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. 2008 Aug 12;105(32):11317-22.
doi: 10.1073/pnas.0801868105. Epub 2008 Aug 6.

Combination Therapy of Established Cancer Using a Histone Deacetylase Inhibitor and a TRAIL Receptor Agonist

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Free PMC article

Combination Therapy of Established Cancer Using a Histone Deacetylase Inhibitor and a TRAIL Receptor Agonist

Ailsa J Frew et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

Conflict of interest statement

Conflict of interest statement: This work was supported in part by a collaborative research grant provided by Merck and Co. to the Johnstone laboratory and the Peter MacCallum Cancer Centre.

Figures

Fig. 1.
Fig. 1.
In vitro sensitivity of tumor lines to Vorinostat and MD5-1. (A) DR5 expression on 4T1.2, A20, MC38, and Renca tumor lines was assessed by using flow cytometry (fluorescence intensity is represented on a logarithmic scale). Shaded histograms represent isotype-stained cells whereas open histograms represent staining with MD5-1 anti-DR5 mAb. (B) 4T1.2 mammary carcinomas were incubated with increasing concentrations of MD5-1 (Upper) and vorinostat (Lower) for 24 h (open diamonds) and 48 h (closed diamonds). (C) Tumor lines were incubated for 24 h with the following concentrations of vorinostat and MD5-1, respectively: 4T1.2 (5 μM, 1 μg/ml), A20 (1.5 μM, 0.5 μg/ml), MC38 (5 μM, 1 μg/ml), and Renca (10 μM, 0.5 μg/ml). A CI <0.5 was found for each tumor line, demonstrating a synergistic apoptotic relationship exists between vorinostat and MD5-1. Apoptotic cells were assessed by annexin V staining. Error bars indicate ± SEM of at least three independent experiments.
Fig. 2.
Fig. 2.
Vorinostat and MD5-1 therapy induces regression of established 4T1.2 mammary carcinomas. (A) BALB/c mice with established s.c. 4T1.2 tumors (>9 mm2, day 6) were treated with control antibody (n = 16), vorinostat (n = 16), MD5-1 (n = 16), or MD5-1 and vorinostat (n = 25). MAbs (50 μg) were given i.p. (4 doses in total, given every 4 d), and vorinostat was injected i.p. daily at 100 mg/kg. Combined mean tumor sizes from three independent experiments are shown. Error bars represent ± SEM. Complete tumor regressions were only observed in mice treated with the vorinostat/MD5-1 combination with 6 of 25 mice tumor-free at the completion of treatment. Percent complete response for each treatment is shown. (B) Images of representative s.c. 4T1.2 tumors during the course of therapy. (C) TUNEL staining of 4T1.2 tumor sections taken from mice after treatment with vehicle, vorinostat (100 mg/kg), MD5-1 (50 μg), or vorinostat and MD5-1 (100 mg/kg and 50 μg, respectively). Tumors were ≈9 mm2 when treatment commenced.
Fig. 3.
Fig. 3.
Mechanisms of combination synergistic antitumor activity by using vorinostat and MD5-1. (A) 4T1.2 cells transduced with MSCV, MSCV-Bcl-2, or MSCV-CrmA were assessed by using Western blot analysis for expression of exogenous proteins. Overexpression of Bcl-2 and CrmA was confirmed by Western blot analysis by using anti-mBcl-2 and anti-CrmA antibodies. Equivalent protein loading was confirmed by reprobing blots with anti-actin mAb. (B) 4T1.2/MSCV, 4T1.2/Bcl-2, and 4T1.2/CrmA cells were assessed for sensitivity to MD5-1 (24 h, Left) and vorinostat (48 h, Right). (C) 4T1.2/MSCV, 4T1.2/Bcl-2, and 4T1.2/CrmA cells were cultured for 24 h with control antibody (1 μg/ml), vorinostat (5 μM), MD5-1 (1 μg/ml), or a combination of vorinostat and MD5-1 (5 μM and 1 μg/ml, respectively). Apoptotic cells were assessed via annexin V/PI staining and flow cytometry. The mean ± SEM of at least three independent experiments is shown. (D) SCID mice with established 4T1.2/MSCV, 4T1.2/Bcl-2, or 4T1.2/CrmA tumors (>9 mm2, day 6) were treated with control antibody (50 μg × 4; n = 6), vorinostat (100 mg/kg/d; n = 6), MD5-1 (50 μg × 4; n = 6), or MD5-1 and Vorinostat (50 μg × 4, 100 mg/kg/d, respectively; n = 8). Representative data from three independent experiments is shown.
Fig. 4.
Fig. 4.
Vorinostat down-regulates c-FLIP expression. (A) 4T1.2 cells were treated with DMSO (D) or 5 μM vorinostat (V) for 24 h. Western blot analysis was performed on whole-cell lysates by using an anti-c-FLIP mAb. Equivalent protein loading was confirmed by reprobing the blot with anti-actin mAb. (B) 4T1.2 cells were treated with DMSO (D) or vorinostat (V) over a 24 h time course. Western blot analysis on whole-cell lysates was performed by using anti-c-FLIP and anti-actin mAbs. The expression of c-FLIP relative to the loading control (actin) was quantitated by using densitometry. Protein expression values were made relative to DMSO (normalized to one) at each of the tested time points. Data shown is the mean ± SEM of three independent biological replicates. (C) BALB/c mice bearing 4T1.2 tumors (≈9 mm2) were treated with vehicle (PEG:DMSO) or vorinostat (100 mg/kg) for 4 h. Tumors were harvested, and whole-cell lysates were produced for use in Western blot analysis as described in A. (D) 4T1.2 were treated with either DMSO or 5 μM vorinostat for the time points shown. Quantitative real time PCR was performed by using primers specific for c-FLIP. Fold change in c-FLIP mRNA levels normalized to the ribosomal protein L32 mRNA levels are shown. Data shown represents mean of three independent experiments ± SEM. (E) 4T1.2 cells were treated in vitro for 8 h with DMSO (D) or 5 μM vorinostat (V) and the proteosome inhibitor MG-132 (0.1 or 10 μM). Western blot analysis was performed on whole-cell lysates as described for A. A representative blot from three independent experiments is shown. Relative c-FLIP protein levels were determined as described in B and are shown.

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