Optimized combination therapy using bortezomib, TRAIL and TLR agonists in established breast tumors

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptosis in various tumor cells by engaging the receptors, DR4 and DR5. Bortezomib (Velcade) is a proteasome inhibitor that has been approved for patients with multiple myeloma. There is some experimental evidence in preclinical models that bortezomib can enhance the susceptibility of tumors to TRAIL-induced apoptosis. In this study, we investigated the effects of TRAIL-induced death using an agonistic antibody to the TRAIL receptor DR5 (alpha-DR5) in combination with bortezomib administered to mice previously injected with breast cancer cells (TUBO). This combination had some beneficial therapeutic effect, which was significantly enhanced by the co-administration of a Toll-like receptor 9 agonist (CpG). In contrast, single agent treatments had little effect on tumor growth. In addition, we evaluated the effect of combination with alpha-DR5, bortezomib, and CpG in the prevention/treatment of spontaneous mammary tumors in Balb-neuT mice. In this model, which is more difficult to treat, we observed dramatic antitumor effects of alpha-DR5, bortezomib and CpG combination therapy. Since such a mouse model more accurately reflects the immunological tolerance that exists in human cancer, our results strongly suggest that these combination strategies could be directly applied to the therapy for cancer patients.

Fig. 1

Induction of apoptosis by α-DR5 and bortezomib. a DR5 expression on TUBO and 4T1-neu tumor cells. The indicated cell lines were stained with either PE-conjugated isotype control antibody (filled histogram) or PE-conjugated anti-DR5 antibody (open histogram). b TUBO cells (white bar) or 4T1-neu cells (black bars) were incubated with increasing concentration of α-DR5 alone. c TUBO cells were incubated with increasing concentration of α-DR5 alone (open circles) or in combination with bortezomib (10 μg/ml) (closed circles) for 24 h. Apoptotic cells were determined by annexin V staining. Percent specific apoptosis was calculated as described in “Materials and methods”

Fig. 2

Partial regression of TUBO tumor after treatment with either α-DR5 alone or combination of bortezomib and α-DR5. Groups of mice were subjected to the following: injected with 3 × 10⁵ TUBO cells on day 0; left untreated (n = 5, closed circles); given α-DR5 (50 μg per injection, i.v. route) for a total of three times every 4 days (lines) (n = 5, closed squares); given bortezomib (50 μg per injection, i.v. route) twice per week, with a 3-day break between injections (arrows) and α-DR5 (50 μg per injection, lines) the next day after bortezomib (n = 5, closed triangles). Significant differences (P < 0.02) between α-DR5 alone and bortezomib + α-DR5-treated mice were observed on day 14 and later. Data represent the results of a single experiment, which was one of three with similar results

Fig. 3

Partial regression of TUBO tumor after treatment with bortezomib alone or combination of bortezomib and different doses of α-DR5. Groups of mice were subjected to the following: injected with 3 × 10⁵ TUBO cells on day 0; left untreated (n = 5, closed circles); given bortezomib alone (25 μg per injection, i.v. route) for a total of three times every 4 days (arrows) (n = 5, closed squares); given bortezomib (25 μg per injection, i.v. route) twice per week, with a 3-day break between injections (arrows) and two injections of α-DR5 (50 μg per injection, i.v. route, lines) between bortezomib injections for two cycles (n = 5, closed triangles). Since we decreased the concentration of bortezomib from 50 to 25 μg, we were able to continue the treatment up to day 21 without toxicity. The injection protocol of mice (n = 5) shown by open circles was identical to those indicated by closed triangle, except for the α-DR5 dose (100 μg per injection, i.v. route). Tumor size (area in mm²) was calculated by multiplying two perpendicular diameters. P < 0.02 between α-DR5 alone (Fig. 2, closed triangles) and bortezomib + 50 μg of α-DR5 (closed triangles), α-DR5 alone (Fig. 2, closed triangles) and bortezomib + 100 μg of α-DR5 (open circles) on day 14 and later. P < 0.02 between bortezomib alone (closed squares) and bortezomib + 50 μg of α-DR5 (closed triangles), bortezomib alone (closed squares) and bortezomib + 100 μg of α-DR5 (open circles) on day 14 and later. Data represent the results of a single experiment, which was one of three with similar results

Fig. 4

Tumor regression after treatment with a combination of bortezomib, α-DR5 and systemic CpG. Groups of mice were challenged with 2 × 10⁵ TUBO cells on day 0. Mice (n = 5) indicated by closed circles were left untreated, and mice were euthanized at different time points based on the tumor size (over 20 mm). Group B and C were challenged with TUBO cells at the same time, but the treatments were different. B (n = 5, open circles) is bortezomib and α-DR5 treatment group. In graph, a indicates the period of treatment with bortezomib and α-DR5 for 3 weekly cycles. The treatment schedule is shown in a. Group C (n = 3) was given bortezomib (arrows), α-DR5 (lines) and CpG (dotted–dashed lines). CpG (50 μg per injection) was given systemically four times per week. All three mice died after treatment of 1 week because of toxicity. The time points were marked by X. Data represent the results of a single experiment, which was one of three with similar results

Fig. 5

Complete tumor regression after treatment with a combination of bortezomib, α-DR5 and intratumoral CpG. Balb/c mice were challenged with 2 × 10⁵ TUBO cells on day 0. Groups of mice were subjected to the following: left untreated (n = 5, closed circles); treated with CpG alone (n = 5, closed triangle); treated with α-DR5 and CpG (n = 5, closed square); treated with bortezomib, α-DR5 and CpG (n = 5, open circles). The schedules for treatment are indicated by arrows (bortezomib), lines (α-DR5) and dotted–dashed lines (CpG). P < 0.02 between control (closed circles) and bortezomib + α-DR5 + intratumoral CpG (open circles) on day 17 and later. Similar results were obtained in six independent experiments

Fig. 6

Antitumor effect in Balb-neuT mice by combination therapy with bortezomib, α-DR5 and CpG. a Regression of tumor size in female Balb-neuT mice. Mice (open circles; n = 5) were given bortezomib (25 μg per injection, i.v. route), α-DR5 (50 μg per injection, i.v. route) and GpG (50 μg per mouse, i.t. route) as shown in Fig 5. Control mice were left untreated (closed circles; n = 5). All animals were monitored for tumor appearance by manual examination of the mammary glands every 5 days. Measurable masses >2 mm diameter were regarded as tumors. b Tumor multiplicity. The mean number of tumors per mouse in each group was calculated. Results were evaluated as described in a. P < 0.02 between untreated group and therapy group on day 7 and day 14 in a. P < 0.02 for comparison of untreated group with therapy group at 20 weeks and later in b. These experiments were repeated twice with similar results