Casey W. Wright

Reawakening the cellular death program in neoplasia through the therapeutic blockade of IAP function

Recent studies have shown that members of the inhibitor of apoptosis (IAP) protein family are highly expressed in several classes of cancer. The primary implication of these findings is that the elevated expression of IAPs is not coincidental but actually participates in oncogenesis by helping to allow the malignant cell to avoid apoptotic cell death. This concept, together with the discovery of several IAP-regulatory proteins that use a conserved mode of action, has stimulated a major effort by many research groups to devise IAP-targeting strategies as a means of developing novel antineoplastic drugs. In this Review, we consider the evidence both for and against the IAPs being valid therapeutic targets, and we describe the types of strategies being used to neutralize their functions.

CD30 activates both the canonical and alternative NF-κB pathways in anaplastic large cell lymphoma cells

CD30 is a member of the tumor necrosis factor receptor superfamily whose expression is up-regulated on anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) cells. Many different outcomes of CD30 stimulation have been reported, including cell cycle arrest, apoptosis, and activation of the prosurvival transcription factor, NF-κB, although this last activity is much less well defined in ALCL cells. In order to better understand the signaling properties of CD30 in cancer, we established a system for the stimulation of CD30 with its physiological ligand. Using this system, CD30 was stimulated on ALCL and HL cells, and the subsequent CD30 signaling properties were characterized. We show that a fraction of ALCL cells rapidly underwent apoptosis following CD30 stimulation, whereas HL cells were unaffected. The surviving ALCL cells exhibited robust activation of both the canonical and alternative NF-κB pathways as measured by nuclear translocation of RelA, p50, RelB, and p52, and this culminated in the transactivation of classical NF-κB-responsive genes. With prolonged CD30 stimulation, ALCL cells underwent cell cycle arrest that correlated with expression of the cell cycle inhibitor p21waf1. Furthermore, p21waf1 expression and cell cycle arrest were found to depend predominantly on the canonical NF-κB pathway, since it was reversed by RNA interference-mediated suppression of RelA. In contrast, suppression of the p100/p52 NF-κB subunit had little effect on p21waf1. These data reveal that in ALCL cells, in contrast to other cell types, CD30 stimulation elicits p21waf1-mediated arrest through the canonical but not the alternative NF-κB pathway.

Cytoprotective effects of IAPs revealed by a small molecule antagonist.

Deregulated expression of members of the IAP (inhibitor of apoptosis) family has been identified in a wide variety of neoplastic cells, and synthetic IAP antagonists represent a promising novel class of chemotherapeutic agents. Early work focused on the ability of these compounds to block the caspase-inhibitory function of XIAP (X-linked IAP). However, recent studies have shown that IAP antagonists, although primarily designed to target XIAP, trigger ubiquitin-mediated degradation of two related proteins, c-IAP (cellular IAP) 1 and c-IAP2, and through this process potentiates the death of tumour cells via autocrine cellular-signalling pathways. In this context, the relative contribution of XIAP as a target of this class of compounds is unclear. In the present study, we examine the involvement of XIAP using a recently described synthetic IAP antagonist, AEG40730, and through comparison of a human XIAP-depleted tumour cell line with its isogenic wild-type control line. Treatment with nanomolar concentrations of AEG40730 resulted in the loss of both XIAP and c-IAP1 proteins, albeit with different kinetics. Although XIAP-deficient HCT116 cells retained some sensitivity to external apoptotic stimuli, the results suggest that IAP antagonists, such as AEG40730, exert their apoptosis-enhancing effects through XIAP in addition to the c-IAPs. These results indicate that IAP antagonists can target multiple IAPs to augment distinct pro-apoptotic signalling pathways, thereby revealing the potential for these compounds in cancer therapy and underscoring the promise of IAP-targeted therapies.

THE ARYL HYDROCARBON NUCLEAR TRANSLOCATOR ALTERS CD30-MEDIATED NF-κB-DEPENDENT TRANSCRIPTION

Expression and signaling of CD30, a tumor necrosis factor receptor family member, is up-regulated in numerous lymphoid-derived neoplasias, most notably anaplastic large-cell lymphoma (ALCL) and Hodgkins lymphoma. To gain insight into the mechanism of CD30 signaling, we used an affinity purification strategy that led to the identification of the aryl hydrocarbon receptor nuclear translocator (ARNT) as a CD30-interacting protein that modulated the activity of the RelB subunit of the transcription factor nuclear factor κB (NF-κB). ALCL cells that were deficient in ARNT exhibited defects in RelB recruitment to NF-κB–responsive promoters, whereas RelA recruitment to the same sites was potentiated, resulting in the augmented expression of these NF-κB–responsive genes. These findings indicate that ARNT functions in concert with RelB in a CD30-induced negative feedback mechanism.

EZH2 regulates the transcription of estrogen-responsive genes through association with REA, an estrogen receptor corepressor

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase polycomb group (PcG) protein, which has been implicated in the process of cellular differentiation and cancer progression for both breast and prostate cancer. Although transcriptional repression by histone modification appears to contribute to the process of cellular differentiation, it is unclear what mediates the specificity of PcG proteins. Since EZH2 requires a binding partner for its histone methyltransferase activity, we surmised that evaluating interacting proteins might shed light on how the activity of EZH2 is regulated. Here we describe the identification of a novel binding partner of EZH2, the repressor of estrogen receptor activity (REA). REA functions as a transcriptional corepressor of the estrogen receptor and can potentiate the effect of anti-estrogens. REA expression levels have also previously been associated with the degree of differentiation of human breast cancers. We show here that EZH2 can also mediate the repression of estrogen-dependent transcription, and that moreover, the ability of both REA and EZH2 to repress estrogen-dependent transcription are mutually dependent. These data suggest that EZH2 may be recruited to specific target genes by its interaction with the estrogen receptor corepressor REA. The identification of a novel interaction between EZH2 and REA, two transcription factors that have been linked to breast cancer carcinogenesis, may lead to further insights into the process of deregulated gene expression in breast cancer.

Two distinct signalling cascades target the NF-κB regulatory factor c-IAP1 for degradation

c-IAP1 (cellular inhibitor of apoptosis 1) has recently emerged as a negative regulator of the non-canonical NF-κB (nuclear factor κB) signalling cascade. Whereas synthetic IAP inhibitors have been shown to trigger the autoubiquitination and degradation of c-IAP1, less is known about the physiological mechanisms by which c-IAP1 stability is regulated. In the present paper, we describe two distinct cellular processes that lead to the targeted loss of c-IAP1. Recruitment of a TRAF2 (tumour necrosis factor receptor-associated factor 2)–c-IAP1 complex to the cytoplasmic domain of the Hodgkins/anaplastic large-cell lymphoma-associated receptor, CD30, leads to the targeting and degradation of the TRAF2–c-IAP1 heterodimer through a mechanism requiring the RING (really interesting new gene) domain of TRAF2, but not c-IAP1. In contrast, the induced autoubiquitination of c-IAP1 by IAP antagonists causes the selective loss of c-IAP1, but not TRAF2, thereby releasing TRAF2. Thus c-IAP1 can be targeted for degradation by two distinct processes, revealing the critical importance of this molecule as a regulator of numerous intracellular signalling cascades.

APOPTOTIC SENSITIVITY OF MURINE IAP-DEFICIENT CELLS

Although numerous studies have implicated the IAPs (inhibitor of apoptosis proteins) in the control of apoptotic cell death, analyses of murine Iap-targeted cells have not revealed significant differences in their susceptibility to apoptosis. In the present study, we show that, under defined conditions, murine cells lacking XIAP (X-linked inhibitor of apoptosis) and c-IAP (cellular IAP) 2, but not c-IAP1, exhibit heightened apoptotic sensitivity to both intrinsic and extrinsic apoptotic stimuli.

Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

Background: NIK stability is necessary for activation of the noncanonical NF-κB pathway. Results: The N terminus of NIK harbors an IBM that binds to c-IAP1 and promotes NIK degradation. Conclusion: The NIK IBM provides substrate recognition for c-IAP1 activity to maximize the suppression of noncanonical NF-κB signaling. Significance: This study reveals new mechanistic insight into the regulation of the c-IAP·TRAF2·TRAF3·NIK complex. Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax−/−Bak−/− cells and better than either Bid−/− or dominant-negative caspase-9-expressing cells. Only Bim−/− and Bax−/−Bak−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid−/− cells, it readily did so in Bim−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-XL with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.