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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Molecular Cancer 1/2017

Overexpression of G protein-coupled receptor GPR87 promotes pancreatic cancer aggressiveness and activates NF-κB signaling pathway

Zeitschrift:
Molecular Cancer > Ausgabe 1/2017
Autoren:
Li Wang, Wei Zhou, Yunfeng Zhong, Yongbao Huo, Ping Fan, Sudong Zhan, Jun Xiao, Xin Jin, Shanmiao Gou, Tao Yin, Heshui Wu, Tao Liu
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12943-017-0627-6) contains supplementary material, which is available to authorized users.

Abstract

Background

Pancreatic cancer is a highly lethal disease and has the worst prognosis of any major malignancy. G protein-coupled receptor GPR87 is reported to be overexpressed in multiple cancers. The clinical significance and biological role of GPR87 in pancreatic cancer, however, remain to be established.

Methods

GPR87 expression in pancreatic cancer cell lines, paired patient tissues were determined using western blotting and Real-time PCR. Ninety-six human pancreatic cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the association between GPR87 expression and the clinicopathological characteristics of pancreatic cancer. Functional assays, such as anchorage-independent growth, chicken chorioallantoic membrane (CAM) assay, transwell matrix penetration assay, and Annexin V-FITC and PI staining and a xenograft tumor model were used to determine the oncogenic role of GPR87 in human pancreatic cancer progression. The effect of GPR87 on NF-κB signaling pathway was further investigated using the luciferase reporter assays, and by detection of the NF-κB signaling downstream genes.

Results

Herein, we reported that GPR87 was markedly overexpressed in pancreatic cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of GPR87 significantly correlated with patients’ clinicopathologic features, including clinical stage and tumor-nodule-metastasis (TNM) classification. Pancreatic cancer patients with higher levels of GPR87 expression had shorter overall survival compared to patients with lower GPR87 levels. We gained valuable insights into the mechanism of GPR87 expression in pancreatic cancer cells by demonstrating that overexpressing GPR87 significantly enhanced, whereas silencing endogenous GPR87 inhibited, the proliferation, angiogenesis and increased resistance to gemcitabine-induced apoptosis of pancreatic cancer in vitro and tumorigenicity of pancreatic cancer cells in vivo. Finally, we demonstrated that GPR87 enhanced pancreatic cancer aggressiveness by activating NF-κB signaling pathway. Conclusions: Taken together, these findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.

Conclusions

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.
Zusatzmaterial
Additional file 1: Figure S1. mRNA expression analysis shows that GPR87 was up-regulated in pancreatic cancer tissues and cell lines. A. Real-time PCR analysis of GPR87 expression in two human pancreatic ductal epithelial cells (HPDECs) and in pancreatic cancer cell lines (AsPC-1, Capan-1, BxPC-3, Capan-2, PANC-1 and MIA PaCa-2). B. Real-time PCR analysis of GPR87 expression in pancreatic cancer tissues (T) with matched adjacent non-tumor tissues (N) from 8 patients. Transcript levels were normalized to GAPDH expression. Each bar represents the mean ± SD of three independent experiments. * p < 0.05. (TIF 110 kb)
12943_2017_627_MOESM1_ESM.tif
Additional file 2: Table S1. Clinicopathological characteristics of studied patients and expression of GPR87 in pancreatic cancer. (DOC 41 kb)
12943_2017_627_MOESM2_ESM.doc
Additional file 3: Table S2. Correlation between the clinicopathological features and expression of GPR87. (DOC 41 kb)
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Additional file 4: Figure S2. Annexin V-FITC and PI staining of indicated cells with or without Fluorouracil treatment. A. Representative images (left panel) and quantification (right panel) of Annexin V-FITC and PI staining of indicated cells with no treatment for 24 h. B. Representative images (left panel) and quantification (right panel) of Annexin V-FITC and PI staining of indicated cells treated with Fluorouracil for 24 h. Each bar represents the mean ± SD of three independent experiments. * p < 0.05. (TIF 508 kb)
12943_2017_627_MOESM4_ESM.tif
Additional file 5: Figure S3. Western blot analysis of GPR87 expression in the indicated xenografts tumors. (TIF 166 kb)
12943_2017_627_MOESM5_ESM.tif
Additional file 6: Figure S4. GPR87 up-regulation activates the NF-κB signaling pathway in pancreatic cancer. GSEA plot, indicating a significant correlation between the mRNA levels of GPR87 expression in pancreatic cancer and the NF-κB-activated gene signatures in multiple published datasets. (TIF 316 kb)
12943_2017_627_MOESM6_ESM.tif
Additional file 7: Figure S5. Effects of Inhibiting NF-κB signaling in the indicated cells. A. Quantification of colony numbers as determined by anchorage-independent growth assay. Colonies larger than 0.1 mm in diameter were scored. B. Quantification of tubule formation by HUVECs cultured in matrigel-coated plates with conditioned media from pancreatic cancer cells transfected with the vector, IκBα-mut or treated with the NF-κB inhibitor (JSH-23). C. Quantification of gemcitabine-induced (1 μM) TUNEL-positive cells in pancreatic cells transfected with vector, IκBα-mut or treated with the NF-κB inhibitor. Each bar represents the mean ± SD of three independent experiments. * p < 0.05. (TIF 288 kb)
12943_2017_627_MOESM7_ESM.tif
Additional file 8: Figure S6. Overexpressing GPR87 promotes proliferation and induces HUVEC tube formation in HPDEC cells and Eca109. A. Western blot of GPR87 expression in HPDEC-1 cells transfected with GPR87 or a vector control. α-tubulin was used as a loading control. B. Quantification of BrdU labeling in HPDEC-1 cells transfected with GPR87 or a vector control. C. Quantification of HUVECs cultured on matrigel-coated plates with conditioned medium from vector control or GPR87 transfected HPDEC-1 cells. D. Western blot of GPR87 expression in Eca109 cells transfected with GPR87, GPR87-RNAi or their corresponding controls. α-tubulin was used as a loading control. E. Quantification of BrdU labeling in indicated cells. F. Quantification of HUVECs cultured on matrigel-coated plates with conditioned medium from indicated cells. (TIF 396 kb)
12943_2017_627_MOESM8_ESM.tif
Literatur
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