Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma

https://doi.org/10.1016/j.jhep.2006.11.021Get rights and content

Background/Aims

Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model.

Methods

Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays.

Results

Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed.

Conclusions

mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.

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

References (31)

  • H.C. Sun et al.

    Microvessel density of hepatocellular carcinoma: its relationship with prognosis

    J Cancer Res Clin Oncol

    (1999)
  • R.T. Poon et al.

    Serum vascular endothelial growth factor predicts venous invasion in hepatocellular carcinoma: a prospective study

    Ann Surg

    (2001)
  • M. Guba et al.

    Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor

    Nat Med

    (2002)
  • D.C. Fingar et al.

    Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression

    Oncogene

    (2004)
  • S. Huang et al.

    Rapamycins: mechanism of action and cellular resistance

    Cancer Biol Ther

    (2003)
  • Cited by (203)

    • Efficacy and safety of sirolimus early conversion protocol in liver transplant patients with hepatocellular carcinoma: A single-arm, multicenter, prospective study

      2022, Hepatobiliary and Pancreatic Diseases International
      Citation Excerpt :

      Sirolimus, the only approved mTOR inhibitor in China, is characterized with antitumorigenic effects and minimal nephrotoxicity. Mechanically, in vivo study shows that sirolimus significantly reduces tumor growth and that this effect is mainly mediated by antiangiogenic effects [8]. In addition, sirolimus inhibits proliferation and migration phenotypes in HCC mice model via modulation of GP73 [9].

    • YY1 and tumor metastasis regulation

      2020, YY1 in the Control of the Pathogenesis and Drug Resistance of Cancer: A Critical Therapeutic Target
    View all citing articles on Scopus

    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.

    View full text