Background
Pancreatic cancer is one of the most aggressive and lethal malignancies and ranks as the eighth most common cause of death from cancer worldwide [
1,
2]. Despite the wide application of surgical resection, chemotherapy and/or chemoradiotherapy, pancreatic cancer still has the worst prognosis of any major malignancy with a 5-year survival rate of <5% and a median survival of <6 months [
3,
4]. Therefore, it will be of great clinical value to identify effective treatment strategies and explore the molecular mechanisms responsible for the pathogenesis of pancreatic cancer to improve the prognosis of pancreatic cancer patients.
Increasing evidence suggests that unsatisfactory therapeutic outcomes and the poor prognosis associated with pancreatic cancer are related to aberrantly activated signaling pathways, including NF-κB signaling [
5,
6]. Constitutive activation of NF-κB signaling pathway plays an important role in the development and progression of pancreatic cancer and contributes to a malignant phenotype. Arlt et al. reported that constitutive NF-κB activity confers resistance to gemcitabine-induced cell death in pancreatic cancer, while inhibition of NF-κB strongly diminishes gemcitabine resistance [
5]. Maier and colleagues showed that NF-κB activation contributes to epithelial-mesenchymal transition, migration and invasion of pancreatic carcinoma cells, while these malignant phenotypes are suppressed upon NF-κB inhibition [
7]. It has been reported that activation of NF-κB signaling pathway also promotes pancreatic tumor angiogenesis [
8] and metastasis [
8,
9]. Conversely, inhibition of NF-κB signaling pathway increases the number of apoptotic pancreatic cells [
10] and inhibiting constitutive NF-κB activity by expressing phosphorylation defective IκBα significantly suppresses pancreatic cancer cell tumorigenesis [
11]. Therefore, identifying novel molecules that modulate the NF-κB signaling pathway could be important for pancreatic cancer therapy.
G protein-coupled receptor (GPCR) 87, a newly identified gene located on chromosome 3q24, encodes a protein that contains an extracellular N terminus, seven helices, three intracellular loops, three extracellular loops and an intracellular C terminus [
12]. It is a cell surface GPCR that is overexpressed in various cancers and plays vital role in tumor cell survival [
13,
14]. A previous study using GPCR-focused Affymetrix microarrays to examine the expression of 929 GPCR transcripts in tissue samples from 10 patients with lung squamous cell carcinoma identified GPR87 as one of five GPCRs significantly overexpressed in this disease [
15]. NII and colleagues reported that overexpression of GPR87 in non-small cell lung carcinoma (NSCLC) is significantly correlated with poorer differentiation and that higher expression of GPR87 is significantly associated with poorer survival for patients with NSCLC [
16]. Moreover, GPR87 is essential for p53-dependent cell survival of RKO and MCF7 cell lines in response to DNA damage induced by doxorubicin or camptothecin and may serve as a novel therapeutic target for cancer treatment and prevention [
17]. Zhang et al. reported that GPR87 is overexpressed in bladder cancer and promotes cell proliferation in bladder cancer cells [
18]. Importantly, overexpression of GPR87 upregulates expression of cancer stem cell marker CD133 and promotes the growth and metastasis of CD133
+ cancer stem-like cells in hepatocellular carcinoma [
19].
Herein, we reported that GPR87 expression was significantly upregulated in pancreatic cancer and clinical tissues, and was correlated with the clinical features of pancreatic cancer. Overexpression of GPR87 promotes proliferation, metastasis, angiogenesis and resistance to apoptosis induced by a chemotherapeutic agent in pancreatic cancer. Our findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.
Discussion
In the present study, we provided evidence for a novel link between GPR87 and the oncogenic NF-κB signaling pathway in pancreatic cancer. IHC analysis revealed that GPR87 was significantly upregulated in pancreatic cancer and correlated with clinical features and a poor prognosis of pancreatic cancer patients. Overexpression of GPR87 dramatically increased the anchorage-independent growth ability and the invasive capacity of pancreatic cells, promoted their ability to induce HUVEC tube formation and CAM neovascularization, and enhanced their resistance to apoptosis, while downregulation of GPR87 expression produced the opposite effects. These findings emphasize the prominent roles of oncogenic GPR87 in promoting carcinogenesis and progression of pancreatic cancer via NF-κB signaling pathway.
It is well established that the NF-κB signaling pathway not only regulates immune and inflammatory responses, but also plays an important role in oncogenesis [
20,
21]. Therapeutic targeting of NF-κB signaling has been aggressively pursued for the treatment of a wide range of malignant pathologies in pancreatic cancer [
5,
22‐
24]. Inhibition of NF-κB pathway by MG132, or sulfasalazine dramatically diminishes resistance to apoptosis induced by gemcitabine in multiple pancreatic cancer cell lines [
5]. Additionally, NF-κB pathway inhibitor sodium salicylate inhibits phosphorylation and degradation of NF-κB negative regulator IκBα and subsequently enhances TNF-α induced apoptosis in BxPC-3 human pancreatic cancer cells [
22]. Treatment with nafamostat mesilate, an NF-κB inhibitor, significantly decreases the volume and weight of nude mice xenografts derived from pancreatic cancer cells compared with the control group [
23]. Furthermore, a nude mouse model was established and revealed that the number of liver metastases derived from pancreatic cancer cells was significantly decreased in the treatment group with the NF-κB inhibitor DHMEQ, compared with the control group [
24]. Collectively, these findings provide a strong rationale for therapeutic targeting NF-κB signaling pathway in pancreatic cancer. Nevertheless, it is still a formidable challenge to archive improved treatment outcomes up to now. Although potential therapeutic approaches, such as the use of NF-κB or IKKβ inhibitors, could potentially exert anti-tumor effects on the cancer-promoting activities of NF-κB, they interfere with its various physiological functions in normal cells, such as functions in immunity and inflammation [
21,
25]. Therefore, more effective therapeutic targets that regulate NF-κB signaling in an appropriate manner is warranted as an alternative to global NF-κB blockade. Herein, our study showed that GPR87 was overexpressed in pancreatic cancer and associated with poor survival of pancreatic cancer patients. Overexpressing GPR87 significantly promoted malignant phenotype including increased proliferation, angiogenesis, invasion and resistance to chemotherapeutic agent via impairing NF-κB signaling, while silencing GPR87 suppressed these effects. Meanwhile, we found that overexpressing GPR87 in HPDEC-1 dramatically increased the BrdU-positive cells and induced HUVEC tube formation (Additional file
8: Figure S6A-C). Furthermore, overexpression of GPR87 could also increase the proliferating rate of the esophageal squamous cell carcinoma (ESCC) cells (Additional file
8: Figure S6D-F), while silencing GPR87 produced the opposite effects, providing further evidence that GPR87 function as an onco-protein.
GPCRs represent the largest family of cell-surface molecules involved in signal transmission by far with more than 800 members, accounting for >2% of the total genes encoded by the human genome. These receptors regulate many cell functions, including cell proliferation, survival and motility, and have recently emerged as key players in processes contributing to tumor progression such as tumor growth, angiogenesis and metastasis [
26]. GPR55, for example, has been reported to be upregulated in human squamous cell carcinomas and to enhance skin cancer cell anchorage-independent growth, invasiveness and tumorigenicity in vivo [
27]. Overexpression of PAR1, a GPCR, promotes invasion and metastasis in breast cancer cells and inhibition of PAR1 signaling suppresses HMGA2-driven invasion in breast cancer cells [
28]. P2Y2, a GPCR, is significantly overexpressed in prostate cancer and promotes cell invasion and metastasis both in vitro and in vivo [
29]. Stevenson et al. reported that silencing the GPCR G-protein–coupled receptor galanin receptor 1 (GalR1) enhanced the effects of chemotherapy in drug-sensitive and resistant colorectal cancer cell lines [
30]. Herein, we found that GPR87 was upregulated in pancreatic cancer and GPR87 overexpression promoted pancreatic tumor progression both in vivo and in vivo, which is in agreement with the oncogenic effects of GPR87 family members.
Accumulating evidence suggests that NF-κB signaling pathway can be activated by members of the GPCR family. Liu et al. reported that somatostatin type 2 receptor (SSTR2), a member of the GPCRs, activates NF-κB signaling pathway by leading to phosphorylation of IKKα/β and degradation of IκBα in pancreatic tumor cell line AR42J [
31]. A previous study revealed that CXCL12 specifically signals through CXCR4, a member of the GPCRs to activate Akt and ERK and subsequently phosphorylate IκBα, causing activation of NF-κB signaling in pancreatic cancer cells [
32]. Additionally, Lee and colleagues reported that activation of the BLT2 (a GPCR)-linked pathway leads to NOX-derived ROS generation and subsequent activation of NF-κB in prostate cancer cells [
33]. Concordant with previous reports, our study showed that GPR87, a member of the GPCRs, could activate NF-κB signaling pathway in pancreatic cancer cells and that GPR87 levels were significantly associated with NF-κB activity in clinical pancreatic cancer tissues. However, the mechanism by which GPR87 activates NF-κB signaling pathway has yet to be elucidated and is the subject of current investigations by our group.
The mechanisms involved in GPR87 upregulation also remain to be characterized. According to TCGA [
34], we found amplification of GPR87 in 25 of 184 cases, suggesting that amplification might account for GPR87 overexpression in 13.59% of pancreatic cancer patients. Interestingly, according to ChIP sequencing tracks in the University of California Santa Cruz (UCSC) genome browser (
http://genome.ucsc.edu/cgi-bin/hgGateway) [
35], the promoter region of GPR87 showed STAT3 binding elements, suggesting that tumor inflammatory microenvironment-mediated hyperactivation of JAK2/STAT3 signaling might contribute to GPR87 upregulation in pancreatic cancer. Thus, it would be of great interest to further investigate whether upregulation of GPR87 in pancreatic cancer is attributed to STAT3-mediated transcriptional upregulation. Apart from overexpression of GPR87, ligands, such as lysophosphatidic acid (LPA), in the tumor microenvironment may stimulate GPR87 and subsequently lead to constitutive activation of cancer-promoting signaling [
36]. LPA is one of the most potent mitogens secreted in the tumor-associated environment including the ascites fluid produced by ovarian cancer cells, stimulating the LPA-sensitive GPCRs that are frequently overexpressed by these tumor cells and thus contributing growth, survival and resistance to chemotherapy [
37]. Meanwhile, a recent study also showed that Gαi and Gαq contribute to GPR87-mediated NF-κB activation in the 293 T cells, which is ligand-independent [
38], further support the notion that functional NF-κB activation is critical for GPR87-mediated cancer progression.
Methods
Cell lines and cell culture
Human pancreatic cancer cell lines, including AsPC-1, Capan-1, BxPC-3, Capan-2, PANC-1 and MIA PaCa-2 were grown in the DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). Primary cultures of normal human pancreatic ductal epithelial cells (HPDECs) were maintained in keratinocyte serum-free medium (KSFM; Invitrogen) with EGF (5 ng/ml) and BPE (50 μg/ml). All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
A total of 96 paraffin-embedded and archived pancreatic cancer samples, which were histopathologically and clinically diagnosed at the Union Hospital, Huazhong University of Science and Technology, from 2001.12 to 2009.01, were examined in this study. All the cases were recruited only based on positive diagnosis of pancreatic cancer without other additional criteria, and all the recruited cases were under standard treatment with chemotherapeutic agent gemcitabine. Survival time months correspond to time after diagnosis. Ten pairs of matched pancreatic cancer/adjacent noncancerous tissues and other 10 pancreatic cancer tissues were frozen and stored in liquid nitrogen until further use. Clinical information on the samples is summarized in Additional file
2: Table S1. The use of human tissue was approved by the local ethics committee (Tongji Medical College, China) and written informed consent was obtained from patients prior to surgery.
Vectors, retroviral infection and transfection
The human GPR87 gene was PCR-amplified from cDNA and cloned into a pMSCV-puro retroviral vector and GPR87-targeting short hairpin RNA (shRNA) oligonucleotides sequences were cloned into pSuper-retro-puro to generate pSuper-retro-GPR87-RNAi(s). pNF-κB-luc and control plasmids (Clontech) were used to examine NF-κB activity. pBabe-puro-IκBα-mut (plasmid#15291) expressing IκBα dominant-negative mutant (IκBα-mut) was purchased from Addgene (Cambridge, MA). Transfection of siRNA or plasmids was performed using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instruction. Stable cell lines expressing GPR87 or GPR87 RNAi were selected for 10 days with 0.5ug/ml puromycin 48 h after infection.
Western blotting analysis
Western blot was performed using anti-GPR87 (Abcam), anti-p-IκBα, IκBα and anti-p-IKKβ, IKKβ, anti-p65, anti-p84 antibodies (Cell Signaling Technology). The membranes were stripped and re-probed with an anti-α-tubulin antibody (Sigma) as a loading control.
Immunohistochemistry (IHC)
IHC analysis was performed to study altered protein expression in 96 paraffin-embedded and archived pancreatic cancer with GPR87 antibody (Abcam, 1:500). The degree of immunostaining of formalin-fixed, paraffin-embedded sections were reviewed and scored separately by two independent pathologists uninformed of the histopathological features and patient data of the samples. The scores were determined by combining the proportion of positively-stained tumor cells and the intensity of staining. The scores given by the two independent pathologists were combined into a mean score for further comparative evaluation. Tumor cell proportions were scored as follows: 0, no positive tumor cells; 1, <10% positive tumor cells; 2, 10%–35% positive tumor cells; 3, 35–75% positive tumor cells; 4, >75% positive tumor cells. Staining intensity was graded according to the following standard: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); 4, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated protein expression in malignant lesions by determining the SI, with possible scores of 0, 2, 3, 4, 6, 8, 9, 12, and 16. Then the median value, SI = 8, was chosen as the cut off value, which samples with a SI ≥ 8 were determined as high expression and samples with a SI < 8 were determined as low expression.
Invasion assay
Transwell inserts for 24-well plates (Corning Costar Corp., Cambridge, MA) were coated with prediluted Matrigel (BD Biosciences, Bedford, MA) and allowed to gel at 37 °C for 30 min. Cells were seeded at a density of 3 × 105 per insert and the lower chamber of the Transwell was filled with 500 μL DMEM supplemented with 10% FBS. After 24 h of incubation, cells remaining on the upper surface of the Transwell membrane were removed by a cotton swab. Cells that had invaded through the Matrigel to the bottom of the insert were fixed, stained, photographed and quantified by counting them in 6 random high-powered fields.
Chicken chorioallantoic membrane (CAM) assay
CAM assay was performed at day 6 of fertilized chicken eggs using a method previously described [
39]. A 1.0-cm diameter window was opened on the egg shell (Huahao Breeding Co. Ltd, Hubei, China). The surface of the dermic sheet on the floor of the air sac was removed to expose the CAM. A 0.5-cm diameter filter paper was first placed on top of the CAM, and 100 μl conditioned medium was added onto the center of the paper. After the window was closed with sterile adhesive tape, the eggs were incubated at 37 °C under 80–90% relative humidity for 4 days. Following fixation with stationary solution (methanol: acetone = 1:1) for 15 min, the CAMs were cut and harvested, and gross photos of each CAM were taken with a digital camera (Panasonic, Osaka, Japan). The effect of conditioned media harvested from different cultured cells was evaluated by the number of second- and third-order vessels.
Briefly, 200 μl of precooled Matrigel (Collaborative Biomedical Products) was transferred into each well of a 24-well plate and polymerized for 30 min at 37 °C. HUVECs (2 × 104) in 200 μl of conditioned medium were added to each well and incubated at 37 °C, 5% CO2 for 20 h. The capillary tube structure was photographed under a 100 × bright-field microscope, and quantified by measuring the total length of the completed tubes. Each condition was assessed in triplicate.
Animal studies, IHC, and H&E staining
BALB/c-nu mice (4–5 weeks of age, 18-20 g) were purchased from Vitalriver (Beijing, China). All experimental procedures were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. The BALB/c nude mice were randomly divided into two groups (n = 6/group). One group of mice was inoculated subcutaneously with PANC-1/Vector cells (2 × 106) in the left dorsal flank and with PANC-1/GPR87 cells (2 × 106) in the right dorsal flank per mouse. Another group was inoculated subcutaneously with PANC-1/RNAi-vector cells (2 × 106) in the left dorsal flank and with PANC-1/GPR87-RNAi cells (2 × 106) in the right dorsal flank. Tumors were examined twice weekly; length and width measurements were obtained with calipers and tumor volumes were calculated using the equation (L × W2)/2. On day 36, tumors were detected by an IVIS imaging system, and animals were euthanized, tumors were excised, weighed and paraffin-embedded. Serial 6.0 μm sections were cut and subjected to IHC analyzed using an anti-Ki67 and anti-CD31 antibodies (Dako). Proliferation index was quantized by counting proportion of Ki67-positive cells. Apoptotic index was measured by percentage of TUNEL-positive cells.
Microarray data process and analysis
The publicly-available gene expression dataset GSE16515 was assessed through the Gene Expression Omnibus (GEO) Datasets website
http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-16515/?query=gse16515. This array consists of 36 tumor samples and 16 normal samples; a total of 52 samples. 16 samples consist of both tumor and normal expression data, whereas 20 samples consist of only tumor data. This microarrays was used to identify the expression differences of genes between the pancreatic tumor and normal samples.
Statistical analysis
Statistical tests for data analysis included Fisher’s exact test, log-rank test, Chi-square test, and Student’s 2-tailed t test. Statistical analyses were performed using the SPSS 11.0 statistical software package. Data represent mean ± SD. p < 0.05 was considered statistically significant.
Acknowledgements
This work was supported by Natural Science Foundation of China (81272658, 81572436), the National Natural Science Foundation of Hubei Province, China (No. 2015CFB621) and the Fundamental Research Funds for the Wuhan Union Hospital, China (No: 02.03.2015-57).