Silencing of Livin inhibits tumorigenesis and metastasis via VEGF and MMPs pathway in lung cancer

Lin,Xian; Li,Hong-Ru; Lin,Xiao-Fen; Yu,Mei-E; Tu,Xun-Wei; Hua,Zhi-Dan; Lin,Ming; Xu,Neng-Luan; Han,Li-Li; Chen,Yu-Sheng

doi:10.3892/ijo.2015.3058

Silencing of Livin inhibits tumorigenesis and metastasis via VEGF and MMPs pathway in lung cancer

Authors:
- Xian Lin
- Hong-Ru Li
- Xiao-Fen Lin
- Mei-E Yu
- Xun-Wei Tu
- Zhi-Dan Hua
- Ming Lin
- Neng-Luan Xu
- Li-Li Han
- Yu-Sheng Chen
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Affiliations: Department of Respiratory Medicine, Fujian Provincial Hospital, Fujian Provincial Clinical Medical College of Fujian Medical University, Fuzhou 350001, P.R. China, Department of Respiratory Medicine, Fujian Zhangzhou First Hospital, Clinical Medical College of Fujian Medical University, Zhangzhou 363000, P.R. China, Cardiovascular Key Laboratory of Fujian Province, Fujian Provincial Hospital, Fujian Provincial Clinical Medical College of Fujian Medical University, Fuzhou 350001, P.R. China
Published online on: June 19, 2015 https://doi.org/10.3892/ijo.2015.3058
Pages: 657-667

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Abstract

Livin, an inhibitor of apoptosis protein (IAP), is overexpressed in various cancers and decreases tumor sensitivity to chemotherapy and radiotherapy. However, the effect of Livin on lung adenocarcinoma metastasis and the specific mechanism involved remain unclear. RNAi technology was used to stably silence Livin in A549 cells in the present study. The effect of Livin on tumor growth and invasion was investigated in lung cancer cells in vitro and animal models were established to determine the anti-metastasis ability of Livin silencing in vivo. The results indicated that Livin knock-down suppressed cell proliferation and inhibited cell invasion, accompanied by downregulation of VEGF and MMP-2/-9. Silencing of Livin resulted in the prevention of xenograft tumor formation. Seventy-five immunodeficient male BALB/C nude mice were randomly divided into three groups, the relative ratio of the areas with pulmonary nodules in the experimental group decreased from 46.71±7.27% to 11.07±2.94% compared with the negative control group (P<0.001), indicating the interaction between Livin, VEGF and MMPs. The xenograft tumor model of intravenous injection of tumor cells were successfully established and applied for the analysis of lung cancer tumorigenesis and metastasis in a time-dependent manner for the first time. Based on the reliable and reproducible animal model, our findings indicate that knock-down of Livin inhibits cell growth and invasion through blockade of the VEGF and MMPs pathways in lung cancer cells in vitro, and inhibits tumorigenesis and metastasis of lung cancer in vivo, suggesting that Livin is a promising antitumor target.

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1	Coleman MP, Forman D, Bryant H, Butler J, Rachet B, Maringe C, Nur U, Tracey E, Coory M, Hatcher J, et al; ICBP Module 1 Working Group. Cancer survival in Australia, Canada, Denmark, Norway, Sweden, and the UK, 1995–2007 (the International Cancer Benchmarking Partnership): An analysis of population-based cancer registry data. Lancet. 377:127–138. 2011. View Article : Google Scholar :
2	Chambers AF, Groom AC and MacDonald IC: Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2:563–572. 2002. View Article : Google Scholar : PubMed/NCBI
3	Bissell MJ and Radisky D: Putting tumours in context. Nat Rev Cancer. 1:46–54. 2001. View Article : Google Scholar
4	Yan B, Kong M, Chen S and Chen YH: VEGF stimulation enhances Livin protein synthesis through mTOR signaling. J Cell Biochem. 111:1114–1124. 2010. View Article : Google Scholar : PubMed/NCBI
5	Funahashi Y, Shawber CJ, Sharma A, Kanamaru E, Choi YK and Kitajewski J: Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity. Vasc Cell. 3:22011. View Article : Google Scholar : PubMed/NCBI
6	Shan B, Li W, Yang SY and Li ZR: Estrogen up-regulates MMP2/9 expression in endometrial epithelial cell via VEGF-ERK1/2 pathway. Asian Pac J Trop Med. 6:826–830. 2013. View Article : Google Scholar : PubMed/NCBI
7	Enciso JM, Gratzinger D, Camenisch TD, Canosa S, Pinter E and Madri JA: Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: Modulation by PECAM-1 and MMP-2. J Cell Biol. 160:605–615. 2003. View Article : Google Scholar : PubMed/NCBI
8	Ebrahem Q, Chaurasia SS, Vasanji A, Qi JH, Klenotic PA, Cutler A, Asosingh K, Erzurum S and Anand-Apte B: Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo. Am J Pathol. 176:496–503. 2010. View Article : Google Scholar :
9	Ou JM, Ye B, Qiu MK, Dai YX, Dong Q, Shen J, Dong P, Wang XF, Liu YB, Quan ZW, et al: Knockdown of Livin inhibits growth and invasion of gastric cancer cells through blockade of the MAPK pathway in vitro and in vivo. Int J Oncol. 44:276–284. 2014.
10	Lazar I, Perlman R, Lotem M, Peretz T, Ben-Yehuda D and Kadouri L: The clinical effect of the inhibitor of apopotosis protein livin in melanoma. Oncology. 82:197–204. 2012. View Article : Google Scholar : PubMed/NCBI
11	Haferkamp A, Bedke J, Vetter C, Pritsch M, Wagener N, Buse S, Crnkovic-Mertens I, Hoppe-Seyler K, Macher-Goeppinger S, Hoppe-Seyler F, et al: High nuclear Livin expression is a favourable prognostic indicator in renal cell carcinoma. BJU Int. 102:1700–1706. 2008. View Article : Google Scholar : PubMed/NCBI
12	Sun JG, Liao RX, Chen ZT, Wang ZX, Zhang Q, Hu YD and Wang DL: Gene transfection of Livin isoforms into A549 cell line and its effect on cell growth and sensitivity to chemotherapy and radiotherapy. Zhonghua Jie He He Hu Xi Za Zhi. 28:836–840. 2005.(In Chinese).
13	Lee DH, Yoon TM, Kim SA, Park YL, Lee KH, Lim SC, Lee JK and Joo YE: Relationship between expression of Livin and the biological behavior of human oral squamous cell carcinoma. Oncol Rep. 32:2453–2460. 2014.PubMed/NCBI
14	Yoon TM, Kim SA, Lee DH, Lee JK, Park YL, Lee KH, Chung IJ, Joo YE and Lim SC: Expression of Livin and the inhibition of tumor progression by Livin silencing in laryngohypopharyngeal cancer. In Vivo. 28:751–759. 2014.PubMed/NCBI
15	Li F, Yin X, Luo X, Li HY, Su X, Wang XY, Chen L, Zheng K and Ren GS: Livin promotes progression of breast cancer through induction of epithelial-mesenchymal transition and activation of AKT signaling. Cell Signal. 25:1413–1422. 2013. View Article : Google Scholar : PubMed/NCBI
16	Li X, Fan S, Li L, Wang L, Fan G, Zhao Q and Li Y: RNA interference-mediated knockdown of Livin suppresses cell proliferation and invasion and enhances the chemosensitivity to cisplatin in human osteosarcoma cells. Int J Oncol. 43:159–168. 2013.PubMed/NCBI
17	Chen F, Yang D, Che X, Wang J, Li X, Zhang Z, Chen X and Song X: Livin mediates tumor cell invasion in the DU-145 cell line via NF-κB. Oncol Rep. 27:2010–2016. 2012.PubMed/NCBI
18	Cho SB, Lee WS, Park YL, Kim N, Oh HH, Kim MY, Oak CY, Chung CY, Park HC, Kim JS, et al: Livin is associated with the invasive and oncogenic phenotypes of human hepatocellular carcinoma cells. Hepatol Res. 45:448–457. 2015. View Article : Google Scholar
19	Myung DS, Park YL, Chung CY, Park HC, Kim JS, Cho SB, Lee WS, Lee KH, Lee JH and Joo YE: Expression of Livin in colorectal cancer and its relationship to tumor cell behavior and prognosis. PLoS One. 8:e732622013. View Article : Google Scholar : PubMed/NCBI
20	Chen YS, Li HR, Lin M, Chen G, Xie BS, Xu NL and Lin LF: Livin abrogates apoptosis of SPC-A1 cell by regulating JNKI signaling pathway. Mol Biol Rep. 37:2241–2247. 2010. View Article : Google Scholar
21	Chen YS, Li HR, Miao Y, Chen WY, Li YT, Wang GQ and Wu ZC: Local injection of lentivirus-delivered livin shRNA suppresses lung adenocarcinoma growth by inducing a G0/G1 phase cell cycle arrest. Int J Clin Exp Pathol. 5:796–805. 2012.
22	Liu B, Han M, Wen JK and Wang L: Livin/ML-IAP as a new target for cancer treatment. Cancer Lett. 250:168–176. 2007. View Article : Google Scholar : PubMed/NCBI
23	Zhao X, Yuan Y, Zhang Z, Feng X, Zhang J, Yuan X and Li J: Effects of shRNA-silenced livin and survivin on lung cancer cell proliferation and apoptosis. J BUON. 19:757–762. 2014.PubMed/NCBI
24	Nachmias B, Ashhab Y, Bucholtz V, Drize O, Kadouri L, Lotem M, Peretz T, Mandelboim O and Ben-Yehuda D: Caspase-mediated cleavage converts Livin from an antiapoptotic to a proapoptotic factor: Implications for drug-resistant melanoma. Cancer Res. 63:6340–6349. 2003.PubMed/NCBI
25	Abd-Elrahman I, Hershko K, Neuman T, Nachmias B, Perlman R and Ben-Yehuda D: The inhibitor of apoptosis protein Livin (ML-IAP) plays a dual role in tumorigenicity. Cancer Res. 69:5475–5480. 2009. View Article : Google Scholar : PubMed/NCBI
26	Shiloach T, Berens C, Danke C, Waiskopf O, Perlman R and Ben-Yehuda D: tLivin displays flexibility by promoting alternative cell death mechanisms. PLoS One. 9:e1010752014. View Article : Google Scholar : PubMed/NCBI
27	Nachmias B, Lazar I, Elmalech M, Abed-El-Rahaman I, Asshab Y, Mandelboim O, Perlman R and Ben-Yehuda D: Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin. Apoptosis. 12:1129–1142. 2007. View Article : Google Scholar : PubMed/NCBI
28	Kafri T, Blömer U, Peterson DA, Gage FH and Verma IM: Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet. 17:314–317. 1997. View Article : Google Scholar : PubMed/NCBI
29	Piacibello W, Bruno S, Sanavio F, Droetto S, Gunetti M, Ailles L, Santoni de Sio F, Viale A, Gammaitoni L, Lombardo A, et al: Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multi-lineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient. Blood. 100:4391–4400. 2002. View Article : Google Scholar : PubMed/NCBI
30	Tabatabai G, Hasenbach K, Herrmann C, Maurer G, Möhle R, Marini P, Grez M, Wick W and Weller M: Glioma tropism of lentivirally transduced hematopoietic progenitor cells. Int J Oncol. 36:1409–1417. 2010.PubMed/NCBI
31	Goel HL and Mercurio AM: VEGF targets the tumour cell. Nat Rev Cancer. 13:871–882. 2013. View Article : Google Scholar : PubMed/NCBI
32	Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP and Iruela-Arispe ML: Autocrine VEGF signaling is required for vascular homeostasis. Cell. 130:691–703. 2007. View Article : Google Scholar : PubMed/NCBI
33	Overall CM and Kleifeld O: Tumour microenvironment -opinion: Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer. 6:227–239. 2006. View Article : Google Scholar : PubMed/NCBI
34	Overall CM and López-Otín C: Strategies for MMP inhibition in cancer: Innovations for the post-trial era. Nat Rev Cancer. 2:657–672. 2002. View Article : Google Scholar : PubMed/NCBI
35	Shiau AL, Teo ML, Chen SY, Wang CR, Hsieh JL, Chang MY, Chang CJ, Chao J, Chao L, Wu CL, et al: Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin. BMC Cancer. 10:2452010. View Article : Google Scholar : PubMed/NCBI
36	Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L and Shim H: Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res. 65:967–971. 2005.PubMed/NCBI
37	Hartman ML and Czyz M: Anti-apoptotic proteins on guard of melanoma cell survival. Cancer Lett. 331:24–34. 2013. View Article : Google Scholar : PubMed/NCBI
38	Rashidi B, Moossa AR and Hoffman RM: Specific route mapping visualized with GFP of single-file streaming contralateral and systemic metastasis of Lewis lung carcinoma cells beginning within hours of orthotopic implantation [correction of implantion]. J Cell Biochem. 114:1738–1743. 2013. View Article : Google Scholar : PubMed/NCBI
39	Yamamoto N, Tsuchiya H and Hoffman RM: Tumor imaging with multicolor fluorescent protein expression. Int J Clin Oncol. 16:84–91. 2011. View Article : Google Scholar : PubMed/NCBI
40	Li X, Wang J, An Z, Yang M, Baranov E, Jiang P, Sun F, Moossa AR and Hoffman RM: Optically imageable metastatic model of human breast cancer. Clin Exp Metastasis. 19:347–350. 2002. View Article : Google Scholar : PubMed/NCBI
41	Li W, Li H, Li J, Wang H, Zhao H, Zhang L, Xia Y, Ye Z, Gao J, Dai J, et al: Self-assembled supramolecular nano vesicles for safe and highly efficient gene delivery to solid tumors. Int J Nanomed. 7:4661–4677. 2012. View Article : Google Scholar
42	Cool SK, Breyne K, Meyer E, De Smedt SC and Sanders NN: Comparison of in vivo optical systems for bioluminescence and fluorescence imaging. J Fluoresc. 23:909–920. 2013. View Article : Google Scholar : PubMed/NCBI
43	Burrell-Saward H, Rodgers J, Bradley B, Croft SL and Ward TH: A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis. J Antimicrob Chemother. 70:510–517. 2015. View Article : Google Scholar
44	Liu HS, Jan MS, Chou CK, Chen PH and Ke NJ: Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 260:712–717. 1999. View Article : Google Scholar : PubMed/NCBI
45	Ejeskär K, Fransson S, Zaibak F and Ioannou PA: Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: Difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP. Int J Mol Med. 17:1011–1016. 2006.PubMed/NCBI