The novel mTOR inhibitor CCI-779 (temsirolimus) induces antiproliferative effects through inhibition of mTOR in Bel-7402 liver cancer cells
- Shuyu Li†1,
- Yan Liang†1,
- Manlin Wu1,
- Xiaojing Wang1,
- Haixia Fu1,
- Yuhao Chen1 and
- Zhigang Wang1Email author
© Li et al.; licensee BioMed Central Ltd. 2013
Received: 28 January 2013
Accepted: 20 March 2013
Published: 28 March 2013
Liver cancer is one of the most frequent cancers in the world. Targeted therapy of cancer with specific inhibitors is developing and has shown promising antitumor efficacy. CCI-779 (temsirolimus), a specific inhibitor of mTOR (mammalian target of rapamycin), can block the mTOR signaling pathway. Here, we systematically examined the expression of mTOR and its downstream targets in liver cancer cells and normal liver cells, then investigated inhibitory effects of CCI-779 on mTOR signaling pathway and its role in regulating liver cancer cell growth.
The expression of mTOR and its downstream targets in Bel-7402 liver cancer cells and HL-7702 normal liver cells were examined by western blot. The mTOR specific inhibitor (CCI-779) was used to treat Bel-7402 cells to identify its effects on Bel-7402 cell growth and activity of mTOR signaling pathway in vitro. Cell viability tests were performed after the treatment of CCI-779. Western blot was applied to assess the changes of mTOR pathway and flow cytometry was used to analyze cell cycle of Bel-7402 cells after the treatment of CCI-779.
mTOR, p70S6K, S6, and 4EBP1 were overexpressed in Bel-7402 cells compared with HL-7702 cells. Bel-7402 cells were sensitive to CCI-779. The survival rate of the cells treated with CCI-779 over 0.312 μM was significantly different compared with that of control (P < 0.05). CCI-779 inhibited the phosphorylation of mTOR (Ser2448), p70S6K (Thr389), S6 (Ser240/244), and 4EBP1 (Thr37/46) in different grades and the expressions of p70S6K, S6, and 4EBP1. As a result, CCI-779 induced a dose-dependent decrease in cell proliferation, G1/S arrest and damage of cell shape.
Taken together, these data showed that CCI-779 can inhibit mTOR signaling and proliferation in Bel-7402 liver cancer cells in vitro. It offers a therapeutic intervention through inhibition of mTOR as a potential strategy for liver cancer.
KeywordsCCI-779 (temsirolimus) mTOR signaling Cell growth Liver cancer cell
Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related death; 55% of cases are in China . Rates of liver cancer in men are typically 2 to 4 times higher than in women . One of the current treatments for liver cancer is liver transplantation. Surgical resection of liver cancer is associated with a high rate of recurrence , due to occult metastases that might exist in the remnant liver at the time of resection or as a result of the ‘field’ effect in the remnant liver . Thus, new therapeutic strategies are needed due to the poor prognosis of patients with liver cancer. Targeting cancers with specific inhibitors is a developing strategy and has shown promising antitumor efficacy.
Increasing knowledge of the signal transduction pathways of growth factors has implicated them as novel targets for cancer therapy. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that belongs to the phosphatidylinositol kinase-related kinase (PIKK) family and functions as a serine/threonine kinase. Mounting evidence has indicated that mTOR and its downstream targets S6K1 and 4EBP1 are linked to tumorigenesis. These proteins are often overexpressed or mutated in various cancers and promote malignant transformation . Thus, inhibition of mTOR signaling is a promising therapeutic anti-cancer strategy.
Rapamycin, the naturally occurring inhibitor of mTOR, and several recently developed rapamycin analogs (RAD001, CCI-779, AP23576, AZD8055) [6, 7] inhibit the growth of cell lines that have been derived from many tumor types in vitro and tumor models in vivo. CCI-779, a dihydroxymethyl propionic acid ester of rapamycin, or temsirolimus, is an effective inhibitor of mTOR. Several groups have studied its suppression of various cancers [9–11], and others have initiated a phase II study of CCI-779 in breast cancer  and glioblastoma multiforme . The clinical function of RAD001 has been studied , but the inhibitive effects and therapeutic value of CCI-779 in liver cancer remains unclear.
In this study, we analyzed the differences in mTOR expression between liver cancer and normal liver cells and treated Bel-7402 liver cancer cells with CCI-779 to show mTOR signaling has an important role in liver cancer cell growth regulation. Our data showed the inhibitive effects of CCI-779 on mTOR signaling and liver cancer cell growth. It offers a therapeutic intervention through inhibition of mTOR as a potential strategy for liver cancer.
Results and Discussion
CCI-779 inhibits proliferation of Bel-7402 liver cancer cells
mTOR, p70S6K, S6, and 4EBP1 are overexpressed in Bel-7402 cells
CCI-779 inhibits activation of mTOR and its downstream targets
CCI-779 induced G1/S cell-cycle arrest in Bel-7402 cells
Morphological changes in Bel-7402 cells after CCI-779 treatment
Conventional therapeutic approaches for tumors include surgery, radiation, and chemotherapy. There has been much recent progress in the development of preventive and therapeutic strategies that target molecules in signal transduction pathways of cell proliferation, migration, and invasion in tumors [15–17]. Many specific inhibitors have been identified and are beginning to be used in clinical trials for cancer. CCI-779 has anti-proliferative effects against a variety of human tumor-derived cell lines [4, 9, 11, 16, 18] and has a good safety profile in clinical trails [12, 13, 19].
We examined the expression of mTOR in Bel-7402 liver cancer cells and HL-7702 normal liver cells by western blot. Consistent with previous data, mTOR levels were elevated in liver cancer cells versus normal liver cells. Further, 4EBP1, p70S6K, and S6 were also overexpressed. Rictor, which complexes with mTOR, is highly expressed in liver cancer . These data indicate that the components of the mTOR pathway are upregulated in tumor cells.
Treatment of Bel-7402 cells with CCI-779 does not affect the phosphorylation of mTOR, p70S6K, or S6 after short (30 min) stimulations . However, we observed that the phosphorylation of mTOR, p70S6K, 4EBP1 and S6 were inhibited after 48 h of treatment with CCI-779. Our data show that CCI-779 affects the activity of mTOR signaling pathway has a time-dependent manner in liver cancer cells. Meanwhile, the expression of p70S6K, 4EBP1 and S6 were inhibited after 48 h of treatment with CCI-779. Similar results also were found in our previous studies on esophageal tumor cells  and other groups [22, 23]. The activity of S6 and 4EBP1 will decreased when mTOR was inhibited by its inhibitors, and then resulting in reduced protein synthesis, but the precise mechanism is unclear now. It is believed that the reduced expression of mTOR, 4EBP1 and S6 will inhibit cell proliferation.
Further, epigenetic mechanisms also govern the development of liver cancer. Several groups have found that histone deacetylase and microRNA mediate the pathogenesis of liver cancer [24, 25]. These data demonstrate the importance of the mTOR signaling pathway in regulating cancer cell proliferation. Targeting proteins in the mTOR signal pathway might be a more effective approach in developing new drugs. For example, FKBP38 is a key regulator of mTOR. The binding of FKBP38 to mTOR inhibits the mTOR pathway; but Rheb can suppress FKBP38 and hence releases the growth signals . These discoveries provide more targets in studying the inhibitors of the mTOR pathway and novel targets for cancer therapies.
mTOR has been proposed to regulate the basic process as a central regulator of cell growth , and there is a relationship between disorganization of the mTOR pathway and tumors [28, 29]. In a previous study, we demonstrated that another clinical analog of rapamycin, RAD001, inhibits the growth of esophageal cancer cells in vitro . Several studies have reviewed mTOR signaling in liver cancer and the occurrence of tumors, therapy, and the design of antitumor drugs [30, 31]. The combination of two or more inhibitors has been proposed to enhance their effects on tumor suppression. Yet, attempts to combine two inhibitors  have not been efficacious. Recent years, transgenic mouse models in the development and progression of liver cancer was developed [33, 34]. Undoubtedly, it will be a powerful tool that can be used to study liver cancer. Although rapamycin and its analogs are well established as anticancer drugs, new inhibitors of mTOR signaling must be identified and designed.
In conclusion, our results demonstrate the importance of the mTOR signaling pathway in regulating liver cancer cell proliferation. CCI-779 has inhibitory effects on Bel-7402 liver cancer cells by suppressing the activity of mTOR and its downstream components. Thus, inhibition of mTOR is a potential therapeutic strategy for liver cancer.
Materials and methods
Cell lines and culture conditions
Bel-7402 human liver cancer cells were grown in 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum. HL-7702 normal liver cells were maintained in 1640 medium, supplemented with 20% heat-inactivated fetal bovine serum. All cell lines were cultured in 5% CO2 at 37°C.
CCI-779 (temsirolimus), a derivative of rapamycin, was synthesized by Selleck Chemicals LLC (Huston, USA) and dissolved in DMSO (Sigma Chemical Corp., St. Louis, MO). The concentration of DMSO in the final solution did not exceed 1% (v/v).
Trypan blue exclusion assay of cell proliferation
The antiproliferative effects of CCI-779 on Bel-7402 cells in culture were measured by trypan blue exclusion assay. Bel-7402 cells were used to seed 24-well culture plates at 1×104 per well 24 h before drug treatment. For sensitivity experiments, subconfluent cells were treated with various concentrations CCI-779 (0.078 μM, 0.016 μM, 0.312 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5.0 μM) and 1% DMSO (v/v) for 48 h; harvested with trypsin; and stained with trypan blue. Cell number was counted using a hematocytometer.
For growth curve experiments, subconfluent cells were treated with various concentrations of CCI-779 (1 μM, 5 μM, 10 μM, and 20 μM) for 48 h, harvested with trypsin on the indicated day after treatment, and stained with trypan blue. Cell number was counted using a hematocytometer.
MTT assay and IC50 calculation
Exponentially growing cells were used to seed 96-well plates at 4×103 cells per well 48 h before drug treatment. Then, cells were incubated with CCI-779 at various concentrations (0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM, 4.0 μM, 8.0 μM, 16.0 μM, 18.0 μM, 24.0 μM, and 28.0 μM) for 48 h. The medium with CCI-779 was absorbed, and fresh medium was added. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 g/l; Sigma-Aldrich, USA) was added to each well and incubated for 4 h at 37°C. The solution was absorbed, and formazan product was dissolved by adding 100 μl DMSO to each well and incubated it for 10 min at 37°C. MTT absorbance was measured at 490/630 nm with a spectrophotometer set (Thermo, Multiskan SX 353, USA). IC50 was calculated by Logit model, based on the data.
Cell cycle analysis by flow cytometry
Bel-7402 cells were plated in 6-well tissue culture plates at 3×105 cells per well and incubated for 48 h at 37°C. For cell cycle analysis, subconfluent cells were treated with 5 μM CCI-779 for 48 h and harvested. Cells were washed with cold PBS and stained with 50 mg/L propidium iodide (PI, Sigma-Aldrich, USA). DNA content was analyzed by flow cytometry (FACS Calibur, Becton-Dickinson Co., USA).
Cell shape assay
Bel-7402 cells were used to seed 6-well culture plates at 3×105 per well 24 h before drug treatment. Cells were treated with various concentrations of CCI-779 (0.1, 1, 10, and 20 μM) for 24 and 48 h and imaged with a digital camera that was mounted on a light microscope (Olympus DP-70, Japan).
Western blot analysis
Bel-7402 cells were plated in 6-well plates at 3×105 per well and incubated for 24 h in medium. On the following day, cells were treated with various concentrations of CCI-779 (1 μM, 5 μM and 10 μM) for 48 h and dissolved in cell lysis buffer (Thermo, USA). The concentrations of protein lysates were measured by Bio-Rad protein determination method (Bio-Rad, CA, USA).
Equal amounts (40 μg) of protein were electrophoresed in SDS-polyacrylamide gels, transferred to Immun-Blot™ PVDF membranes (Bio-RAD, CA, USA), and incubated with primary antibodies overnight at 4°C and peroxidase-linked secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence (ECL) (GE Healthcare, UK) was used to detect the signals. The primary antibodies were against mTOR, phosphor-mTOR (Ser2448), phosphor-p70S6K (Thr389) (Abcam, UK); S6, phosphor-S6 (Ser240/244), phosphor-4EBP1 (Thr37/46) (Cell Signaling Technology, Inc., MA, USA); p70S6K, 4EBP1 (Santa Cruz Biotechnology, Inc., CA, USA), and β-actin (Sigma-Aldrich, USA).
Descriptive statistics were generated for all quantitative data and expressed as mean ± SD. Proliferation of cells that were exposed to the drugs was compared with that of the negative control. Statistical significance was defined as *p < 0.05.
Mammalian target of rapamycin
Ribosomal protein S6
p70 ribosomal S6 kinase
Eukaryotic translation initiation factor 4E-binding protein 1
Insulin receptor substrate-1
This work was supported by a grant from the Natural Sciences Foundation of China (No. 31160469) and Natural Sciences Foundation of Inner Mongolia, China (No. 2011MS0521).
- Jemal A, Bray F, Center MM, Ferlay J, Ward EForman D: Global cancer statistics. CA Cancer J Clin. 2011, 61 (2): 69-90. 10.3322/caac.20107.View ArticlePubMedGoogle Scholar
- Bosch FX, Ribes J, Diaz MCleries R: Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004, 127 (5): S5-S16. 10.1053/j.gastro.2004.09.011.View ArticlePubMedGoogle Scholar
- Shah SA, Wei AC, Cleary SP, Yang I, McGilvray ID, Gallinger S, Grant DRGreig PD: Prognosis and results after resection of very large (≥ 10 cm) hepatocellular carcinoma. J Gastrointest Surg. 2007, 11 (5): 589-595. 10.1007/s11605-007-0154-7.View ArticlePubMedGoogle Scholar
- Hui ICF, Tung EKK, Sze KMF, Ching YPNg IOL: Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma. Liver Int. 2010, 30 (1): 65-75. 10.1111/j.1478-3231.2009.02117.x.View ArticlePubMedGoogle Scholar
- Petroulakis E, Mamane Y, Le Bacquer O, Shahbazian D, Sonenberg N: mTOR signaling: implications for cancer and anticancer therapy. Br J Cancer. 2005, 94 (2): 195-199.View ArticleGoogle Scholar
- Vignot S, Faivre S, Aguirre DRaymond E: mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol. 2005, 16 (4): 525-537. 10.1093/annonc/mdi113.View ArticlePubMedGoogle Scholar
- Chresta CM, Davies BR, Hickson I, Harding T, Cosulich S, Critchlow SE, Vincent JP, Ellston R, Jones D, Sini P, James D, Howard Z, Dudley P, Hughes G, Smith L, Maguire S, Hummersone M, Malagu K, Menear K, Jenkins R, Jacobsen M, Smith GC, Guichard S, Pass M: AZD8055 Is a Potent, Selective, and Orally Bioavailable ATP-Competitive Mammalian Target of Rapamycin Kinase Inhibitor with In vitro and In vivo Antitumor Activity. Cancer Res. 2010, 70 (1): 288-298. 10.1158/0008-5472.CAN-09-1751.View ArticlePubMedGoogle Scholar
- Easton J, Houghton P: mTOR and cancer therapy. Oncogene. 2006, 25 (48): 6436-6446. 10.1038/sj.onc.1209886.View ArticlePubMedGoogle Scholar
- Peralba JM, de Graffenried L, Friedrichs W, Fulcher L, Grunwald V, Weiss GHidalgo M: Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res. 2003, 9 (8): 2887-2892.PubMedGoogle Scholar
- Raymond E, Alexandre J, Faivre S, Vera K, Materman E, Boni J, Leister C, Korth-Bradley J, Hanauske A, Armand JP: Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer. J Clin Oncol. 2004, 22 (12): 2336-2347. 10.1200/JCO.2004.08.116.View ArticlePubMedGoogle Scholar
- Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, Staroslawska E, Sosman J, McDermott D, Bodrogi I, Kovacevic Z, Lesovoy V, Schmidt-Wolf IG, Barbarash O, Gokmen E, O’Toole T, Lustgarten S, Moore L, Motzer RJ: Global ARCC Trial. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med. 2007, 356 (22): 2271-2781. 10.1056/NEJMoa066838.View ArticlePubMedGoogle Scholar
- Chan S, Scheulen ME, Johnston S, Mross K, Cardoso F, Dittrich C, Eiermann W, Hess D, Morant R, Semiglazov V: Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol. 2005, 23 (23): 5314-5322. 10.1200/JCO.2005.66.130.View ArticlePubMedGoogle Scholar
- Chang SM, Wen P, Cloughesy T, Greenberg H, Schiff D, Conrad C, Fink K, Robins HI, De Angelis L, Raizer J, Hess K, Aldape K, Lamborn KR, Kuhn J, Dancey J, Prados MD: North American Brain Tumor Consortium and the National Cancer Institute. Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Invest New Drugs. 2005, 23 (4): 357-361. 10.1007/s10637-005-1444-0.View ArticlePubMedGoogle Scholar
- Zhu AX, Abrams TA, Miksad R, Blaszkowsky LS, Meyerhardt JA, Zheng H, Muzikansky A, Clark JW, Kwak EL, Schrag D, Jors KR, Fuchs CS, Iafrate AJ, Borger D, RRyan DP: Phase 1/2 study of everolimus in advanced hepatocellular carcinoma. Cancer. 2011, 117 (22): 5094-5102. 10.1002/cncr.26165.PubMed CentralView ArticlePubMedGoogle Scholar
- Wan X, Helman LJ: The biology behind mTOR inhibition in sarcoma. Oncologist. 2007, 12 (8): 1007-1018. 10.1634/theoncologist.12-8-1007.View ArticlePubMedGoogle Scholar
- Fasolo A, Sessa C: mTOR inhibitors in the treatment of cancer. Expert Opin Investig Drugs. 2008, 17 (11): 1717-1734. 10.1517/135437220.127.116.117.View ArticlePubMedGoogle Scholar
- Sarkar FH, LI Y: Targeting multiple signal pathways by chemopreventive agents for cancer prevention and therapy1. Acta Pharmacol Sin. 2007, 28 (9): 1305-1315. 10.1111/j.1745-7254.2007.00689.x.View ArticlePubMedGoogle Scholar
- Yu K, Toral-Barza L, Discafani C, Zhang W, Skotnicki J, Frost PGibbons J: mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer. 2001, 8 (3): 249-258. 10.1677/erc.0.0080249.View ArticlePubMedGoogle Scholar
- Buckner JC, Forouzesh B, Erlichman C, Hidalgo M, Boni JP, Dukart G, Berkenblit A, Rowinsky EK: Phase I, pharmacokinetic study of temsirolimus administered orally to patients with advanced cancer. Invest New Drugs. 2010, 28 (3): 334-342. 10.1007/s10637-009-9257-1.View ArticlePubMedGoogle Scholar
- Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, Tovar V, Roayaie S, Minguez B, Sole M: Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008, 135 (6): 1972-1983. 10.1053/j.gastro.2008.08.008.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang ZG, Fukazawa T, Nishikawa T, Watanabe N, Sakurama K, Motoki T, Hatakeyama S, Omori O, Ohara T, Tanabe S, Fujiwara Y, Takaoka M, Shirakawa Y, Yamatsuji T, Tanaka N, Naomoto Y: RAD001 offers a therapeutic intervention through inhibition of mTOR as a potential strategy for esophageal cancer. Oncol Rep. 2010, 23 (4): 1167-1172.View ArticlePubMedGoogle Scholar
- Batista A, Barata JT, Raderschall E, Sallan SE, Carlesso N, Nadler LM, Cardoso AA: Targeting of active mTOR inhibits primary leukemia T cells and synergizes with cytotoxic drugs and signaling inhibitors. Exp Hematol. 2011, 39 (4): 457-472. 10.1016/j.exphem.2011.01.005.View ArticlePubMedGoogle Scholar
- Lee KH, Hur HS, Im SA, Lee J, Kim HP, Yoon YK, Han SW, Song SH, Oh DY, Kim TY, Bang YJ: RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase. Cancer Lett. 2010, 299 (1): 22-28. 10.1016/j.canlet.2010.07.020.View ArticlePubMedGoogle Scholar
- Pineau P, Volinia S, McJunkin K, Marchio A, Battiston C, Terris B, Mazzaferro V, Lowe SW, Croce CM, Dejean A: miR-221 overexpression contributes to liver tumorigenesis. Proc Natl Acad Sci USA. 2010, 107 (1): 264-269. 10.1073/pnas.0907904107.PubMed CentralView ArticlePubMedGoogle Scholar
- Shao H, Gao C, Tang H, Zhang H, Roberts LR, Hylander BL, Repasky EA, Ma WW, Qiu J, Adjei AA, Dy GK, Yu C: Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated anti-tumor efficacy in primary HCC cancer in vitro and in vivo. J Hepatol. 2012, 56 (1): 176-183.View ArticlePubMedGoogle Scholar
- Bai X, Ma D, Liu A, Shen X, Wang QJ, Liu Y, Jiang Y: Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science. 2007, 318 (5852): 977-980. 10.1126/science.1147379.View ArticlePubMedGoogle Scholar
- Schmelzle T, Hall MN: TOR, a central controller of cell growth. Cell. 2000, 103 (2): 253-262. 10.1016/S0092-8674(00)00117-3.View ArticlePubMedGoogle Scholar
- Albanell J, Dalmases A, Rovira A, Rojo F: mTOR signaling in human cancer. Clin Transl Oncol. 2007, 9 (8): 484-493. 10.1007/s12094-007-0092-6.View ArticlePubMedGoogle Scholar
- Hidalgo M, Rowinsky EK: The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene. 2000, 19 (56): 6680-6686. 10.1038/sj.onc.1204091.View ArticlePubMedGoogle Scholar
- Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and metabolism. Cell. 2006, 124 (3): 471-484. 10.1016/j.cell.2006.01.016.View ArticlePubMedGoogle Scholar
- Shaw RJ, Cantley LC: Ras, PI (3) K and mTOR signalling controls tumour cell growth. Nature. 2006, 441 (7092): 424-430. 10.1038/nature04869.View ArticlePubMedGoogle Scholar
- Jiang Q, Weiss JM, Back T, Chan T, Ortaldo JR, Guichard S, Wiltrout RH: mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment. Cancer Res. 2011, 71 (12): 4074-4084. 10.1158/0008-5472.CAN-10-3968.PubMed CentralView ArticlePubMedGoogle Scholar
- French DM, Lin BC, Wang M, Adams C, Shek T, Hötzel K, Bolon B, Ferrando R, Blackmore C, Schroeder K, Rodriguez LA, Hristopoulos M, Venook R, Ashkenazi A, Desnoyers LR: Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models. PLoS One. 2012, 7 (5): e36713-10.1371/journal.pone.0036713.PubMed CentralView ArticlePubMedGoogle Scholar
- Fausto N, Campbell JS: Mouse models of hepatocellular carcinoma. Semin Liver Dis. 2010, 30 (1): 87-98. 10.1055/s-0030-1247135.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.