Inhibition of TACE activity enhances the susceptibility of myeloma cells to TRAIL

Abstract

Background: TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. However, the susceptibility of MM cells to TRAIL is largely low in most of MM cells by yet largely unknown mechanisms. Because TNF-α converting enzyme (TACE) can cleave some TNF receptor family members, in the present study we explored the roles of proteolytic modulation by TACE in TRAIL receptor expression and TRAIL-mediated cytotoxicity in MM cells.

Methodology/principal findings: MM cells preferentially expressed death receptor 4 (DR4) but not DR5 on their surface along with TACE. Conditioned media from RPMI8226 and U266 cells contained a soluble form of DR4. The DR4 levels in these conditioned media were reduced by TACE inhibition by the TACE inhibitor TAPI-0 as well as TACE siRNA. Conversely, the TACE inhibition restored surface levels of DR4 but not DR5 in these cells without affecting DR4 mRNA levels. The TACE inhibition was able to restore cell surface DR4 expression in MM cells even in the presence of bone marrow stromal cells or osteoclasts, and enhanced the cytotoxic effects of recombinant TRAIL and an agonistic antibody against DR4 on MM cells.

Conclusions/significance: These results demonstrate that MM cells post-translationally down-modulate the cell surface expression of DR4 through ectodomain shedding by endogenous TACE, and that TACE inhibition is able to restore cell surface DR4 levels and the susceptibility of MM cells to TRAIL or an agonistic antibody against DR4. Thus, TACE may protect MM cells from TRAIL-mediated death through down-modulation of cell-surface DR4. It can be envisaged that TACE inhibition augments clinical efficacy of TRAIL-based immunotherapy against MM, which eventually becomes resistant to the present therapeutic modalities.

Figure 1. The expression of TACE and TIMP-3 in various types of hematopietic malignant cells.

TACE and TIMP-3 mRNA expression was analysed by RT-PCR in MM, B-lymphoid and leukemic cell lines as indicated as well as normal PBMCs. Human GAPDH was used as a housekeeping gene for quantity normalization.

Figure 2. Up-modulation of cell surface DR4 levels by TACE inhibition.

A. RPMI8226 and U266 cells were cultured for 24 hours in the absence (dotted lines) or presence (solid lines) of TAPI-0, a TACE inhibitor, at 10 µM. Surface expression of DR4, DR5 and Fas was analyzed by flow cytometry. B. Surface expression of DR4 was analyzed by flow cytometry in various types of cell lines as indicated as well as primary MM cells after culturing for 24 hours in the absence (dotted lines) or presence (solid lines) of TAPI-0 at 10 µM. Background staining with normal IgG is shown in gray.

Figure 3. TACE is responsible for ectodomain shedding of DR4.

A. Cell lysates and culture supernatants were harvested after culturing RPMI8226 and U266 cells for 48 hours in the absence or presence of TAPI-0 at 10 µM. DR4 immunoreactivity was analyzed by immunobloting with an antibody against an DR4 extracellular domain. B. RPMI8226 and U266 cells were cultured for different hours as indicated in the absence or presence of TAPI-0 at 10 µM. DR4 mRNA expression was analysed by RT-PCR in RPMI8226 and U266 cells. GAPDH was used for quantity normalisation. C. RPMI8226 and U266 cells seeded in 24-well plates were transfected with TACE or control scrambled siRNA. After culturing for 48 hours, cell lysates were harvested. DR4 immunoreactivity was analysed by immunobloting. β-actin was used as a loading control.

Figure 4. TACE inhibition enhances the cytotoxic effects of TRAIL and R1-B12 on MM cells.

A. MM.1S and RPMI8226 cells were cultured in quadruplicate in 24-well plates in the absence or presence of recombinant TRAIL at 20 ng/ml. TAPI-0 and rh osteoprotegerin (OPG) was added at 10 µM and 1 µg/ml, respectively, as indicated. B. RPMI8226 and U266 cells were cultured in quadruplicate in 24-well plates in the absence or presence of the anti-DR4 agonistic antibody R1-B12 (□) or the anti-DR5 agonistic antibody R2-E11 (▪) at the different concentrations as indicated. C. RPMI8226, UTMC-2, MM.1S and OPC cells were cultured in quadruplicate in 24-well plates in the absence or presence of R1-B12. R1-B12 was added at the different concentrations as indicated. TAPI-0 was added at 10 µM. After culturing for 24 hours, cell viability was determined by WST-8 assay. The results are expressed as % change from controls without the treatment (mean +/− SD). *, p<0.05. D. Per cent distribution of CD138-positive MM cells. MM cell fractions were gated according to their expression of CD138, a specific marker for MM cells, as marked in the histograms. X and Y axis represents a side scatter (SS) and forward scatter (FS), respectively. CD138 staining within the gated cells in the control (left lower) indicates that MM cell fraction was correctly gated. Per cent distribution of CD138-positive MM cells was counted within whole bone marrow cells in in the histograms.

Figure 5. TACE inhibition enhances anti-MM effects of R1-B12 in the presence of bone marrow stromal cells.

A. Surface DR4 levels on MM cells in cocultures with bone marrow stromal cells or osteoclasts. MM cell lines as indicated were cultured alone (dotted line) or cocultured (solid line) for 2 days with bone marrow stromal cells or osteoclasts (OCs). B. The effects of TAPI-0 on surface DR4 levels on MM cells in the cocultures. MM cell lines as indicated were cultured alone (solid line) or cocultured (dotted line) with bone marrow stromal cells or osteoclasts (OCs). TAPI-0 was added at 10 µM into the cocultures as indicated. After culturing for 2 days, MM cells were harvested, and surface expression of DR4 was analyzed by flow cytometry. C. RPMI8226 and OPC cells were cocultured with bone marrow stromal cells. The anti-DR4 agonistic antibody R1-B12 at the different concentrations and TAPI-0 at 10 µM were added as indicated. After culturing for 2 days, the viability of was RPMI8226 cells analysed by WST-8 assay. Data are expressed as the change in viable cell numbers relative to a control without any agents (mean +/− SD). *, p<0.05. D. Colony forming cell assays. CD138-negative bone marrow cells were cultured in the absence or presence of R1-B12 or R1-B12 plus TAPI-0 for 14 days, and BFU-E and CFU-GM were counted. E. The effects of R1-B12 plus TAPI-0 on normal hematopoietic cells. CD138-negative normal hematopoietic cells were isolated from the bone marrow sample from a patient with MM, and treated for 2 days with R1-B12 or TAPI-0 alone, or both in combination. The cells were then subject to annexin V-FITC and PI dual staining.