Sensitization of human breast cancer cells to natural killer cell-mediated cytotoxicity by proteasome inhibition

Abstract

The proteasome inhibitor, bortezomib, has direct anti-tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor-related apoptosis-inducing ligand-mediated apoptosis. Natural killer (NK) cells are effective mediators of anti-tumour responses, both through cytotoxic granule killing and apoptosis-inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK-92. Bortezomib was unable to sensitize MDA-231 breast cancer cells to NK cell-mediated killing in short-term in vitro assays. However, bortezomib did cause these cells to up-regulate apoptosis-related mRNA as well as death receptors on the cell surface. In a long-term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell-mediated killing, which led to greater anti-tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB-17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK-92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour-bearing mice. However, while bortezomib was highly cytotoxic to NK-92 cells in vitro, bortezomib treatment in vivo did not decrease NK-92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti-tumour responses than either therapy alone.

Figures

Fig. 1

Bortezomib administration in vitro causes a slight but significant increase in apoptosis and a decrease in proliferation in MDA-231 breast cancer cells. (a) MDA-231 cells were incubated with varying concentrations of bortezomib for 24 h, then stained with annexin-V and propidium iodide to determine the percentage of apoptotic cells. (b) MDA-231 cells were exposed to 0, 5, 10 or 20 nM bortezomib. After 24 and 48 h, the number of viable cells was determined through trypan blue staining. *P < 0·01 as determined by one-way analysis of variance (anova) with Dunnett's post-test in (a), two-way anova with Bonferroni post-test in (b).

Fig. 2

Sensitization of MDA-231 cells to natural killer (NK)-92 killing is not shown in short-term chromium release assays. MDA-231 cells were placed in a culture flask and treated with bortezomib at either 10 (▴) or 20 (♦) nM or left untreated (□) for 48 h. The MDA-231 cells were then washed twice, resuspended in media and used in a 7-h chromium-release assay. The NK-92 effector cells were added at the indicated effector-to-target (E : T) ratios, with a maximum E : T of 32:1. No significant difference was found between the treatments, as determined by a two-way analysis of variance with a Bonferroni post-test.

Fig. 3

Bortezomib pretreatment on MDA-231 cells increases the mRNA of proteins associated with apoptosis. MDA-231 cells were incubated with 0, 5 or 10 nM of bortezomib for 24 h. RNA was then isolated and a ribonuclease protection assay as performed to determine the amount of mRNA present from apoptosis-related proteins. The ratio of caspase 8 (a), Fas (b), death receptor 3 (DR3) (c), DR5 (d), DR4 (e), the p55 tumour necrosis factor (TNF) receptor (f), TNF receptor-associated death domain TRADD (g) and receptor-interacting protein (RIP) (h) was determined through comparison with the L32 ribosomal protein. Although all proteins examined displayed a marked increase, only the increases in the indicated panels proved to be significant. *P < 0·05 by one-way analysis of variance with Bonferroni post-test.

Fig. 4

Bortezomib pretreatment increases the surface expression of death receptor 5 (DR5) and Fas and decreases the expression of human leucocyte antigen (HLA) molecules. MDA-231 cells were treated with 20 nM bortezomib or left untreated for 24 h. Cells were stained with fluorescent-conjugated anti-DR5, anti-Fas and anti-HLA-A, B, C and analysed by flow cytometry. The mean fluorescent intensity was calculated in order to determine the relative amount of the indicated surface protein. The P-values shown were calculated by a two-tailed Student's t-test.

Fig. 5

A long-term tumour outgrowth assay displays a synergistic increase in natural killer (NK)-mediated killing of MDA-231 cells after bortezomib pretreatment; 5 × 105 BT-20 (a), MCF-7 (a) or MDA-231 (c) cells were incubated with bortezomib (BT-20, 20 nM; MCF-7 and MDA-231, 10 nM) for 48 h. Target cells were then washed, labelled with 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE), and allowed another 24 h to adhere to wells. In some wells, NK-92 cells were then added at a 1:1 effector-to-target ratio. Plates were incubated for an additional 6 days then analysed by flow cytometry for CFSE and 7-amino-actinomycin D (7-AAD). The mean fluorescence intensity of 7-AAD was determined on CFSE-positive cells.

Fig. 6

Natural killer (NK)-92 cells are sensitive to bortezomib in vitro. 1 × 105 NK-92 were exposed to 0, 5, 10 or 20 nM bortezomib for 48 h, then pulsed with [3H]-thymidine to determine proliferation. The doses of 5, 10 and 20 nM each displayed a significant decrease in proliferation when compared with the untreated control cells (P < 0·001) as determined by one-way analysis of variance with Dunnett's post-test.

Fig. 7

Administration of natural killer (NK)-92 cells increases survival and decreases lung metastases in xenogeneic breast cancer studies while bortezomib displayed no effect. (a) MDA-231 cells were administered intravenously in SCID mice prior to treatment with bortezomib, NK-92 cells and interleukin (IL)-2, or a combination of both as described in Methods. Mice were monitored for survival and the experiment was terminated on day 95. (b) An MDA-231 tumour burden was established in SCID mice with treatments of bortezomib, NK-92 and IL-2, or a combination of both, as described in Methods. Mice were killed on day 49 and lungs were examined to determine the number of tumour metastases. *P < 0·01 by log-rank test compared with MDA-231 alone.