Journal of Translational Medicine
Tang et al. J Transl Med (2017) 15:238 DOI 10.1186/s12967-017-1342-1
Open Access
RESEARCH
Autocrine parathyroid hormone‑like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK‑ATF2‑cyclinD1 signaling Jing Tang1†, Yan Liao1†, Shuying He1†, Jie Shi1†, Liang Peng1†, Xiaoping Xu1, Fang Xie1, Na Diao1, Jinlan Huang2, Qian Xie1, Chuang Lin4, Xiaoying Luo1, Kaili Liao1, Juanjuan Ma3, Jingyi Li1, Daichao Zhou1, Zhijun Li1, Jun Xu1, Chao Zhong1, Guozhen Wang1 and Lan Bai1*
Abstract Background and aims: Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development. Methods: The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2). Results: PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists. Conclusions: Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients. Keywords: Parathyroid hormone-like hormone, Activating transcription factor-2, Proliferation, Intrahepatic cholangiocarcinoma
*Correspondence:
[email protected];
[email protected] † Jing Tang, Yan Liao, Shuying He, Jie Shi and Liang Peng contributed equally to this work 1 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou Avenue North, Baiyun District, Guangzhou, Guangdong, China Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Tang et al. J Transl Med (2017) 15:238
Background Intrahepatic cholangiocarcinoma arises from epithelial cells lining the bile duct and occurs proximally at the second degree bile ducts within the liver. The condition is commonly associated with cirrhosis, viral hepatitis B and C [1]. ICC displays a feature of rapid progression and a poor outcome, and its global disease incidence has been rapidly increasing [2]. Radical resection and curative liver transplantation are preferred surgical treatments for ICC, however, the patients with vascular and lymph nodes metastases are not eligible for surgical therapy. Although the chemotherapy regimen of gemcitabine and cisplatin and locoregional therapies are additional options for inoperable ICC patients, the 5-year survival rates are very low. An enhanced understanding of the biology pathological progress and the interaction with tumor microenvironment of ICC is needed to improve patient survival. PTHLH, also referred to parathyroid hormone-related protein (PTHrP), has emerged as an important cytokine with diverse cell functions, including growth, survival, migration, and differentiation [3]. Tumor-derived PTHLH participates in the bone metastatic processes of breast cancer via an intracrine fashion [4]. In addition, PTHLH supports colorectal cancer cell to form distant lung metastatic processes via inducing caspase-independent death in human lung vasculature endothelial cells [5]. A previous report demonstrated that PTHLH produced by proliferating bile duct epithelial cells and may interact with growth factors and hormones to form complex loops that promotes proliferation [6]. Growing evidences indicate that PTHLH-producing cholangiocarcinoma (CHO) patients suffer from humoral hypercalcemia of malignancy [7–9], but litter is known regarding PTHLH’s effect on ICC cells growth. MAP kinase pathways are involved in the process through the PTHLH-induced activation of PTH1R to activate downstream effectors [10–12]. ATF2, as a downstream effector of MAPK in response to cytokines, is phosphorylated on Thr69 and/or Thr71 by either JNK or p38 [13, 14], and is also activated by the ERK1/2 pathway in two step [13]. Several observations support that ATF2 regulates cell cycle progression via controlling the transcriptional output of several key genes, including CyclinD1, CyclinA and RB1 [15–18]. However, no studies have documented a role for the PTHLH-MAPK-ATF2-CyclinD1 signaling axis in the regulation of ICC cells growth. This study aims to elucidate the role and clinical significance of the PTHLH-MAPK-ATF2-CyclinD1 axis in ICC cell cycle progression. Methods Patients, tissue samples and microarrays
59 ICC samples and paired non-tumor tissues and 10 normal tissues were obtained from the Department of
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Hepatobiliary Surgery, NanFang Hospital, Southern Medical University between 2014 and 2016. All patients signed informed consent for therapy and subsequent tissue studies, which were approved by the NanFang Hospital Institutional Review Board. The ICC tissue microarrays, which contained 100 cases, and the extrahepatic cholangiocarcinoma (ECC) tissue microarrays, which contained 27 cases were purchased from Shanghai Outdo Biotech Inc. (Shanghai, China). All tumors were defined as a primary tumor arising from the bile ducts and diagnosed as adenocarcinomas. Tumor stage was defined according to the seventh edition of American Joint Committee on Cancer/International Union against Cancer (AJCC/UICC). All specimens were used for routine pathological processing with comparable clinicopathological features, and complete follow-up data were obtained. Western blot, real‑time PCR analysis, immunohistochemistry and immunofluorescence
RNA and protein lysate extraction, cDNA synthesis, final real-time PCR and western blots were performed according to general protocols. ICC cells were processed for immunofluorescence (IF) using target antibodies with optimized conditions. In addition, human samples and ICC microarrays were subjected to IHC staining to evaluate the expression of relative proteins. Cell counting kit‑8 assay, colony formation assays, cell cycle analysis, cell migration and invasion assay
Cell counting kit-8 assay, colony formation assays, cell cycle analysis, cell migration and invasion assay and Annexin V apoptosis assay were performed according to general protocols and can be found in Additional file 1. Dual‑luciferase reporter gene assay
To determine the effect of ATF2 on PTHLH transcription, RBE cells were transfected with pGL3 as vehicle control, pGL3-PTHLH or pGL3-MUT-PTHLH using Lipofectamine 3000. Firefly and Renilla luciferase activities were measured separately on a fluorescence spectrophotometer (FlOUstar omega, BMG Labtech, Germany) in triplicate according to the manufacturer’s instructions for the dual-luciferase reporter assay kit (Promega). The relative transcriptional activity was normalized by the corresponding vehicle control value. Chromatin immunoprecipitation (ChIP) assay
Genomic DNA prepared from RBE cells transfected with shControl was crosslinked with 1% formaldehyde and fragmented into 500 ± 100-bp fragments by sonication. Soluble chromatin was then incubated overnight with anti-ATF2 antibodies. Finally, the immunoprecipitated
Tang et al. J Transl Med (2017) 15:238
DNA fragments were amplified and quantified using realtime PCR using the following PCR primers specific to the human PTHLH promoter region. Establishment of a subcutaneous tumor xenograft
RBE cells (shCtrl or shPTHLHx) (1 × 107) were injected subcutaneously into the groins of BALB/c nude mice (6 weeks old, male, n = 5 for each group). Tumor growth was monitored at 2 or 3-day intervals. When the mice were sacrificed after 25 days, tumor weight and size were measured, and the tumor was fixed for additional experimental use. Statistical analyses
Different statistical analysis methods were used to compare different groups or different categories of data. Extended details regarding materials and methods can be found in Additional file 1.
Results PTHLH is highly expressed in human CHO tissues specimens and ICC cell lines
To identify the potential role of PTHLH in CHO, we evaluated 59 ICC samples and paired non-tumor tissues from NanFang Hospital. We also screened an additional 10 samples of normal liver tissues for comparison. IHC analyses of ICC tumor regions revealed strong staining of PTHLH compared with that in adjacent regions in the same patients (Fig. 1a, top panel and b). Immunostaining of PTHLP protein was located in the cytoplasm and nucleus of ICC cells. We also observed weak staining in the bile duct of adjacent and normal tissue samples (Fig. 1a, top panel). In addition, we detected the expression of PTH1R, a specific receptor for PTHLH, in membranes of ICC cells (Fig. 1a, middle panel). Cytokeratin19 (CK) staining revealed the presence of adenocarcinoma cells and biliary epithelial cells (Fig. 1a, bottom panel). In addition, to further confirm the expression of PTHLH in CHO, we screened ICC microarrays that contained 100 cases and the ECC microarrays that contained 27 cases (Fig. 1c and Additional file 1: Figure S1). Our results were consistent with the conclusion above that PTHLH was highly expressed in CHO cells. In addition, PTHLH protein expression was examined in ICC cell lines by IF microscopy (Fig. 1e). Microscopy analysis detected cytosolic and nucleus expression of PTHLH in RBE and HCCC-9810 cells, which is consistent with previous observations. Overexpression PTHLH is positively correlated with poor pathological differentiation in ICC patients
To investigate the clinical significance of PTHLH upregulation in CHO, we further analyzed the relationship
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between clinicopathological features and PTHLH expression levels in CHO cases. These patients were divided into high- (score, 2–3) or low- (score, 0–1) PTHLH expression groups according to the immunostaining scores (Fig. 1c, d). Scoring was conducted according to the ratio and intensity of positive-staining cells: 0–5% scored 0; 6–35% scored 1; 36–70% scored 2; more than 70% scored 3. The final score was designated as low or high expression group as follows: score 0–1, low expression, score 2–3, high expression. A high expression of PTHLH was positively correlated with poor pathological differentiation (p
