Nutlin's two roads toward apoptosis

Abstract

Commentary to:

Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma: Evidence for p53-transcription-dependent and -independent pathways

Manujendra N. Saha, Hua Jiang and Hong Chang

The applications of genotoxic chemotherapies are often limited by their systemic toxicity and the development of multidrug resistance. One of the current major objectives of cancer research is, therefore, to discover non-genotoxic drugs that are able to effectively eliminate tumor cells with minimum side effects to patients. It's no surprise then that Nutlin, which was the first potent MDM2-p53 inhibitor, reported by Vassilev, et al. in 2004,1 and represents non-genotoxic drugs, has been the focus of active, intense and ongoing research in the field of p53 and cancer therapy. The p53 tumor suppressor protein can inhibit tumor formation by activating cell cycle checkpoints to prevent damaged cells from proliferation (cell cycle arrest and DNA repair), promoting senescence (permanent cell cycle arrest), and/or triggering programmed cell death (apoptosis),2,3 whereas p53 inactivation promotes genome instability and tumor progression and occurs via various direct or indirect mechanisms in most of human cancers. Hence, re-activating p53 in tumors that harbor wild-type p53 has been a promising strategy for cancer therapy.4 The crucial event in the induction of the p53 pathway, in addition to biological or pathological stress signals including DNA damage and overexpressed oncongenes, such as c-MYC, is to uncouple p53 from its key negative regulators, principally MDM2, consequently leading to the accumulation of stable and active p53.5 Nutlin, a novel cis-imidazoline small molecule that mimics a MDM2-binding p53 peptide, binds to MDM2, interferes with its interaction with p53 and robustly induces p53 without causing a genotoxic stress stimulus.1 Studies over the last six years have underscored the central importance of the non-genotoxic p53 activation by Nutlin in the efficacy of cancer therapy.4,6 In pre-clinical trials, Nutlin has displayed increasing potential for the treatment of human cancers, particularly in blood malignancies, such as acute myeloid leukemia (AML), B-chronic lymphocytic leukemia (B-CLL) and multiple myeloma (MM), which possess infrequent p53 mutation/deletion.4,7 Notably, rapid, but transient, p53 activation by Nutlin causes cell cycle arrest in both normal and tumor cells, with selective apoptosis in the latter.8 Combining DNA-damaging drugs with Nutlin has been shown to have synergistic effects on p53 activation, maximally inducing p53-dependent apoptosis and completely inhibiting tumor growth with a lower genotoxic burden.8–11 In spite of significant progress in understanding the biological importance of Nutlin in p53 activation and tumor suppression, detailed mechanisms underlying the action of this small molecule on p53 cellular functions have been open questions, in particular as to whether it induces transcriptiondependent or transcription-independent apoptosis of p53 or both in a specific type of cancer.

While it is well-established that p53 induces apoptosis by activating the transcription of its pro-apoptotic target genes (e.g., BAX, PUMA, NOXA, BAD, FAS, PERP, p53AIP1 and DRAM) in the nucleus, accumulating evidence also boosts that notion that p53 can induce apoptosis via a transcriptionindependent mechanism.12 p53 can also promote apoptosis directly through the mitochondria.13–15 Following an apoptotic stimulus, a fraction of cellular p53 molecules localizes to the mitochondria, physically and functionally interacting with various Bcl-2 family members to promote the disruption of mitochondrial membrane integrity and subsequent cytochrome c release. Michael Andreeff 's group initially found that Nutlin-activated p53 can use both transcription-dependent and transcription-independent pathways to induce apoptosis in AML and CLL cells.16,17 Ute Moll and her colleagues later showed that mitochondrial p53 is a major contributor to Nutlin-induced apoptosis in acute myeloid leukemia (ML-1) and colorectal carcinoma (RKO) cells.14 There are at least three lines of evidence supporting their view. First, the release of cytochrome c in response to Nutlin was rapid and preceded the induction of the p53 target gene, p21. Second and intriguingly, Nutlin-treated cells were characterized by a significant increase in p53 monoubiquitination, a post-translational modification that is essential for p53 to re-locate to the mitochondria. This could be partly due to the possibility that Nutlin may not completely disrupt the MDM2-p53 complex in cells or that monoubiquitination might not require a direct binding between MDM2 and p53 in cells so that the MDM2 E3 ligase may be able to mediate p53 mono-, but not poly-, ubiquitination. Third, the chemical p53 inhibitor pifithrinµ (PFTµ), which specifically interferes with the interaction between p53 and its mitochondrial binding partners, markedly decreased Nutlin-induced translocation of p53 to mitochondria and apoptosis as measured by PARP cleavage and TUNEL assays. Furthermore and surprisingly, disabling the transcriptional activity of p53 with PFTα, a selective p53 transcription inhibitor, not only failed to protect ML-1 cells from Nutlin-induced apoptosis, but actually potentiated the lethal effects of Nutlin. Therefore, there might be instances in which the p53-mediated transcriptome could oppose the extranuclear p53-elicited pro-apoptotic effects,18 probably by inducing the expression of p21, which was previously shown to inhibit apoptosis.19,20

The new study by Hong Chang and his colleagues in this issue of Cancer Biology & Therapy consolidates the notion that Nutlin can induce p53-dependent apoptosis via both transcription-dependent and independent mechanisms in MM cells.21 In their previous work, the authors found that Nutlin, in conjunction with velcade (a proteasome inhibitor), displayed a synergistic response in MM.22 The cytotoxic effects of velcade in MM cell lines and primary MM samples were significantly increased by Nutlin-3. However, it was unclear whether Nutlin kills MM cells via nuclear p53 or mitochondrial p53. Clarifying this would be crucial to increase our understanding of how Nutlin activates p53-dependent apoptosis as well as for directing future clinical applications to specific cancers. Employing the previously characterized approaches, they demonstrate that Nutlin can utilize both transcription-dependent and transcription-independent mechanisms to trigger p53-mediated apoptosis in MM cells.

The regulation of the expression of apoptotic genes is one characteristic of the well-established transcription-mediated route of apoptosis that is exerted by nuclear p53.23 Indeed, once freed from MDM2 by Nutlin, p53 accumulated in the nuclei of MM cells, activated pro-apoptotic genes such as PUMA, Bax and Bak, as well as repressed the pro-survival genes Bcl2 and surviving.21 However, consistent with previous reports, specifically blockading the transcriptional activity of p53 by PFT-α not only inhibited Nutlin-induced upregulation of p53-transcriptional target genes, such as p21, MDM2 and PUMA, but also enhanced the apoptotic activity of Nutlin in MM cells. One possible explanation for the increase in the apoptotic rate after PFT-α treatment might be the anti-apoptotic action of some p53 transcriptional targets. The most studied one is p21, which has been shown to block cell cycle progression as well as to inhibit apoptosis, in part by blocking the activation of procaspase-3.21 Therefore, removal of p21 can enhance p53-induced cell death. In support of this hypothesis, a recent report provided evidence showing that Nutlin drastically enhances imatinibinduced apoptosis in imatinib-resistant leukemic cells.9 Imatinib does not significantly affect the Nutlin-3-induced level of p53, but abrogated that of p21. The activation of Bax as well as caspase-3 induced by a combined treatment with imatinib and Nutlin-3 was observed preferentially in cells expressing less p21.9 By microarray analysis and qRT-PCR, the current study also showed that two of the putative candidate target genes, MYC and MAF, are negatively regulated by PFT-α. This finding is somewhat surprising as c-MYC has been reported to induce apoptosis in response to cellular stress via both p53-dependent and p53-independent mechanisms.24,25 Similarly, MAF has also been shown to increase apoptosis in peripheral CD8 cells by transactivating Caspase 6.26 This apparent contradiction might suggest that these two proteins are not involved in Nutlin-induced apoptosis in MM cells and also implies that c-MYC or MAF might regulate apoptosis in a cell- or microenvironment-specific fashion. Regardless of this discrepancy, future studies on specific modulations in gene expression by PFT-α in Nutlin-treated cells would provide more clues for rational design of therapeutic strategies of using Nutlin for different types of cancers.

The observation of the augmentation of apoptosis in MM cells by PFT-α also leads the authors to investigate the role of mitochondrial function in p53 induced apoptosis. First, by two independent experiments, immunofluorescencebased confocal microscopy and western blot analysis of subcellular fractions, they found that p53 is co-localized with the mitochondrial marker, COXIV, while p53 protein level is elevated in mitochondrial fractions of the Nutlin-treated cells. Second, co-immunoprecipitation experiments provided evidence of p53-Bcl2 interactions. Based on these results, they propose that the formation of p53-Bcl2 complexes neutralizes the inhibitory effect of Bcl2 on Bax/Bak, leading to the activation of Bax/Bak. However, mitochondrial p53, although necessary, was not sufficient to promote apoptotic response to Nutlin, as the inhibition of mitochondrial translocation of p53 by PFT-µ did not prevent Nutlin-induced apoptosis in MM cells. This finding is contrary to the previous observations that the p53's transcription-independent pathway is the major route for Nutlin-induced apoptosis in AML and colorectal carcinoma (RKO) cells, and demonstrates that Nutlin could utilize both p53's transcription-dependent and -independent pathways to promote apoptosis in MM cells. Although much more need to be investigated as to how transcription-independent functions are controlled,23,27 this study re-verifies the concept that the transcriptional and mitochondrial functions of p53 are equally important for Nutlin-triggered apoptosis, perhaps depending on cancer cell types and their local microenvironments. This feature should be considered when designing a Nutlin-based cancer therapy for different types of cancers in the near future.

Acknowledgements

H.L. was supported by NIH-NCI grants CA127724, CA095441 and CA129828.

Commentary to: