastasis are gradually being revealed, less is known concerning the characteristics of HCC cells with metastatic potential. Clarification of the HCC lymphatic metastasis mechanism is essential for developing novel therapeutics to sensitize hepatoma cells to radiation and improve patient survival. Cancer stem cells (CSCs) are rapidly proliferating and highly invasive, and so are likely the critical drivers of tumor growth. It is becoming increasingly evident that cancer treatments that fail to eliminate CSC may allow regrowth of the tumor. In this paper, highly metastatic Hca-F cells and poorly lymphatic metastatic Hca-P cells were used to explore the cellular properties that distinguish liver CSCs and contribute to tumor aggression and LN metastasis. We were able to isolate a subpopulation of CD133+ cells from both lines using MACS and demonstrated that these CD133+ cells proliferated and migrated more rapidly than CD133– cells within the total Hca-F and Hca-P cell populations, consistent with studies showing that CD133+ tumor cells exhibit properties of CSCs in HCC [34–37]. Moreover, the line with a greater proportion of CD133+ cells, Hca-F, formed a higher number of CSC spheres after 10 days’ cultivation in FBS-free media (Fig. 1B, C). Thus, the line with the higher lymphatic metastatic potential also had a higher clonogenic capacity. The glycoprotein CD133 is a CSC maker and an independent predictor of survival in patients with gastric cancer [31]. Thus, the present data from liver cancer cells are consistent with these earlier findings; the subpopulation of CD133+ cells show greater capacity for self-renewal (clonal expansion) and so are the major drivers of tumorigenicity. The results from in vitro colony formation and in vivo tumorigenicity assays suggested that Hca-F cells exhibited stronger stem cell-like properties than Hca-P cells. While CD133+ cells from both lines showed enhanced proliferation and migration compared to CD133– cells, cell expansion and migration/invasion were strongest in CD133+ cells from the highly metastatic Hca-F line. The stem cell properties of CD133+ cancer cells may be responsible for tumor initiation and progression [38]. Indeed, CD133+ liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal [39]. Similarly, CD133+ cells from medulloblastoma and glioblastoma lines exhibit stem cell properties in vitro [40,41]. A recent study proposed the existence of CSCs with high invasive capacity in pancreatic cancer [42]. Metastasis and invasion are not random processes but are highly organ-specific and pathophysiologically organized, involving multiple steps and numerous interactions between cancer cells and the host. Our in vivo tumorigenicity assays revealed that CD133+ Hca-F cells also proliferated faster in mice after inoculation, resulting in larger tumors than those derived from Hca-P injection (Fig. 4A, B). In both lines, however, the CD133+ subpopulation (presumed CSCs) showed greater tumorigenic capacity than CD133– cells. It is known that CD133+ HCCs possess high tumorigenicity [17], as well as greater colony-forming efficiency, proliferation, and tumorigenesis in vivo [43]. Tumor formation was also higher in mice injected with pre-irradiated CD133+ cells than in mice injected with unirradiated CD133– cells, suggesting that CD133 expression contributes to radioresistance [44]. The stronger CSC-like properties of Hca-F cells suggests that Hca-F cells may possess greater lymphatic metastasis potential than Hca-P cells, likely because the Hca-F line is enriched in metastatic CSCs. Furthermore, a subpopulation of CD133+/CXCR4+ CSCs has been reported with increased metastatic potential in pancreatic cancer [42]. Both HcaF and Hca-P demonstrated migration and invasiveness, so both may possess subpopulation(s) of metastatic CSCs (mCSCs). It is not

known if the mCSC subpopulation corresponds completely to the CD133+ population or is a unique population within the CSCs. The unique gene expression patterns of CSCs and non-CSCs within tumors have rarely been addressed. Our study demonstrated an inverse relationship between CD133 expression and the expression levels of annexin VII, CDH1, and JNK1. Specifically, these genes were underexpressed in cells with the greatest tumor lymphatic metastasis potential, CD133+ Hca-F cells. Thus, CD133 may be a potential target to improve the specificity of HCC therapy. Analysis of the JNK network revealed an indirect relationship between E-cadherin and annexin VII through JNK1. Indeed, annexin VII and JNK1 are involved in LNM-associated HCC [22–24]. Thus, it is likely that both JNK/P53/annexin VII and JNK/ ATF-2/CDH1/annexin VII signaling pathways are involved in tumor lymphatic metastasis. Annexin VII is an indirect substrate of p53 and ATF-2, which are both activated by JNK. E-cadherin (CDH1) is an epithelial cell adhesion molecule, and decreased CDH1 expression in HCC is associated with poor prognosis [45]. Hence, these findings implicate JNK1, annexin VII, and CDH1 as key regulators of lymphatic metastasis in HCC. Taken together, our data establish a potentially important role for JNK1 in tumor lymphatic metastasis and identify annexin VII and CDH1 as novel downstream regulators. These findings suggest that therapeutic targeting of the JNK/P53/annexin VII and (or) JNK/ ATF-2/CDH1/annexin VII signaling pathway may suppress lymphatic metastasis. 5. Conclusion We reported the identification of a CSC subpopulation derived from HCC cells characterized by a CD133+ phenotype. These CD133+ cells exhibited much stronger stem cell-like properties than CD133– cells. Moreover, the highly metastatic Hca-F cell line exhibited stronger stem cell properties than the Hca-P line with low-metastatic potential. Thus, CD133+ cancer stem cells may be major contributors to tumorigenicity and liver cancer lymphatic metastasis. The correlation between CD133 expression and underexpression of JNK1, annexin VII, and E-cadherin suggests that the JNK/P53/annexin VII and JNK/ATF-2/CDH1/annexin VII signal transduction pathways may regulate cellular metastatic potential and so determine patient prognosis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by The National Natural Science Foundation of China (grant number 81272430, 30772468 and 81071725) and The Educational Department of Liaoning Province (grant number 2008225010-3, 2007T024 and 2009S028). This work was also supported by Tumor Stem cell Research Key Laboratory of Liaoning Province and Key Laboratory of Tumor Metastasis of Liaoning Province University. We would like to thank the Thomson Reuters of authorized trial for building network and pathway. References [1] Ferna´ndez M, Semela D, Bruix J, Colle I, Pinzani M, Bosch J. Angiogenesis in liver disease. J Hepatol 2009;50:604–20. [2] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362:1907–17. [3] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009;59:225–49.

Y. Jin et al. / Biomedicine & Pharmacotherapy 67 (2013) 337–345 [4] Croker AK, Allan AL. Cancer stem cells: implications for the progression and treatment of metastatic disease. J Cell Mol Med 2008;12:374–90. [5] Chen HC, Chou AS, Liu YC, Hsieh CH, Kang CC, Pang ST, et al. Induction of metastatic cancer stem cells from the NK/LAK-resistant floating, but not adherent, subset of the UP-LN1 carcinoma cell line by IFN-g. Lab Invest 2011;91:1502–13. [6] Hou L, Li Y, Jia YH, Wang B, Xin Y, Ling MY, et al. Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice. World J Gastroenterol 2001;7:532–6. [7] Song B, Tang JW, Wang B, Cui XN, Hou L, Sun L, et al. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip. World J Gastroenterol 2005;11:1463–72. [8] Liu S, Sun MZ, Tang JW, Wang Z, Sun C, Greenaway FT, et al. High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers. Rapid Commun Mass Spectrom 2008;22:3172–8. [9] Sun MZ, Liu S, Tang J, Wang Z, Gong X, Sun C, et al. Proteomics analysis of two mice hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastasis rates provide potential protein markers for tumor malignancy attributes to lymphatic metastasis. Proteomics 2009;9:3285–302. [10] Zhang J, Song M, Wang J, Sun M, Wang B, Li R, et al. Enoyl coenzyme A hydratase 1 is an important factor in the lymphatic metastasis of tumors. Biomed Pharmacother 2011;65:157–62. [11] Song MY, Tang JW, Sun MZ, Liu SQ, Wang B. Localization and expression of CLIC1 in hepatocarcinoma ascites cell lines with high or low potentials of lymphatic spread. Zhonghua Bing Li Xue Za Zhi 2010;39:463–6. [12] He L, Zhang J, Jiang L, Jin C, Zhao Y, Yang G, et al. Differential expression of Axl in hepatocellular carcinoma and correlation with tumor lymphatic metastasis. Mol Carcinog 2010;49:882–91. [13] Cui XN, Tang JW, Hou L, Song B, Ban LY. Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. World J Gastroenterol 2006;12:6893–7. [14] Chu H, Zhou H, Liu Y, Liu X, Hu Y, Zhang J. Functional expression of CXC chemokine recepter-4 mediates the secretion of matrix metalloproteinases from mouse hepatocarcinoma cell lines with different lymphatic metastasis ability. Int J Biochem Cell Biol 2007;39:197–205. [15] Zhou H, Jia L, Wang S, Wang H, Chu H, Hu Y, et al. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability. Biochem Biophys Res Commun 2006;345:486–94. [16] Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, Moriwaki H. Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells. Biochem Biophys Res Commun 2006;351:820–4. [17] Yin S, Li J, Hu C, Chen X, Yao M, Yan M, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007; 120:1444–50. [18] Ma S. Biology and clinical implications of CD133(+) liver cancer stem cells. Exp Cell Res 2013;319:126–32. [19] Zhang J, Luo N, Luo Y, Peng Z, Zhang T, Li S. microRNA-150 inhibits human CD133-positive liver cancer stem cells through negative regulation of the transcription factor c-Myb. Int J Oncol 2012;40:747–56. [20] Ma S, Tang KH, Chan YP, Lee TK, Kwan PS, Castilho A, et al. miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1. Cell Stem Cell 2010;7:694–707. [21] Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al. Cancer stem cells – perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006;66:9339–44. [22] Zhang YH, Wang SQ, Sun CR, Wang M, Wang B, Tang JW. Inhibition of JNK1 expression decreases migration and invasion of mouse hepatocellular carcinoma cell line in vitro. Med Oncol 2011;28:966–72. [23] Qazi AS, Sun M, Huang Y, Wei Y, Tang J. Subcellular proteomics: determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation. Biomed Pharmacother 2011;65:407–16.

345

[24] Jin YL, Wang ZQ, Qu H, Wang HX, et al. Annexin A7 gene is an important factor in the lymphatic metastasis of tumors. Biomed Pharmacother [in press]. [25] van der Wurff AA, Arends JW, van der Linden EP, ten Kate J, Bosman FT. L-CAM expression in lymph node and liver metastases of colorectal carcinomas. J Pathol 1994;172:177–81. [26] Yang Z, Zhang L, Ma A, Liu L, Li J, Gu J, et al. Transient mTOR inhibition facilitates continuous growth of liver tumors by modulating the maintenance of CD133+ cell populations. PLoS One 2011;6:e28405. [27] Curtin JC, Lorenzi MV. Drug discovery approaches to target Wnt signaling in cancer stem cells. Oncotarget 2010;1:563–77. [28] Dubrovska A, Kim S, Salamone RJ, Walker JR, Maira SM, Garcı´a-Echeverrı´a C, et al. The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci U S A 2009; 106:268–73. [29] Ou J, Pan F, Geng P, Wei X, Xie G, Deng J, et al. Silencing fibronectin extra domain A enhances radiosensitivity in nasopharyngeal carcinomas involving an FAK/Akt/JNK pathway. Int J Radiat Oncol Biol Phys 2012;82:e685–91. [30] Veit C, Genze F, Menke A, Hoeffert S, Gress TM, Gierschik P, et al. Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for glial cell line-derived neurotrophic factor-induced migration and invasion of pancreatic carcinoma cells. Cancer Res 2004;64:5291–300. [31] Wakamatsu Y, Sakamoto N, Oo HZ, Naito Y, Uraoka N, Anami K, et al. Expression of cancer stem cell markers ALDH1, CD44 and CD133 in primary tumor and lymph node metastasis of gastric cancer. Pathol Int 2012;62:112–9. [32] Neuzil J, Stantic M, Zobalova R, Chladova J, Wang X, Prochazka L, et al. Tumourinitiating cells vs. cancer ‘‘stem’’ cells and CD133: what’s in the name? Biochem Biophys Res Commun 2007;355:855–9. [33] Wei XD, Zhou L, Cheng L, Tian J, Jiang JJ, Maccallum J. In vivo investigation of CD133 as a putative marker of cancer stem cells in Hep-2 cell line. Head Neck 2009;31:94–101. [34] You H, Ding W, Rountree CB. Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-beta. Hepatology 2010;51:1635–44. [35] Ding W, Mouzaki M, You H, Laird JC, Mato J, Lu SC, et al. CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis. Hepatology 2009;49:1277–86. [36] Rountree CB, Barsky L, Ge S, Zhu J, Senadheera S, Crooks GM. A CD133expressing murine liver oval cell population with bilineage potential. Stem Cells 2007;25:2419–29. [37] Rountree CB, Ding W, He L, Stiles B. Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice. Stem Cells 2009;27:290–9. [38] Rountree CB, Senadheera S, Mato JM, Crooks GM, Lu SC. Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice. Hepatology 2008;47:1288–97. [39] Tang KH, Ma S, Lee TK, Chan YP, Kwan PS, Tong CM, et al. CD133(+) liver tumorinitiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling. Hepatology 2012;55:807–20. [40] Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003;63:5821–8. [41] Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature 2004;432:396–401. [42] Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;13:313–23. [43] Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007;132:2542–56. [44] Piao LS, Hur W, Kim TK, Hong SW, Kim SW, Choi JE, et al. CD133+ liver cancer stem cells modulate radioresistance in human hepatocellular carcinoma. Cancer Lett 2012;315:129–37. [45] Chien MH, Yeh KT, Li YC, Hsieh YH, Lin CH, Weng MS, et al. Effects of E-cadherin (CDH1) gene promoter polymorphisms on the risk and clinicopathological development of hepatocellular carcinoma. J Surg Oncol 2011;104:299–304.