HHLA2 promotes tumor progression by long non‑coding RNA H19 in human gallbladder cancer

Zhang,Yizhou; Li,Hanrong; Lv,Chao; Wu,Baokang; Yu,Yang; Zhong,Chongli; Lang,Qi; Liang,Zhiyun; Li,Yang; Shi,Yu; Zhang,Jian; Xu,Feng; Tian,Yu

doi:10.3892/ijo.2022.5402

HHLA2 promotes tumor progression by long non‑coding RNA H19 in human gallbladder cancer

Authors:
- Yizhou Zhang
- Hanrong Li
- Chao Lv
- Baokang Wu
- Yang Yu
- Chongli Zhong
- Qi Lang
- Zhiyun Liang
- Yang Li
- Yu Shi
- Jian Zhang
- Feng Xu
- Yu Tian
View Affiliations

Affiliations: Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China, Department of Ophthalmology, Fourth Affiliated Hospital of China Medical University, Shenyang, Liaoning 110005, P.R. China, Department of Surgery, Jinzhou Medical University, Jinzhou, Liaoning 121001, P.R. China, Department of Oncology, Liaoyang Central Hospital of China Medical University, Liaoyang, Liaoning 111010, P.R. China
Published online on: August 1, 2022 https://doi.org/10.3892/ijo.2022.5402
Article Number: 112
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Advanced gallbladder cancer (GBC) is one of the most malignant of all types of biliary tract cancers that is associated with poor prognosis and high mortality. Accumulating evidence suggest that the B7 family of proteins serve an essential role in various types of cancers, including GBC. However, the potential function and regulatory mechanism of human endogenous retrovirus‑H long terminal repeat‑associating protein 2 (HHLA2; also known as B7‑H7 or B7H5) in GBC remain poorly understood. In the present study, immunohistochemistry was used to examine the expression pattern of HHLA2 in samples from 89 patients with GBC. The possible association between HHLA2 expression and the clinicopathological parameters, including prognosis, were then assessed. Using lentiviruses, overexpression of HHLA2 plasmid or short‑hairpin RNA (shRNA) of HHLA2 were transfected into GBC‑SD cells to overexpress or knock down HHLA2 expression, respectively. The effects of HHLA2 overexpression and knockdown on the epithelial‑mesenchymal transition (EMT) process on GBC‑SD cells were measured by the western blotting and immunofluorescence staining of collagen I, N‑cadherin, E‑cadherin, vimentin and α‑SMA. By contrast, changes in cell proliferation were measured using EdU assay. Cell invasion and migration were assessed using Transwell and wound‑healing assays, respectively. In addition, a xenograft mouse model was established to evaluate the tumorigenic ability of the GBC cell line in vivo after stable transfection with lentivirus for HHLA2 overexpression or shRNA for HHLA2 knockdown. The regulatory relationships among TGF‑β1, long non‑coding RNA (lncRNA) H19 (H19) and HHLA2 were then investigated. The mRNA expression of lncRNA H19 were assessed using reverse transcription‑quantitative PCR, whereas the expression levels of HHLA2 were detected by western blotting and immunofluorescence staining. HHLA2 expression was found to gradually increase as the stages of the GBC samples become more advanced. In addition, the expression level of HHLA2 was calculated to be positively associated with the Nevin stage, American Joint Committee on Cancer stage, tumor invasion and regional lymph node metastasis but was negatively associated with the overall patient survival (OS). In vitro experiments demonstrated that overexpression of HHLA2 promoted GBC migration, invasion, proliferation and EMT, whereas in vivo experiments found a promoting role of HHLA2 overexpression on GBC tumor growth. After transfection with lentiviruses encoding the overexpression plasmid of lncRNA H19, GBC migration, invasion, proliferation and EMT were increased. By contrast, knocking down HHLA2 expression suppressed TGF‑β1‑ or lncRNA H19 overexpression‑induced GBC migration, invasion, proliferation and EMT. In addition, HHLA2 knockdown significantly reduced the sizes of the GBC tumors in vivo. These results suggest that HHLA2 overexpression can promote GBC progression. Conversely, ablation of HHLA2 expression inhibited both TGF‑β1‑ and lncRNA H19‑induced GBC progression, suggesting that HHLA2 is a potential therapeutic target for this disease.

View Figures

View References

1	Castro FA, Koshiol J, Hsing AW and Devesa SD: Biliary tract cancer incidence in the United States-Demographic and temporal variations by anatomic site. Int J Cancer. 133:1664–1671. 2013. View Article : Google Scholar : PubMed/NCBI
2	Acharya M, Patkar S, Parray A and Goel M: Management of gallbladder cancer in India. Chin Clin Oncol. 8:352019. View Article : Google Scholar : PubMed/NCBI
3	Song X, Hu Y, Li Y, Shao R, Liu F and Liu Y: Overview of current targeted therapy in gallbladder cancer. Signal Transduct Targeted Ther. 5:2302020. View Article : Google Scholar
4	Jin L, Cai Q, Wang S, Wang S, Wang J and Quan Z: Long noncoding RNA PVT1 promoted gallbladder cancer proliferation by epigenetically suppressing miR-18b-5p via DNA methylation. Cell Death Dis. 11:8712020. View Article : Google Scholar : PubMed/NCBI
5	McNamara M, Lopes A, Wasan H, Malka D, Goldstein D, Shannon J, Okusaka T, Knox JJ, Wagner AD, André T, et al: Landmark survival analysis and impact of anatomic site of origin in prospective clinical trials of biliary tract cancer. J Hepatol. 73:1109–1117. 2020. View Article : Google Scholar : PubMed/NCBI
6	Jackson S, Adami H, Andreotti G, Beane-Freeman LE, de González AB, Buring JE, Fraser GE, Freedman ND, Gapstur SM, Gierach G, et al: Associations between reproductive factors and biliary tract cancers in women from the biliary tract cancers pooling project. J Hepatol. 73:863–872. 2020. View Article : Google Scholar : PubMed/NCBI
7	Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68:394–424. 2018. View Article : Google Scholar : PubMed/NCBI
8	Regmi P, Hu HJ, Chang-Hao Y, Liu F, Ma WJ, Ran CD, Wang JK, Paudyal A, Cheng NS and Li FY: Laparoscopic surgery for oncologic extended resection of T1b and T2 incidental gallbladder carcinoma at a high-volume center: A single-center experience in China. Surg Endos. 35:6505–6512. 2020. View Article : Google Scholar
9	Rizzo A, Tavolari S, Ricci AD, Frega G, Palloni A, Relli V, Salati M, Fenocchio E, Massa A, Aglietta M and Brandi G: Molecular features and targeted therapies in extrahepatic cholangiocarcinoma: Promises and failures. Cancers (Basel). 12. pp. 32562020, View Article : Google Scholar
10	Valle JW, Kelley RK, Nervi B, Oh DY and Zhu AX: Biliary tract cancer. Lancet. 397:428–444. 2021. View Article : Google Scholar : PubMed/NCBI
11	Ebata N, Fujita M, Sasagawa S, Maejima K, Okawa Y, Hatanaka Y, Mitsuhashi T, Oosawa-Tatsuguchi A, Tanaka H, Miyano S, et al: Molecular classification and tumor microenvironment characterization of gallbladder cancer by comprehensive genomic and transcriptomic analysis. Cancers. 13:7332021. View Article : Google Scholar : PubMed/NCBI
12	Nepal C, Zhu B, O'Rourke C, Bhatt DK, Lee D, Song L, Wang D, Van Dyke AL, Choo-Wosoba H, Liu Z, et al: Integrative molecular characterisation of gallbladder cancer reveals micro-environment-associated subtypes. J Hepatol. 74:1132–1144. 2021. View Article : Google Scholar
13	Zou W and Chen L: Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 8:467–477. 2008. View Article : Google Scholar : PubMed/NCBI
14	Zhu Y, Yao S, Iliopoulou BP, Han X, Augustine MM, Xu H, Phennicie RT, Flies SJ, Broadwater M, Ruff W, et al: B7-H5 costimulates human T cells via CD28H. Nat Commun. 4:20432013. View Article : Google Scholar : PubMed/NCBI
15	Crespo J, Vatan L, Maj T, Liu R, Kryczek I and Zou W: Phenotype and tissue distribution of CD28H immune cell subsets. Oncoimmunology. 6:e13625292017. View Article : Google Scholar
16	Bhatt R, Berjis A, Konge JC, Mahoney KM, Klee AN, Freeman SS, Chen CH, Jegede OA, Catalano PJ, Pignon JC, et al: KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1. Cancer Immunol Res. 9:156–169. 2021. View Article : Google Scholar :
17	Dong Z, Zhang L, Xu W and Zhang G: EGFR may participate in immune evasion through regulation of B7-H5 expression in non-small cell lung carcinoma. Mol Med Rep. 18:3769–3779. 2018.PubMed/NCBI
18	Luo M, Lin Y, Liang R, Li Y and Ge L: Clinical significance of the HHLA2 protein in hepatocellular carcinoma and the tumor microenvironment. J Inflamm Res. 14:4217–4228. 2021. View Article : Google Scholar : PubMed/NCBI
19	Zhou Q, Li K, Lai Y, Yao K, Wang Q, Zhan X, Peng S, Cai W, Yao W, Zang X, et al: B7 score and T cell infiltration stratify immune status in prostate cancer. J Immunother Cancer. 9:e0024552021. View Article : Google Scholar : PubMed/NCBI
20	Xu G, Shi Y, Ling X, Wang D, Liu Y, Lu H, Peng Y and Zhang B: HHLA2 predicts better survival and exhibits inhibited proliferation in epithelial ovarian cancer. Cancer Cell Int. 21:2522021. View Article : Google Scholar : PubMed/NCBI
21	Winkle M, El-Daly SM, Fabbri M and Calin GA: Noncoding RNA therapeutics-challenges and potential solutions. Nat Rev Drug Discov. 20:629–651. 2021. View Article : Google Scholar : PubMed/NCBI
22	Rainier S, Johnson LA, Dobry CJ, Ping AJ, Grundy PE and Feinberg AP: Relaxation of imprinted genes in human cancer. Nature. 362:747–749. 1993. View Article : Google Scholar : PubMed/NCBI
23	Liu SJ, Dang HX, Lim DA, Feng FY and Maher CA: Long noncoding RNAs in cancer metastasis. Nat Rev Cancer. 21:446–460. 2021. View Article : Google Scholar : PubMed/NCBI
24	Wang SH, Ma F, Tang ZH, Wu XC, Cai Q, Zhang MD, Weng MZ, Zhou D, Wang JD and Quan ZW: Long non-coding RNA H19 regulates FOXM1 expression by competitively binding endogenous miR-342-3p in gallbladder cancer. J Exp Clin Cancer Res. 35:1602016. View Article : Google Scholar : PubMed/NCBI
25	Wang SH, Wu XC, Zhang MD, Weng MZ, Zhou D and Quan ZW: Upregulation of H19 indicates a poor prognosis in gallbladder carcinoma and promotes epithelial-mesenchymal transition. Am J Cancer Res. 6:15–26. 2016.PubMed/NCBI
26	Giannis D, Cerullo M, Moris D, Shah KN, Herbert G, Zani S, Blazer DG III, Allen PJ and Lidsky ME: Validation of the 8th edition American joint commission on cancer (AJCC) gallbladder cancer staging system: Prognostic discrimination and identification of key predictive factors. Cancers. 13:5472021. View Article : Google Scholar : PubMed/NCBI
27	Nagtegaal ID, Odze RD, Klimstra D, Paradis V, Rugge M, Schirmacher P, Washington KM and Carneiro F: The 2019 WHO classification of tumours of the digestive system. Histopathology. 76:182–188. 2020. View Article : Google Scholar :
28	Xu X, He M, Wang H, Zhan M and Yang L: Development and validation of a prognostic nomogram for gallbladder cancer patients after surgery. BMC Gastroenterol. 22:2002022. View Article : Google Scholar : PubMed/NCBI
29	Camp RL, Dolled-Filhart M and Rimm DL: X-tile: A new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 10:7252–7259. 2004. View Article : Google Scholar : PubMed/NCBI
30	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
31	Clark JM and Sun D: Guidelines for the ethical review of laboratory animal welfare People's Republic of China National Standard GB/T 35892-2018[Issued 6 February 2018 Effective from 1 September 2018]. Animal Models Exp Med. 3:103–113. 2020. View Article : Google Scholar
32	Cheng H, Janakiram M, Borczuk A, Lin J, Qiu W, Liu H, Chinai JM, Halmos B, Perez-Soler R and Zang X: HHLA2, a new immune checkpoint member of the B7 family, is widely expressed in human lung cancer and associated with EGFR mutational status. Clin Cancer Res. 23:825–832. 2017. View Article : Google Scholar :
33	Boor P, Sideras K, Biermann K, Aziz MH, Levink IJM, Mancham S, Erler NS, Tang X, van Eijck CH, Bruno MJ, et al: HHLA2 is expressed in pancreatic and ampullary cancers and increased expression is associated with better post-surgical prognosis. Br J Cancer. 122:1211–1218. 2020. View Article : Google Scholar : PubMed/NCBI
34	Zhong C, Lang Q, Yu J, Wu S, Xu F and Tian Y: Phenotypical and potential functional characteristics of different immune cells expressing CD28H/B7-H5 and their relationship with cancer prognosis. Clin Exp Immunol. 200:12–21. 2020. View Article : Google Scholar : PubMed/NCBI
35	Seaman S, Zhu Z, Saha S, Zhang XM, Yang MY, Hilton MB, Morris K, Szot C, Morris H, Swing DA, et al: Eradication of tumors through simultaneous ablation of CD276/B7-H3-positive tumor cells and tumor vasculature. Cancer Cell. 31:501–515.e8. 2017. View Article : Google Scholar : PubMed/NCBI
36	Schildberg FA, Klein SR, Freeman GJ and Sharpe AH: Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity. 44:955–972. 2016. View Article : Google Scholar : PubMed/NCBI
37	Marangoni F, Zhakyp A, Corsini M, Geels SN, Carrizosa E, Thelen M, Mani V, Prüßmann JN, Warner RD, Ozga AJ, et al: Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop. Cell. 184:3998–4015.e19. 2021. View Article : Google Scholar : PubMed/NCBI
38	Nishimura CD, Pulanco MC, Cui W, Lu L and Zang X: PD-L1 and B7-1 cis-interaction: New mechanisms in immune checkpoints and immunotherapies. Trends Mol Med. 27:207–219. 2021. View Article : Google Scholar
39	Chen L, Gibbons DL, Goswami S, Cortez MA, Ahn YH, Byers LA, Zhang X, Yi X, Dwyer D, Lin W, et al: Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun. 5:52412014. View Article : Google Scholar : PubMed/NCBI
40	Lee Y, Shin J, Longmire M, Wang H, Kohrt HE, Chang HY and Sunwoo JB: CD44+ cells in head and neck squamous cell carcinoma suppress T-cell-mediated immunity by selective constitutive and inducible expression of PD-L1. Clin Cancer Res. 22:3571–3581. 2016. View Article : Google Scholar : PubMed/NCBI
41	Alsuliman A, Colak D, Al-Harazi O, Fitwi H, Tulbah A, Al-Tweigeri T, Al-Alwan M and Ghebeh H: Bidirectional cross-talk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells. Mol Cancer. 14:1492015. View Article : Google Scholar
42	Hsu JM, Xia W, Hsu YH, Chan L, Yu WH, Cha JH, Chen CT, Liao HW, Kuo CW, Khoo KH, et al: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion. Nat Commun. 9:19082018. View Article : Google Scholar : PubMed/NCBI
43	Xu S, Zhan M and Wang J: Epithelial-to-mesenchymal transition in gallbladder cancer: From clinical evidence to cellular regulatory networks. Cell Death Discov. 3:170692017. View Article : Google Scholar : PubMed/NCBI
44	Krebs AM, Mitschke J, Losada MA, Schmalhofer O, Boerries M, Busch H, Boettcher M, Mougiakakos D, Reichardt W, Bronsert P, et al: The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer. Nat Cell Biol. 19:518–529. 2017. View Article : Google Scholar : PubMed/NCBI
45	Cao L, Bridle KR, Shrestha R, Prithviraj P, Crawford DHG and Jayachandran A: CD73 and PD-L1 as potential therapeutic targets in gallbladder cancer. Int J Mol Sci. 23:15652022. View Article : Google Scholar : PubMed/NCBI
46	Shi Y, Sun L, Zhang R, Hu Y, Wu Y, Dong X, Dong D, Chen C, Geng Z, Li E and Fan Y: Thrombospondin 4/integrin α2/HSF1 axis promotes proliferation and cancer stem-like traits of gallbladder cancer by enhancing reciprocal crosstalk between cancer-associated fibroblasts and tumor cells. J Exp Clin Cancer Res. 40:142021. View Article : Google Scholar
47	Yin Y, Shi L, Yang J, Wang H, Yang H and Wang Q: B7 family member H4 induces epithelial-mesenchymal transition and promotes the proliferation, migration and invasion of colorectal cancer cells. Bioengineered. 13:107–118. 2022. View Article : Google Scholar :
48	Kang JH, Jung MY and Leof EB: B7-1 drives TGF-β stimulated pancreatic carcinoma cell migration and expression of EMT target genes. PLoS One. 14:e02220832019. View Article : Google Scholar
49	Kang F-B, Wang L, Jia H-C, Li D, Li H-J, Zhang Y-G and Sun D-X: B7-H3 promotes aggression and invasion of hepatocellular carcinoma by targeting epithelial-to-mesenchymal transition via JAK2/STAT3/Slug signaling pathway. Cancer Cell Int. 15:452015. View Article : Google Scholar : PubMed/NCBI
50	Lee DW, Lee SH, Kim JS, Park J, Cho YL, Kim KS, Jo DY, Song IC, Kim N, Yun HJ, et al: Loss of NDRG2 promotes epithelial-mesenchymal transition of gallbladder carcinoma cells through MMP-19-mediated Slug expression. J Hepatol. 63:1429–1439. 2015. View Article : Google Scholar : PubMed/NCBI
51	Wu K, Liang W, Feng L, Pang JX, Waye MMY, Zhang JF and Fu WM: H19 mediates methotrexate resistance in colorectal cancer through activating Wnt/β-catenin pathway. Exp Cell Res. 350:312–317. 2017. View Article : Google Scholar
52	Yan L, Yang S, Yue CX, Wei XY, Peng W, Dong ZY, Xu HN, Chen SL, Wang WR, Chen CJ and Yang QL: Long noncoding RNA H19 acts as a miR-340-3p sponge to promote epithelial-mesenchymal transition by regulating YWHAZ expression in paclitaxel-resistant breast cancer cells. Environ Toxicol. 35:1015–1028. 2020. View Article : Google Scholar : PubMed/NCBI
53	Wu B, Zhang Y, Yu Y, Zhong C, Lang Q, Liang Z, Lv C, Xu F and Tian Y: ViaLong noncoding RNA H19: A novel therapeutic target emerging in oncology regulating oncogenic signaling pathways. Front Cell Dev Biol. 9:7967402021. View Article : Google Scholar
54	Jang H-R, Shin S-B, Kim C-H, Won J-Y, Xu R, Kim D-E and Yim H: PLK1/vimentin signaling facilitates immune escape by recruiting Smad2/3 to PD-L1 promoter in metastatic lung adenocarcinoma. Cell Death Diff. 28:2745–2764. 2021. View Article : Google Scholar