Adrenomedullin promotes intrahepatic cholangiocellular carcinoma metastasis and invasion by inducing epithelial-mesenchymal transition

Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its etiology and molecular pathogenesis remain largely unknown. The present study aimed to investigate the association between adrenomedullin (ADM) and epithelial-mesenchymal transition (EMT) in ICC and to elucidate the underlying signaling pathway. We evaluated the clinical significance of ADM in 133 ICC patients using tissue microarray analysis of ICC tissues. We also investigated the mechanisms of ADM in ICC EMT-mediated metastasis in cholangiocarci-noma cell lines in vitro. The results revealed that ADM was upregulated in human ICC tissues (73/133) compared with that in healthy controls. ADM expression was positively correlated with shorter overall survival (P<0.01). The characteristics of EMT were induced in vitro by adenoviral transduction of ADM into HuCCT1 cells, resulting in the downregulation of E-cadherin and ZO-1, and the concomitant upregulation of N-cadherin and vimentin. Knockdown of ADM by short hairpin RNA in HUH28 cells expressing high levels of ADM was associated with the reversal of EMT. Functional studies revealed that ADM regulated the activation of ZEB1, which subsequently mediated EMT. The results of the present study suggest that ADM plays an important role in ICC metastasis, and that ADM signaling of EMT may represent a valuable therapeutic target in cancer patients.


Introduction
Intrahepatic cholangiocarcinoma (ICC) is defined as a cholangiocarcinoma located proximally to the second-degree bile ducts. It is an aggressive neoplasm associated with extremely poor survival. The incidence and mortality of ICC have increased worldwide over the past two decades (1)(2)(3). Surgical treatment is the only curative treatment option for ICC, and the 5-year survival rate for patients with unresectable ICC is currently <5% (4), compared with 20-44% in patients undergoing resection at early T1-T2 stages. Tumor recurrence and metastasis are common in ICC (5), and the prevention of recurrence is thus the key to improving patient overall survival (OS). Adrenomedullin (ADM) is a multifunctional regulatory and vasoactive peptide originally isolated from human pheochromocytoma (6). ADM overexpression has been detected in human breast, lung, ovarian, pancreatic, prostate and renal cancers (7)(8)(9). However, the physiologic significance of ADM in ICC metastasis and its underlying molecular mechanism are largely unknown. A better understanding of the biological characteristics of ICC that contribute to tumor invasion and metastasis is paramount for developing novel strategies to treat this cancer.

Materials and methods
Clinical samples and cell lines. This study included 80 men and 53 women with an average age of 55.6±12. 8  RT-PCR was performed using specific TaqMan probes and primer sets to examine ADM RNA expression levels. TATA-box-binding protein (TBP), which is considered to be a reliable reference gene for quantitative PCR normalization in HCC specimens, was used as a control (10). Commercialized probes and primer sets specific for ADM and TBP were purchased from Applied Biosystems (Foster City, CA, USA). TaqMan primers and probes for ADM and TBP were designed using Primer Express (Applied Biosystems). The transcripts were amplified with the TaqMan One-Step RT-PCR Master Mix reagent and ABI Prism 7900HT sequence detection system (both from Applied Biosystems). The expression levels of the tested genes were quantified in relation to the expression of TBP using sequence detector software and the relative quantification method (Applied Biosystems). The relative ADM mRNA levels were determined using the 2 -ΔΔCT method (11).
Tissue microarray (TMA) and IHC. A tissue microarray was constructed as described previously (12). Samples were taken from each representative tumor tissue and from liver tissue adjacent to the tumor (within 10 mm) to construct TMA slides (in collaboration with the Shanghai Biochip Company Ltd., Shanghai, China). Duplicate tissue cylinders were obtained from intratumoral and peritumoral areas (a total of 4 punches for each patient). Tissues were incubated with primary rabbit anti-ADM monoclonal antibody (1:200; Abcam, Cambridge, MA, USA), according to previously described IHC protocols (13), using the EnVision Plus detection system (EnVision; Dako, Carpinteria, CA, USA). Reaction products were visualized by incubation with 3,3-diaminobenzidine. Semi-quantitative analysis of IHC staining was performed by two experienced pathologists in two sections of each specimen in 10 fields from each section (magnification, x200). Immunostaining scoring was based on the intensity of staining and the percentage of positively stained cells: negative (-), 0-5%; intermediate (+), >5-10%; moderate (++), >10-25%; strong (+++), >25%. ADM staining ≥5% was considered positive.

Construction of recombinant plasmids and transfection.
Full-length human ADM cDNA was amplified by PCR and cloned into the pEGFP-N1 expression vector (Clontech, Palo Alto, CA, USA) to construct pEGFP-N1-ADM, and then transfected into HuCCT1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with pEGFP-N1 were used as a negative control. Stable ADM-expressing clones were selected using geneticin (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 500 µg/ml.

Establishment of ADM-knockdown cells.
Lentivirus containing short hairpin RNAs targeting ADM was purchased from GeneCopoeia (Rockville, MD, USA) and transfected into HUH28 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with the empty vector were used as controls. Stable clones were selected using puromycin (final concentration, 2 µg/ml).
In vitro cell behavior assay. Cellular proliferation was assayed in cells seeded at a density of 5x10 3 cells/well in 96-well plates. The proliferation of the transfected cells was measured

Results
Expression of ADM in human ICC. The significance of ADM expression in ICC patients was investigated by IHC (Fig. 1). ADM staining was mainly located in the cytoplasm of tumor cells. Most stromal cells were negative for ADM. As shown in Fig. 2, ADM mRNA expression was significantly higher in the ICC tumor tissues compared with that in the peritumoral and healthy liver tissues, according to RT-PCR. We investigated the clinical significance of ADM overexpression in ICC by tissue microarray analysis of ICC tissues from 133 patients who underwent resection (Table I). Kaplan-Meier analysis revealed that patients with high ADM expression had poorer OS and shorter time to recurrence (TTR) (both P<0.01, Fig. 3). Univariate Cox regression analysis identified differentiation, encapsulation and ADM overexpression as factors significantly associated with OS, while multivariate Cox proportional hazards regression analysis identified differentiation and ADM overexpression as independent prognostic factors for OS in ICC patients (Table II).

Effect of ADM on ICC cell growth.
To confirm the involvement of ADM in the growth of ICC cells, we transfected HuCCT1 cells with ADM and silenced HUH28 cells with short hairpin RNA, respectively. ADM expression was determined by western blotting (Fig. 4A). The tumorigenicity of ADM was  determined by functional assays. The cell growth rates in the ADM-transfected cells were significantly higher than the rates in the control cells (P<0.01, Fig. 4B). ADM-transfected cells also formed significantly more and larger colonies (P<0.01) than the control cells (Fig. 4C).

Effects of ADM on ICC cell migration and tumor metastasis.
The roles of ADM in tumor cell migration and invasion were investigated by wound-healing and Transwell invasion assays. ADM-transfected cells achieved faster closure of the scratched ̔wounds' than this rate in the control cells (Fig. 5A). Furthermore, cell motility and invasion in the Transwell invasion assays were significantly increased by ADM transfection (P<0.05, Fig. 5B). Opposite results were obtained in the ADM-silenced cells.

Effect of ADM on epithelial-mesenchymal transition (EMT) in ICC.
EMT is one of the key events in tumor invasion and metastasis. We therefore investigated the effects of ADM on EMT by analyzing the expression levels of EMT markers and EMT-related transcription factors, and by morphological changes in the ICC cell lines. Western blotting demonstrated decreased expression levels of the epithelial markers E-cadherin and ZO-1, and increased expression of the mesenchymal markers vimentin and N-cadherin, and the EMT-related transcription factors ZEB1 and ZEB2 in the ADM-transfected cells (Fig. 6), compared with the control cells. Opposite expression patterns of these genes were observed in the ADM-silenced cells.

Discussion
ADM expression has been demonstrated in many human malignant cells (14,15), but to the best of our knowledge, the present study is the first to report ADM expression and its specific mechanisms in ICC. ADM protein was highly expressed in ICCs according to IHC, while ADM mRNA expression levels were higher in the tumor tissues compared with that in the peritumoral and healthy tissues. Overexpression of ADM was significantly associated with poorer OS (P<0.001) and tumor recurrence (P<0.01). These results suggest that ADM acts as an oncogene with an important role in ICC progression.
EMT is considered to be a critical mechanism involved in cancer metastasis (16,17); however, compared with other types of human cancers, few studies have focused on the significance of EMT in ICC (18,19). Our functional studies demonstrated that ADM had strong tumorigenicity, with its overexpression promoting cell growth, migration and invasion. These results Hazard ratio (95% CI) P-value a Hazard ratio (95% CI) P-value a were in agreement with those of previous studies (8,20). The two main types of primary liver cancer are HCC and ICC. Although ADM may promote metastasis in both ICC and HCC, the mechanisms appear to differ, possibly because of the different origins of the two tumors. ICC results from the malignant transformation of cholangiocytes. Gene expression profiles analyzed by microarray identified ADM as a metastasis-associated gene.
Previous IHC analysis showed that HCC samples with intrahepatic metastasis expressed strong ADM immunoreactivity in the cytoplasm of tumor cells (21). ADM signaling has been reported to be hypoxia-inducible and functionally active in HCCs (20). In the present study, morphological changes in ICC cells suggest a link between the biological function of ADM and EMT induction. As anticipated, mesenchymal markers were significantly upregulated in stable ADM transfectants, whereas epithelial markers were significantly downregulated. In addition, ADM silencing was associated with increased expression of epithelial markers and decreased levels of mesenchymal markers. ZEBl is the most important transcription factor in the regulation of EMT in epithelial cells (22,23). The present study provides the first evidence demonstrating that ADM induces EMT through ZEB1. Our study had some limitations. First, although animal models offer an opportunity to bridge the gap between in vitro   findings and clinical applicability, such animal experiments were not performed in the present study because of a lack of time and experimental conditions. Second, we focused mainly on patients with resectable ICC at a single institution, and further multicenter studies are needed to validate the findings.
In conclusion, overexpression of ADM in human ICC cells led to increased growth, invasion and metastasis in vitro. High ADM levels in clinical ICC specimens correlated with poor prognosis. These findings suggest that ADM should be evaluated as a potential novel therapeutic target in ICC with the aim of improving the currently poor outcome of this disease.