Gene therapy targeting hepatocellular carcinoma by a dual-regulated oncolytic adenovirus harboring the focal adhesion kinase shRNA

Gao,Yang; Zhu,Yayun; Huang,Xinyu; Ai,Kaixing; Zheng,Qi; Yuan,Zhou

doi:10.3892/ijo.2015.3047

Gene therapy targeting hepatocellular carcinoma by a dual-regulated oncolytic adenovirus harboring the focal adhesion kinase shRNA

Authors:
- Yang Gao
- Yayun Zhu
- Xinyu Huang
- Kaixing Ai
- Qi Zheng
- Zhou Yuan
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Affiliations: Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, P.R. China
Published online on: June 12, 2015 https://doi.org/10.3892/ijo.2015.3047
Pages: 668-678

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Abstract

Cancer targeting gene-viro-therapy (CTGVT) approach has become a hotspot and a trend in the field of cancer biotherapy and oncolytic adenovirus is an ideal vector to carry the targeting genes. In this study, we used human telomerase reverse transcriptase (hTERT) promoter to control the adenovirus early region 1a (E1A) and the human α-fetoprotein (AFP) promoter integrated with hypoxia response element (HRE) to control the adenovirus early region 1b (E1B). Then the novel double-regulated adenovirus Ad-hTERT-HREAF (named SG505) was engineered. The short-hairpin RNA against focal adhesion kinase (FAK) was inserted into SG505 and thus forming Ad-hTERT-HREAF-shRNA (called SG505‑siFAK). Then various oncolytic adenoviruses were examined to verify whether they could suppress liver cancer cells selectively and efficiently both in vitro and in vivo. Both replicative and replication-defective adenoviruses carrying FAK-shRNA significantly inhibited the expression of FAK in Hep3B and SMMC-7721 cell lines and efficiently suppressed the growth of liver cancer cell lines with minor effect to normal cells. Furthermore, the recombined oncolytic adenoviruses, SG505-siFAK, SG505-EGFP and SG505 were able to selectively propagate in AFP-positive liver cancer cells in vitro and the SG505-siFAK efficiently suppressed the expression of FAK. SG505-siFAK showed the most potent tumor inhibition capability among the three recombined adenovirus with IC50 levels of 0.092±0.009 and 0.424±0.414 pfu/cell in the Hep3B and HepG2 cell line, respectively. Animal experiment further confirmed that SG505-siFAK achieved the most significant tumor inhibition of Hep3B liver cancer xenografted growth by intratumoral injection comparing to the intravenous injection among the three recombined viruses. Immunohistochemical results indicated that FAK expression was downregulated significantly in the tumors treated with SG505-siFAK. The dual-regulated oncolytic adenovirus SG505-siFAK was proven to inhibit the growth of liver cancer cells selectively and efficiently, therefore SG505-siFAK could be a potential agent for future clinical trials of hepatocellular carcinoma.

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1	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI
2	Tabrizian P, Roayaie S and Schwartz ME: Current management of hepatocellular carcinoma. World J Gastroenterol. 20:10223–10237. 2014. View Article : Google Scholar : PubMed/NCBI
3	Llovet JM, Fuster J and Bruix J: Intention-to-treat analysis of surgical treatment for early hepatocellular carcinoma: Resection versus transplantation. Hepatology. 30:1434–1440. 1999. View Article : Google Scholar : PubMed/NCBI
4	Bai W, Wang YJ, Zhao Y, Qi XS, Yin ZX, He CY, Li RJ, Wu KC, Xia JL, Fan DM, et al: Sorafenib in combination with transarterial chemoembolization improves the survival of patients with unresectable hepatocellular carcinoma: A propensity score matching study. J Dig Dis. 14:181–190. 2013. View Article : Google Scholar : PubMed/NCBI
5	Graf D, Vallböhmer D, Knoefel WT, Kröpil P, Antoch G, Sagir A and Häussinger D: Multimodal treatment of hepatocellular carcinoma. Eur J Intern Med. 25:430–437. 2014. View Article : Google Scholar : PubMed/NCBI
6	Russell SJ, Peng KW and Bell JC: Oncolytic virotherapy. Nat Biotechnol. 30:658–670. 2012. View Article : Google Scholar : PubMed/NCBI
7	Cao X, Yang M, Wei RC, Zeng Y, Gu JF, Huang WD, Yang DQ, Li HL, Ding M, Wei N, et al: Cancer targeting gene-viro-therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene. Gene Ther. 18:765–777. 2011. View Article : Google Scholar : PubMed/NCBI
8	Wei RC, Cao X, Gui JH, Zhou XM, Zhong D, Yan QL, Huang WD, Qian QJ, Zhao FL and Liu XY: Augmenting the antitumor effect of TRAIL by SOCS3 with double-regulated replicating oncolytic adenovirus in hepatocellular carcinoma. Hum Gene Ther. 22:1109–1119. 2011. View Article : Google Scholar : PubMed/NCBI
9	Hu ZB, Wu CT, Wang H, Zhang QW, Wang L, Wang RL, Lu ZZ and Wang LS: A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes. Cancer Gene Ther. 15:173–182. 2008. View Article : Google Scholar
10	Kasala D, Choi JW, Kim SW and Yun CO: Utilizing adenovirus vectors for gene delivery in cancer. Expert Opin Drug Deliv. 11:379–392. 2014. View Article : Google Scholar : PubMed/NCBI
11	Liu XY, Gu JF and Shi WF: Targeting gene-virotherapy for cancer. Acta Biochim Biophys Sin. 37:581–587. 2005. View Article : Google Scholar : PubMed/NCBI
12	Huang Q, Zhang X, Wang H, et al: A novel conditionally replicative adenovirus vector targeting telomerase-positive tumor cells. Clin Cancer Res. 10:1439–1445. 2004. View Article : Google Scholar : PubMed/NCBI
13	Hahn WC: Targeting cancer with telomerase: commentary re Q. Huang et al:, a novel conditionally replicative adenovirus vector targeting telomerase-positive tumor cells. Clin Cancer Res, 10: 1439–1445, 2004. Clin Cancer Res. 10:1203–1205. 2004. View Article : Google Scholar : PubMed/NCBI
14	Tangkijvanich P, Anukulkarnkusol N, Suwangool P, Lertmaharit S, Hanvivatvong O, Kullavanijaya P and Poovorawan Y: Clinical characteristics and prognosis of hepatocellular carcinoma: Analysis based on serum alpha-fetoprotein levels. J Clin Gastroenterol. 31:302–308. 2000. View Article : Google Scholar : PubMed/NCBI
15	Li Y, Yu DC, Chen Y, Amin P, Zhang H, Nguyen N and Henderson DR: A hepatocellular carcinoma-specific adenovirus variant, CV890, eliminates distant human liver tumors in combination with doxorubicin. Cancer Res. 61:6428–6436. 2001.PubMed/NCBI
16	Ido A, Uto H, Moriuchi A, Nagata K, Onaga Y, Onaga M, Hori T, Hirono S, Hayashi K, Tamaoki T, et al: Gene therapy targeting for hepatocellular carcinoma: Selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter. Cancer Res. 61:3016–3021. 2001.PubMed/NCBI
17	Kwon OJ, Kim PH, Huyn S, Wu L, Kim M and Yun CO: A hypoxia- and {alpha}-fetoprotein-dependent oncolytic adenovirus exhibits specific killing of hepatocellular carcinomas. Clin Cancer Res. 16:6071–6082. 2010. View Article : Google Scholar : PubMed/NCBI
18	Huang TG, Savontaus MJ, Shinozaki K, Sauter BV and Woo SL: Telomerase-dependent oncolytic adenovirus for cancer treatment. Gene Ther. 10:1241–1247. 2003. View Article : Google Scholar : PubMed/NCBI
19	Yuan Z, Zheng Q, Fan J, Ai KX, Chen J and Huang XY: Expression and prognostic significance of focal adhesion kinase in hepatocellular carcinoma. J Cancer Res Clin Oncol. 136:1489–1496. 2010. View Article : Google Scholar : PubMed/NCBI
20	Fujii T, Koshikawa K, Nomoto S, Okochi O, Kaneko T, Inoue S, Yatabe Y, Takeda S and Nakao A: Focal adhesion kinase is over-expressed in hepatocellular carcinoma and can be served as an independent prognostic factor. J Hepatol. 41:104–111. 2004. View Article : Google Scholar : PubMed/NCBI
21	Golubovskaya VM: Focal adhesion kinase as a cancer therapy target. Anticancer Agents Med Chem. 10:735–741. 2010. View Article : Google Scholar
22	He D, Sun L, Li C, Hu N, Sheng Y, Chen Z, Li X, Chi B and Jin N: Anti-tumor effects of an oncolytic adenovirus expressing hemagglutinin-neuraminidase of Newcastle disease virus in vitro and in vivo. Viruses. 6:856–874. 2014. View Article : Google Scholar : PubMed/NCBI
23	Boncler M, Różalski M, Krajewska U, Podsędek A and Watala C: Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells. J Pharmacol Toxicol Methods. 69:9–16. 2014. View Article : Google Scholar
24	Ford AL, An H, Kong L, Zhu H, Vo KD, Powers WJ, Lin W and Lee JM: Clinically relevant reperfusion in acute ischemic stroke: MTT performs better than Tmax and TTP. Transl Stroke Res. 5:415–421. 2014. View Article : Google Scholar : PubMed/NCBI
25	Liu L, Wu W, Zhu G, Liu L, Guan G, Li X, Jin N and Chi B: Therapeutic efficacy of an hTERT promoter-driven oncolytic adenovirus that expresses apoptin in gastric carcinoma. Int J Mol Med. 30:747–754. 2012.PubMed/NCBI
26	Itoh S, Maeda T, Shimada M, Aishima S, Shirabe K, Tanaka S and Maehara Y: Role of expression of focal adhesion kinase in progression of hepatocellular carcinoma. Clin Cancer Res. 10:2812–2817. 2004. View Article : Google Scholar : PubMed/NCBI
27	Yu DC, Chen Y, Seng M, Dilley J and Henderson DR: The addition of adenovirus type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts. Cancer Res. 59:4200–4203. 1999.PubMed/NCBI
28	Nettelbeck DM, Rivera AA, Balagué C, Alemany R and Curiel DT: Novel oncolytic adenoviruses targeted to melanoma: Specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter. Cancer Res. 62:4663–4670. 2002.PubMed/NCBI
29	Leitner S, Sweeney K, Oberg D, Davies D, Miranda E, Lemoine NR and Halldén G: Oncolytic adenoviral mutants with E1B19K gene deletions enhance gemcitabine-induced apoptosis in pancreatic carcinoma cells and anti-tumor efficacy in vivo. Clin Cancer Res. 15:1730–1740. 2009. View Article : Google Scholar : PubMed/NCBI
30	Dong X, Qu W, Ma S, Zhu Z, Zheng C, He A, Karlsson A, Xu K and Zheng X: Potent antitumoral effects of targeted promoter-driven oncolytic adenovirus armed with Dm-dNK for breast cancer in vitro and in vivo. Cancer Lett. 328:95–103. 2013. View Article : Google Scholar
31	Wang L, Zhang Y, Zhao J, Xiao E, Lu J, Fu S and Wang Z: Combination of bladder cancer-specific oncolytic adenovirus gene therapy with cisplatin on bladder cancer in vitro. Tumour Biol. 35:10879–10890. 2014. View Article : Google Scholar : PubMed/NCBI
32	Liu XR, Cai Y, Cao X, Wei RC, Li HL, Zhou XM, Zhang KJ, Wu S, Qian QJ, Cheng B, et al: A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect. J Cell Mol Med. 16:1298–1309. 2012. View Article : Google Scholar
33	He B, Huang X, Liu X and Xu B: Cancer targeting gene-viro-therapy for pancreatic cancer using oncolytic adenovirus ZD55-IL-24 in immune-competent mice. Mol Biol Rep. 40:5397–5405. 2013. View Article : Google Scholar : PubMed/NCBI
34	Shang N, Arteaga M, Zaidi A, Stauffer J, Cotler SJ, Zeleznik-Le NJ, Zhang J and Qiu W: FAK is required for c-Met/beta-catenin-driven hepatocarcinogenesis. Hepatology. 61:214–226. 2014. View Article : Google Scholar
35	Dai Z, Zhou SL, Zhou ZJ, Bai DS, Xu XY, Fu XT, Chen Q, Zhao YM, Zhu K, Yu L, et al: Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK-Src signalling pathways. J Pathol. 234:316–328. 2014. View Article : Google Scholar : PubMed/NCBI
36	Ko BS, Jan YJ, Chang TC, Liang SM, Chen SC, Liu TA, Wu YM, Wang J and Liou JY: Upregulation of focal adhesion kinase by 14-3-3ɛ via NFκB activation in hepatocellular carcinoma. Anticancer Agents Med Chem. 13:555–562. 2013. View Article : Google Scholar
37	Cai L, Han J, Zhuo X, Xiong Y, Dong J and Li X: Overexpression and significance of focal adhesion kinase in hepatocellular carcinoma and its relationship with HBV infection. Med Oncol. 26:409–414. 2009. View Article : Google Scholar
38	Takeshita F and Ochiya T: Therapeutic potential of RNA interference against cancer. Cancer Sci. 97:689–696. 2006. View Article : Google Scholar : PubMed/NCBI
39	Liu XY: Targeting gene-virotherapy of cancer and its prosperity. Cell Res. 16:879–886. 2006. View Article : Google Scholar : PubMed/NCBI
40	Grünweller A, Wyszko E, Bieber B, Jahnel R, Erdmann VA and Kurreck J: Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res. 31:3185–3193. 2003. View Article : Google Scholar
41	Prados J, Melguizo C, Roldan H, Alvarez PJ, Ortiz R, Arias JL and Aranega A: RNA interference in the treatment of colon cancer. BioDrugs. 27:317–327. 2013. View Article : Google Scholar : PubMed/NCBI
42	Liu W, Zhu F, Jiang Y, Sun D, Yang B and Yan H: siRNA targeting survivin inhibits the growth and enhances the chemosensitivity of hepatocellular carcinoma cells. Oncol Rep. 29:1183–1188. 2013.
43	Pei Z, Chu L, Zou W, Zhang Z, Qiu S, Qi R, Gu J, Qian C and Liu X: An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice. Hepatology. 39:1371–1381. 2004. View Article : Google Scholar : PubMed/NCBI
44	Zhang J, Gan Y, Gu J, Hu J, Liu X and Zhao X: Potent anti-hepatoma efficacy of HCCS1 via dual tumor-targeting gene-virotherapy strategy. Oncol Rep. 20:1035–1040. 2008.PubMed/NCBI
45	Wu WY, Kim H, Zhang CL, Meng XL and Wu ZS: Loss of suppressors of cytokine signaling 3 promotes aggressiveness in hepatocellular carcinoma. J Invest Surg. 27:197–204. 2014. View Article : Google Scholar : PubMed/NCBI
46	Guo X, Wang W, Zhou F, Lu Z, Fang R, Jia F, Bu X, Li R, Zhang B, Wu M, et al: siRNA-mediated inhibition of hTERT enhances chemosensitivity of hepatocellular carcinoma. Cancer Biol Ther. 7:1555–1560. 2008. View Article : Google Scholar : PubMed/NCBI