Proteasome inhibitors are known to suppress NFκB activity and induce apoptosis by inhibiting the 26S proteasome-mediated degradation of IκBα in stimulated cells [1
]. Thus, modulation of proteasome function has emerged as a novel target for the treatment of multiple cancers as well as inflammatory and immune disorders [35
]. In this study, we have shown that proteasome inhibitors suppress the constitutive NFκB DNA binding activity in metastatic prostate cancer cells by a novel mechanism, which consists of increasing the cellular levels of IκBα, which then translocates from the cytoplasm to the nucleus (). The nuclear accumulation of IκBα, induced by proteasome inhibition, results in the nuclear IκBα-p65 NFκB association, inhibition of NFκB DNA binding activity and induction of apoptosis of prostate cancer cells. Since NFκB activity is constitutively increased in many human cancers as well as in inflammatory disorders, the proteasome inhibition-induced nuclear accumulation of IκBα could thus provide a new therapeutic strategy aimed at the specific inhibition of NFκB activity by the nuclear IκBα.
Current model of the regulation of nuclear IκBα translocation by proteasome inhibition
The 26S proteasome is a large, multi-subunit protein complex that selectively degrades cellular proteins, present both in the cytoplasm and in the nucleus. Our data show that although proteasome inhibition resulted in the translocation of IκBα from the cytoplasm to the nucleus, it did not change the cellular localization on NFκB p50 and p65 proteins, nor induced nuclear translocation of LDH (). Interestingly, however, proteasome inhibition has been also shown to induce the nuclear translocation of GR [26
], aryl hydrocarbon receptor [25
], and varicella zoster DNA-binding protein ORF29p [27
]. All these proteins are transcriptional regulators that can be specifically degraded by the 26S proteasome. Although the exact mechanisms by which the proteasome inhibition induces their nuclear translocation have not been fully elucidated, studies suggest an involvement of protein stabilization resulting in increased total cellular levels, and post-translational modification by phosphorylation by protein kinase C [25
Our data demonstrate that the nuclear translocation of IκBα induced by proteasome inhibition in metastatic prostate cancer cells correlates with the stabilization of IκBα, resulting in the increased total cellular levels of IκBα (). Even though in prostate cancer cells, the proteasome inhibition induces nuclear translocation of IκBα that is phosphorylated by IKK on Ser-32 (), this phosphorylation does not seem to be necessary for the IκBα nuclear translocation in response to proteasome inhibition. First, the nuclear translocation of the endogenous IκBα is not prevented by the inhibition of IKK (), and second, the nuclear translocation of IκBα occurs also in cells that exhibit low levels of constitutive IKK activity and IκBα Ser-32 phosphorylation: HeLa cells () and HL-60 cells (not shown). These data suggest that there might be an IKK-independent mechanism that targets IκBα to the proteasome in these cells. In this context, several mechanisms have been described that lead to ubiquitin-mediated proteasome-dependent NFκB activation that occurs in the absence of IκBα phosphorylation on Ser-32 and Ser-36 [39
]. These IKK-independent mechanisms include the UV-induced NFκB activation [39
], NFκB activation induced by hepatitis B virus X protein [42
], and the IκBα degradation and NFκB activation induced by prolonged cell treatment with a chemotherapeutic agent, doxorubicin [43
]. Although the precise mechanisms by which these slow-activating agents induce the proteasomal IκBα degradation have not been identified, they seem to involve phosphatidylinositol 3-kinases, a serine-threonine kinase Akt, and the extracellular signal regulated kinase ERK [43
Since IκBα is a protein that has a high rate of metabolic turnover, our data indicate that proteasome inhibition prevents degradation of the cytoplasmic IκBα, which then translocates to the nucleus, and by binding to p65 NFκB inhibits NFκB DNA binding activity. These data are also consistent with our results showing that new protein synthesis is required in order for IκBα to translocate to the nucleus (). The lack of IκBα nuclear translocation in response to proteasome inhibition in CHX-treated cells could be explained by two mutually non-exclusive mechanisms. In the first model, treatment with CHX might prevent resynthesis of a protein that is otherwise necessary for the nuclear translocation of IκBα in MG132-treated cells, but has a short half-life; thus, treatment with CHX would significantly decrease its level. Alternatively, the nuclear translocation of IκBα in MG132-treated cells may require that the cellular (cytoplasmic) level of IκBα increases above certain threshold level. When the cells are treated with CHX, de-novo
synthesis of IκBα is inhibited, and IκBα never reaches this threshold level, even after the degradation of IκBα is blocked by MG132. Similar mechanism has been suggested to account for the nuclear accumulation of GR [26
] and the varicella-zoster virus DNA binding protein ORF29p [27
]. In this model, the nuclear translocation of IκBα, triggered by the increased cytoplasmic level of IκBα, could be explained by saturating a cytoplasmic protein, or a cellular structure, which binds and anchors IκBα in the cytoplasm. This model is also supported by previous studies that used cells transfected with constructs expressing wild type (wt) IκBα and demonstrated that when the wt IκBα protein is over-expressed, it localizes in the nucleus [45
]. These studies indicated that IκBα is retained in the cytoplasm through its association with the NFκB proteins; however, when not bound to NFκB, the free IκBα translocates to the nucleus [45
The nuclear translocation of IκBα is mediated by the second ankyrin repeat of IκBα that also mediates interaction with NFκB proteins [49
]. As a result, in most unstimulated cells, IκBα is localized in the cytoplasm bound to NFκB proteins, which can translocate to the nucleus only after IκBα had been degraded by proteasome upon stimulation [1
]. However, during post-induction repression in continuously stimulated cells, since the NFκB proteins are in the nucleus, the newly synthesized IκBα is not bound to NFκB in the cytoplasm, resulting in IκBα translocation to the nucleus and terminating NFκB-dependent transcription [51
]. Interestingly, however, we have found that in the metastatic prostate cancer PC-3 cells, IκBα is localized in the cytoplasm, while the NFκB proteins are predominantly in the nucleus (). Thus, the mechanisms that regulate the cytoplasmic localization of IκBα in PC-3 cells, and its MG132-induced nuclear translocation, are clearly different from the IκBα nuclear shuttling during post-induction repression.
In addition, the cytoplasmic localization of IκBα in PC-3 cells raises a question of the mechanisms responsible for its cytoplasmic retention in these cells. Since IκBα contains nuclear export sequences [52
], one possibility might be that IκBα continuously shuttles in the prostate cancer cells, and the nuclear export is dominant over the nuclear import, as has been described in most unstimulated cells [46
]. However, this does not seem very likely, since our data indicate that once in the nucleus, IκBα binds to p65 NFκB, inhibits NFκB DNA binding activity and induces apoptosis of the androgen-independent PC-3 cells ( and ). Alternatively, the cytoplasmic retention of IκBα in untreated PC-3 cells can be explained by binding of IκBα to cytoplasmic proteins or components. This hypothesis seems to be supported by our CHX data (), suggesting that the nuclear translocation of IκBα is regulated by its total cytoplasmic levels, as well as by previous study by Prigent et al
demonstrating that in HeLa cells, IκBα is retained in the cytoplasm by binding to protein G3BP2 [48
]. In addition, even though the cytoplasmic level of p65 NFκB in PC-3 cells is relatively low, it might be sufficient to retain IκBα in the cytoplasm. However, if the concentration of free cytoplasmic IκBα is increased by blocking the proteasome-dependent IκBα degradation, IκBα translocates and accumulates in the nucleus.
Inhibitors of the 26S proteasome have been used in the treatment of patients with multiple myeloma, and are undergoing evaluation in clinical trials in a variety of malignancies, including the metastatic, androgen independent prostate cancer [55
]. In summary, our results show that the proteasome inhibitors have a novel, previously unrecognized effect: they induce the nuclear translocation and accumulation of IκBα. In the metastatic prostate cancer cells, this results in the inhibition of the constitutive NFκB activity and induction of apoptosis. Future studies should address the specific mechanisms by which the increased stability of IκBα leads to its translocation to the nucleus. The induction of nuclear accumulation of IκBα could provide a basis for novel therapies in disorders characterized by high levels of constitutive NFκB activity, such as certain cancers and inflammatory disorders.