Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma☆
Introduction
There are more than half a million new cases of hepatocellular carcinoma (HCC) each year making it the fifth most common tumor worldwide and the third cause of cancer-related deaths [1]. The incidence of HCC is increasing and mortality has nearly doubled over the last 20 years [2]. Curative treatments such as liver resection and transplantation are only possible if the tumor is detected at an early stage [3]. Prognosis of HCC remains poor since only a minority of patients qualifies for these treatments. Systemic chemotherapy is not effective in HCC and local treatments such as chemoembolization have shown only limited survival benefit in selected patients [3]. Innovative therapeutic approaches are urgently needed.
HCC displays a characteristic hypervascularity and depends on angiogenesis for tumor growth [4]. Intratumoral microvessel density (MVD) is a predicting factor of disease-free survival after curative resection of HCC [5]. A prospective study with HCC patients found that high serum levels of vascular endothelial growth factor (VEGF), the most important growth factor for endothelial cells, are associated with absence of a tumor capsule, with tumor invasion and postoperative recurrence [6].
Recently, Geissler and coworkers reported that pharmacological inhibition of the kinase mammalian target of rapamycin (mTOR) with sirolimus (rapamycin) impairs tumor growth by an antiangiogenic mechanism. Sirolimus was shown to inhibit VEGF secretion and VEGF signaling in endothelial cells [7]. mTOR is a highly conserved serine–threonine kinase and plays a central role in modulating cell growth and proliferation [8]. In response to cellular nutritional status and activation of the upstream PI-3 kinase/Akt pathway, mTOR enhances mRNA translation and protein synthesis by phosphorylating its downstream targets p70 S6 kinase (S6K) and the translation initiation factor 4E-binding protein 1 (4E-BP1) [9]. Inhibition of mTOR induces arrest of the cell cycle in G1 phase [10] and interrupts downstream propagation of PI-3 kinase/Akt-mediated proliferative signals [7]. mTOR-dependent signaling was found to be activated in several tumor types including HCC [11], [12]. Immunohistochemical studies revealed that 45% of HCC displayed an increased expression of S6K which correlates with tumor nuclear grade whereas hepatic adenomas did not have increased S6K expression [11].
The immunosuppressive drugs, sirolimus (rapamycin), temsirolimus (CCI-779) and everolimus, are inhibitors of mTOR and possess antiproliferative and antiangiogenic properties [12]. These drugs are currently being evaluated in clinical trials for various malignancies [12], but not yet for HCC. We tested the antitumoral properties of mTOR inhibition in a HCC model and investigated the antiangiogenic effects of this treatment. Our results provide the first in vivo data in HCC and a rationale to test pharmacological mTOR inhibition in patients with HCC.
Section snippets
Cells and culture conditions
Morris Hepatoma McA-RH7777 (MH) cells were obtained from the German Cancer Research Center (DKFZ; Heidelberg, Germany) and were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum, l-glutamine, penicillin and streptomycin. Rat aortic endothelial cells were isolated from thoracic aorta of ACI rats by collagenase and cultured in F12-K medium (Gibco, Basel, Switzerland) with 10% fetal bovine serum (Sigma–Aldrich Chemie GmbH, Munich, Germany), penicillin and streptomycin,
Results
All rats (n = 8/group) developed HCC and finished 4 weeks of treatment (Fig. 1). Animals treated with sirolimus developed significantly smaller tumors than untreated animals as assessed by repetitive MR imaging (Fig. 2a and b). Tumor sizes before harvest after 4 weeks of treatment were 775 mm3 ± 214 in the sirolimus treated group and 1538 mm3 ± 884 in the control group (p < 0.05). Survival was significantly longer in treated animals than in controls (mean survival 50.5 vs. 42.0 days; p = 0.0122, n = 10/group) (
Discussion
Our results show that mTOR inhibition prevents the progression of HCC and improves survival. In this orthotopic, syngeneic model, rats treated with sirolimus had slower HCC growth, less metastases and less ascites than untreated animals. Histological examination revealed decreased intratumoral MVD and larger areas of central necrosis in animals receiving sirolimus. Inhibition of mTOR primarily affected proliferation of endothelial cells rather than proliferation of the malignant cells and
Acknowledgements
We thank M. Ledermann for expert technical assistance in tissue handling and immunohistochemistry, K. Matozan and Dr. R. Rieben for endothelial cell isolation, F. Theiler for MR imaging and Dr. C. Redaelli for animal surgery. We are indebted to Drs. M. Schneider and C. Winnips (Wyeth Pharmaceuticals AG) for their interest in these experiments and for providing temsirolimus.
Financial support: This work was supported by the Bernische Krebsliga, the Stanley Thomas Johnson Foundation, the Swiss
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The authors who have taken part in this study declared that they have not a relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research. The authors did not receive funding from any source to carry out this study.