Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer

Abstract

NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα) or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030) inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic approach.

Fig 1. mIκBα transduction of BCPAP cells causes IκBα accumulation (A) and inhibition of both baseline and TNFα-stimulated p65 DNA binding (B).

Data are normalized to the vehicle treated vector group. *—p<0.01 for the effect of TNFα in the vector group (two-way ANOVA followed by Bonferroni posttest).

Fig 2. Docetaxel activates NF-κB signaling in thyroid cancer cell lines BCPAP (A,B) and 8505C (C,D).

Proteasome inhibitor bortezomib and IKK complex inhibitor GO-Y030 counteract NF-κB activation by docetaxel. Cells were incubated with drugs for 48 h prior to reporter luciferase activity measurements. The differences between groups are highly significant (p<0.0001, one-way ANOVA). Asterisks indicate significant change (p<0.05, Newman-Keuls posttest) when compared to the corresponding control group (TNFα, docetaxel alone and bortezomib alone were compared to vehicle; docetaxel and bortezomib/GO-Y030 combinations were compared to the docetaxel only group).

Fig 3. The ionizing radiation activates NF-κB signaling in BCPAP (A,C) and 8505C (B) thyroid cancer cells as measured by dual luciferase reporter assay (A, B) and p65 DNA binding ELISA (C).

p65 DNA binding experiment was performed 72 h after radiation exposure. Data are normalized to non-irradiated group (at a time 0 h for panels A and B). *—p<0.05, (two-way ANOVA, Bonferroni posttest).

Fig 4. The caspase-3/7 activity is increased in BCPAP (A) and 8505C (B) thyroid cancer cells by bortezomib and a combination of bortezomib and docetaxel but not when treated with docetaxel alone.

Asterisks point to a statistical significance when compared to a lowest dose group (p<0.05, one-way ANOVA, Newman-Keuls posttest).

Fig 5. The docetaxel and bortezomib synergistically decrease thyroid cancer cell line 8505C invasion in vitro.

Asterisks indicate a significant effect of the drug combination (p<0.05, repeated measurements one-way ANOVA, Newman-Keuls posttest). The effect of the drugs on the invasion of BCPAP and THJ16T cells was not significant. BI—Bliss independence index.

Fig 6. NF-κB signaling inhibition with mIκBα potentiates cytotoxic/anti-proliferative effect of SAHA in BCPAP (A) but not 8505C (B) and THJ16T (C) thyroid cancer cells.

The differential response to SAHA in BCPAP cells was significant at p <0.0001 (two-way ANOVA).