NF- κ B and chemoresistance: potentiation of cancer drugs via inhibition of NF- κ B

he transcription factor NF-κB is an intensively studied regulator of gene expression involved in immune and inflammatory responses. In most cells, the bulk of NF-κB is found in the cytoplasm associated with an inhibitor known as IκB. In response to a variety of stimuli, including the proinflammatory cytokine TNFα, IκB becomes phosphorylated which leads to its ubiquitination and subsequent degradation by the proteasome, allowing for the nuclear translocation of NF-κB. Mutation of the serines (ser32 and ser36) on IκBα that are inducibly phosphorylated creates a so-called ‘super-repressor’ IκBα which strongly blocks NF-κB activation. Expression of the super-repressor is one approach to block NF-κB activation in cells. A report from Beg et al. established the anti-apoptotic nature of NF-κB when it was shown that animals null for the p65/RelA subunit of NF-κB die embryonically due to extensive liver apoptosis.A series of papers establishing the antiapoptotic role for NF-κB in response to B-cell stimulation, TNFα, chemotherapy and radiation were subsequently published. Several of these reports utilized the super-repressor form IκBα to block NF-κB while others utilized p65/RelA null cells to show that NF-κB functions in an anti-apoptotic manner. Numerous papers and reports at meetings and in abstracts have shown that activation of NF-κB in response to TNF and other stimuli blocks apoptosis. In fact, reports from several groups have identified some of the key genes regulated by NF-κB to suppress apoptosis (clearly establishing the anti-apoptotic nature of NF-κB). It should be noted that under some conditions NF-κB may serve a pro-apoptotic role. Although several of the reports indicating that NF-κB can function pro-apoptotically have used non-specific inhibitors of NF-κB to draw their conclusions or have not distinguished between the roles of different dimeric forms of NF-κB,evidence that NF-κB can function pro-apoptotically in T cells through the upregulation of Fas ligand transcription is compelling. In this regard, NF-κB can be viewed as a stress response factor where cell-type mechanisms or signalingtype mechanisms utilize NF-κB either to be anti-apoptotic or pro-apoptotic. The realizations that NF-κB can be activated by chemotherapy, that chemotherapy induces cell death through apoptotic mechanisms, and that NF-κB can function T anti-apoptotically led us to explore whether the treatment of cancer cells with chemotherapy in parallel with NF-κB inhibition would enhance apoptosis.Thus, we established stable HT1080 fibrosarcoma cell lines expressing the super-repressor IκBα. Daunorubicin was shown to activate NF-κB in these cells and inhibition of NF-κB lead to dramatically improved cell death response as compared to the parental cell line HT1080.The specificity of this response was shown in an experiment in which staurosporine-induced cell death was not strongly enhanced by inhibition of NF-κB.We have subsequently made several different stable cell lines expressing the super-repressor IκBα and have found that the apoptotic potential of TNFα and of several chemotherapies is strongly enhanced in these cell lines as compared to the control cells. In order not to depend on in vitro studies, we analyzed tumors generated in nude mice.This recent study showed that the adenovirus-mediated delivery of super-repressor IκBα into tumors of colorectal and fibrosarcoma origin strongly enhanced apoptosis in response to the topoisomerase I inhibitor CPT-11. In fact, certain tumors could be completely eliminated when this combined approach was used.This result is a cause for optimism for the potential use of NF-κB inhibitors in cancer patients. Recently, Bentires-Alj et al. published a paper entitled ‘Stable inhibition of NF-κB in cancer cells does not increase sensitivity to cytotoxic drugs’.This group analyzed four cell lines: MCF-7 breast cancer cells, OVCAR-3 ovarian carcinoma cells, HPB lymphoid T cells, and HCT116 colon carcinoma cells. In order to inhibit NF-κB, they expressed the superrepressor IκBα stably in these cells. In agreement with our publications and those of others, this group showed that chemotherapy activates NF-κB, but they did not find that stable inhibition of NF-κB enhanced cytotoxicity to chemotherapy or to TNFα. In contrast with the overall conclusion of the Bentires-Alj et al. work, each of the stable cancer cell lines that we have created (fibrosarcoma, myeloid and non-small cell carcinoma) are strongly induced to undergo apoptosis when NFκB is inhibited and when the cells are treated with either TNFα or chemotherapy. However, we and others have observed that there can be cell death when some cancer cells are transfected with IκBα. Thus, it is possible that at least some of the stable cells used in the Bentires-Alj et al. paper are clonal variants that have upregulated an NF-κBindependent anti-apoptotic mechanism to overcome cell death induced in response to NF-κB inhibition. In such a hypothetical situation, the inhibition of the activation of NFκB in response to chemotherapy may not enhance cell death beyond the response obtained with chemotherapy alone because the cancer cell has already upregulated another antiapoptotic pathway. Based on the possibility that some stable IκBα transfectants do not show the same properties as the untransfected cell population,we have tested two of the cell lines studied by BentiresAlj et al. (namely, HCT116 and MCF-7). In contrast with the conclusion of that paper and consistent with the hypothesis outlined above, we find that the transient inhibition of NF-κB by adenoviral delivery of super-repressor IκBα strongly enhanced HCT116 and MCF-7 cell death in the presence of