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Combination Strategy Targeting the Hypoxia Inducible Factor-1 with Mammalian Target of Rapamycin and Histone Deacetylase Inhibitors

Authors' Affiliations: ¹ Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore Maryland; ² The University Medical Center Utrecht, Utrecht, The Netherlands; and ³ Novartis Institute for Biomedical Research, East Hanover, New Jersey

Requests for reprints: Roberto Pili, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Cancer Research Building I, 1M52, Baltimore, MD 21231. Phone: 410-502-7482; Fax: 410-614-8160; E-mail: piliro{at}jhimi.edu.

Purpose: The hypoxia-inducible factor-1 (HIF-) is a key regulator of tumor angiogenesis. Mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors suppress tumor-induced angiogenesis by reducing tumor HIF-1 protein expression. Thus, we hypothesized that combination treatment of rapamycin and the HDAC inhibitor LBH589 has greater antiangiogenic and antitumor activity compared with single agents.

Experimental Design: To evaluate the effect of LBH589 and rapamycin on HIF-1 in human prostate PC3, renal C2 carcinoma cell lines, and endothelial cells (human umbilical vein endothelial cells), we did Western blot analysis. To determine the antitumor activity of LBH589 and rapamycin, cell proliferation assays and xenograft experiments were conducted.

Results: Western blotting showed that combination treatment of human umbilical vein endothelial cells, C2 and PC3, significantly reduced HIF-1 protein expression compared with single agents. Treatment with rapamycin resulted in inhibition of the downstream signals of the mTOR pathway and increased phosphorylation of Akt in C2 cells, whereas the constitutively activated Akt in PC3 cells was not modulated. LBH589 decreased both constitutively expressed and rapamycin-induced phosphorylated Akt levels in PC3 and C2 cell lines. In clonogenic assays, the combination treatment had a greater inhibitory effect in PC3 cells (93 ± 1.4%) compared with single agents (66 ± 9% rapamycin and 43 ± 4% LBH589). Combination of rapamycin and LBH589 significantly inhibited PC3 and C2 in vivo tumor growth and angiogenesis as measured by tumor weight and microvessel density.

Conclusions: Combination treatment of mTOR and HDAC inhibitors represents a rational therapeutic strategy targeting HIF-1 that warrants clinical testing.