Combined modality therapy with TRAIL or agonistic death receptor antibodies

Abstract

Molecularly targeted therapies, such as antibodies and small molecule inhibitors have emerged as an important breakthrough in the treatment of many human cancers. One targeted therapy under development is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to its ability to induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in most normal cells. TRAIL and apoptosis-inducing agonistic antibodies to the TRAIL death receptors have been the subject of many preclinical and clinical studies in the past decade. However, the sensitivity of individual cancer cell lines of a particular tumor type to these agents varies from highly sensitive to resistant. Various chemotherapy agents have been shown to enhance the apoptosis-inducing capacity of TRAIL receptor-targeted therapies and induce sensitization of TRAIL-resistant cells. This review provides an overview of the mechanisms associated with chemotherapy enhancement of TRAIL receptor-targeted therapies including modulation of the apoptotic (death receptor expression, FLIP, and Bcl-2 or inhibitors of apoptosis (IAP) families) as well as cell signaling (NFκB, Akt, p53) pathways. These mechanisms will be important in establishing effective combinations to pursue clinically and in determining relevant targets for future cancer therapies.

Figure 1

TRAIL receptors. Ligand binds to cysteine-rich domains. The death receptors have death domains that provide interactions with intercellular proteins to induce apoptotic signaling. Figure based on Almodovar et al.

Figure 2

The death receptor induced extrinsic and intrinsic apoptotic pathways. Each pathway begins with caspase-8 activation. Extrinsic pathway proceeds with the direct activation of caspase-3 by activated caspase-8. Intrinsic or mitochondrial pathway activation involves the cleavage of Bid to activate Bcl-2 family members to depolarize the mitochondrial membrane and release cytochrome c and Smac/DIABLO into the cytosol. Once released these molecules interact with Apaf-1 to activate caspase-9, which activates effector caspase-3. In some cells, the extrinsic pathway is reported to be sufficient for TRAIL-induced apoptosis, while in other cells both pathways are activated. Chemotherapy drugs may increase activation of the intrinsic pathway to enhance TRAIL receptor targeted therapies.

Figure 3

TRA-8 induced cytotoxicity against A549 lung carcinoma cells was enhanced by chemotherapy pretreatment. Cells (1,000 per well) were plated in 96 well plates and incubated at 37°C for 24 h. Then cells were treated with doxorubicin, docetaxel or bortezomib for 24 h prior to the addition of TRA-8. Cell viability was determined via ATPLite assay after 24 h of TRA-8 treatment. ATP levels are reported relative to untreated control cells and represent the mean and SD of quadruplicate samples from three independent experiments.

Figure 4

TRA-8 or TRAIL induced cytotoxicity was enhanced by doxorubicin pretreatment. Cells (1,000 per well) were plated in 96 well plates and incubated at 37°C for 24 h. Then cells were treated with DOX for 24 h prior to the addition of TRA-8 or TRAIL. Cell viability was determined via ATPLite assay after 24 h of TRA-8 or TRAIL treatment. ATP levels are reported relative to untreated control cells and represent the mean and SD of quadruplicate samples from three independent experiments.

Figure 5

Caspase-3, -8, -9 and PARP cleavage and decreased Bid and XIAP levels were induced by TRA-8 or TRAIL in sensitize cells and with combination treatment in resistant cells. 2LMP and BT-474 cells were pretreated for 24 h with DOX (50 and 5,000 nM, respectively) before the addition of TRA-8 or TRAIL (125 and 1,000 ng/ml; TRA-8, 0.84 and 6.7 nM; TRAIL, 5 and 40 nM) for 3 h. Whole cell lysates were analyzed by western blot analysis using antibodies specific for the identified protein: caspase-3 (Stressgen); caspase-8, PARP (BD PharMingen); caspase-9, Bid (cell signaling) and XIAP (R&D Systems). Actin (Sigma) was used as a loading control.

Figure 6

NFκB pathway activation via death receptor signaling. TRAIL has been reported to induce the canonical NFκB signaling pathway, which involves the inactivation of IκB subunits by the IKK complex to release active NFκB homodimers or heterodimers. Generally, these active NFκB dimers translocate to the nucleus and induce gene transcription. NFκB may have different functions depending on the NFκB subunits present and their binding partners. Chen et al. reported the p65 subunit to be anti-apoptotic and cRel to be pro-apoptotic.

Figure 7

Expression of IκBα or p65 after exposure to TRA-8 and doxorubicin. BT-474 cells were treated with DOX (5 µM for 24 h samples and 3 µM for 48 h samples) and TRA-8 (1,000 ng/ml) prior to western blot analysis with antibodies to IκBα (Cell Signaling) and p65 (Santa Cruz).

Figure 8

Inhibition of NFκB did not enhance cytotoxicity of TRA-8 against BT-474 breast cancer cells. Cells were pretreated with the NFκB translocation inhibitor SN50 or p65 specific siRNA for 24 h prior to the addition of TRA-8. Cell viability was determined via ATPLite assay after 24 h of TRA-8 treatment.

Figure 9

Akt activity in breast cancer cell lines. 2LMP and BT-474 cells were treated with TRA-8 (125 and 1,000 ng/ml) for 3 h following pretreatment with DOX (50 and 5,000 nM) for 24 h. Phospho-Akt (Ser473) and Akt were detected with specific primary antibodies (cell signaling) in whole cell lysates.

Figure 10

Pretreatment with Akt inhibitors did not enhance cytotoxicity of TRA-8 against BT-474 breast cancer cells. Cells were treated with the PI3K inhibitor, LY294002 or an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, for 24 h prior to the addition of TRA-8. Cell viability was determined via ATPLite assay after 24 h of TRA-8 treatment.