Wataru Nakajima

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

Paclitaxel-Induced Apoptosis Is BAK-Dependent, but BAX and BIM-Independent in Breast Tumor

Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim−/− MEFs (mouse embryonic fibroblasts), the bim−/− mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak −/− MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax−/− MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion

The tumor suppressor p53 induces activation of the mitochondrial protease HtrA2/Omi and prevents Ras-driven invasion by modulating the actin cytoskeleton.

Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex.

5-Aza-2'-deoxycytidine restores proapoptotic function of p53 in cancer cells resistant to p53-induced apoptosis.

The expression of p53-target genes encoding the proapoptotic factor Noxa, but not PUMA, was not induced by p53 in HCT116 and SW480 cells, which show resistance to apoptosis in response to p53 overexpression. The lack of p53 inducibility of Noxa was restored by treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-aza-CdR). Furthermore, p53 induced apoptosis in HCT116 and SW480 cells treated with 5-aza-CdR. Moreover, the inhibition of Noxa expression by RNAi in 5-aza-CdR-treated HCT116 cells resulted in the partial inhibition of p53-induced apoptosis. These results suggest that epigenetic cancer therapy is possible for some cancers in combination with forced p53 activation.

p53‐independent Noxa induction by cisplatin is regulated by ATF3/ATF4 in head and neck squamous cell carcinoma cells

The platinum‐based DNA damaging agent cisplatin is used as a standard therapy for locally advanced head and neck squamous cell carcinoma (HNSCC). However, the mechanisms underpinning the cytotoxic effects of this compound are not entirely elucidated. Cisplatin produces anticancer effects primarily via activation of the DNA damage response, followed by inducing BCL‐2 family dependent mitochondrial apoptosis. We have previously demonstrated that cisplatin induces the expression of proapoptotic BCL‐2 family protein, Noxa, that can bind to the prosurvival BCL‐2 family protein, MCL‐1, to inactivate its function and induce cell death. Here, we show that the upregulation of Noxa is critical for cisplatin‐induced apoptosis in p53‐null HNSCC cells. This induction is regulated at the transcriptional level. With a series of Noxa promoter‐luciferase reporter assays, we find that the CRE (cAMP response element) in the promoter is critical for the Noxa induction by cisplatin treatment. Among the CREB/ATF transcription factors, ATF3 and ATF4 are induced by cisplatin, and downregulation of ATF3 or ATF4 reduced cisplatin‐induced Noxa. ATF3 and ATF4 bind to and cooperatively activate the Noxa promoter. Furthermore, ERK1 is involved in cisplatin‐induced ATF4 and Noxa induction. In conclusion, ATF3 and ATF4 are important regulators that induce Noxa by cisplatin treatment in a p53‐independent manner.

BH3 mimetics: Their action and efficacy in cancer chemotherapy

Evading apoptosis is a hallmark of cancer, and anti-apoptotic BCL-2 family proteins are frequently highly expressed in cancers. In cancer cells, aberrant DNA replication invokes replication-associated DNA damage signaling in cancer cells; however, DNA damage-induced apoptotic signals are masked by such apoptosis evasion systems. Therefore, it is considered that targeting of apoptosis is efficient for cancer cell-selective therapeutic methods. BCL-2 family proteins are critical regulators of mitochondrion-mediated apoptosis, and ‘BH3-only’ subfamily proteins induce apoptosis by binding to anti-apoptotic BCL-2 family proteins via their BH3 domain. BH3 mimetics are small molecules that mimic BH3-proteins by binding to anti-apoptotic BCL-2 family proteins. To date, more than 20 compounds have been identified, and their effects in cancer therapy have been analyzed. In this review, their efficacy in cancer chemotherapy will be discussed. Introduction Mechanistic-based molecular-targeted therapies to treat human cancers have undergone rapid development [1]. For example, small molecules specifically blocking certain aspects of signaling pathways associated with tumor growth are widely utilized for cancer chemotherapy, such as cancer-associated tyrosine kinase inhibitors [24]. Furthermore, drugs targeting chromatin modifiers, cancer-specific metabolic regulators, telomerase regulators and immune checkpoint regulators have also been investigated [5-8]. In addition to these approaches, targeting apoptosis regulators has been considered as an attractive option in cancer therapy. BH3 mimetics are small compounds that antagonize antiapoptotic BCL-2 family proteins, resulting in apoptosis induction in cancer cells [9,10]. Recently, several BH3 mimetics were identified, and accumulating evidence has shown their efficacy in cancer therapy. However, at present, their effects are limited by their target specificity and adverse drug reactions. We present here the recent advances in BH3 mimetics research, and discuss their efficacy and limitations. Regulation of apoptosis by BCL-2 family proteins Apoptosis is a major barrier to cancer that must be circumvented, and evasion of apoptosis is a hallmark of cancer, causing resistance to cancer chemotherapy [1,11,12]. Therefore, therapeutic agents that can overcome the effect of evading apoptosis may be utilized for cancer therapy. BCL-2 family proteins are critical regulators of apoptosis and function immediately upstream of mitochondria. BCL-2 family proteins possess conserved BCL-2 homology (BH) domains and are classified into antiand pro-apoptotic members that are further subdivided into ‘multidomain’ proteins, which contain four BH domains (BH1 to BH4), and ‘BH3-only’ proteins [10,13,14]. Among these proteins, the pro-apoptotic multidomain members BAX and BAK function as mitochondrial executioners and directly open pores in the mitochondrial outer membrane, resulting in the release of the apoptogenic factors such as cytochrome c and Smac/Diabro. Studies in mice lacking both Bax and Bak showed that Bax and Bak are essential inducers of mitochondrion-mediated apoptosis in response to various stimuli, including DNA damage. In contrast, anti-apoptotic multidomain members, Bcl-2, Bcl-XL and Mcl-1, inhibit the pore formation of Bax and Bak through direct binding [10,13,14]. BH3-only proteins are critical for initiating apoptosis, functioning immediately upstream of multidomain members, and activate Bax and Bak through direct and/or indirect activation [13,15]. Quadruple deficiency of Bim, Bid, Puma and Noxa abrogates apoptosis induced by various stimuli, suggesting the importance of these direct activator type BH3-only proteins in triggering Bax/Bak-mediated apoptosis induction [16]. In addition to their direct effect, BH3-only proteins also inactivate antiapoptotic multidomain proteins, resulting in indirect activation of Bax and Bak [15,16]. Among BH3-only proteins, BIM and PUMA appear to bind to all anti-apoptotic multidomain proteins with equal affinity, whereas the other members display differential affinity. Particularly, NOXA, an inducer of tumor suppressor p53-mediated apoptosis [17], shows a unique feature in that it does not bind to BCL-2, BCLXL or BCL-W but does bind to MCL-1 and A1 with high affinity [15]. Therefore, it is possible that differences in BH3 domain structure control altered apoptosis-induction pathways. BH3 mimetics and their action The pro-apoptotic BH3 domain consists of an amphipathic α-helix and binds to the hydrophobic groove, which contains BH1, -3 and -4, of anti-apoptotic multidomain proteins, resulting in the release of sequestered pro-apoptotic proteins BAX, BAK, and the activator type BH3-only proteins [10,18]. Released BAX and BAK activate themselves Correspondence to: Nobuyuki Tanaka, Department of Molecular Oncology, Institute for Advanced Medical Sciences, Nippon Medical School, 1-396 Kosugicho, Nakahara-ku, Kawasaki 211-8533, Japan, Tel: +81-44-733-1860, Fax: +8144-733-1821, E-mail: nobuta@nms.ac.jp

The anti-apoptotic protein MCL1, a novel target of lung cancer therapy

Evasion of apoptosis is one of the typical hallmarks of cancer and a major mechanism for cancer development, tumor growth, and acquisition of resistance to chemotherapy. The anti-apoptotic Bcl-2 protein family, particularly MCL1 and BCL-XL, play an important role in acquisition of apoptosis evasion. MCL1 is a highly unstable protein that is constantly degraded by the ubiquitin-proteasome system. An increase in MCL1 protein has been reported in many cancers, including lung cancer, through high mRNA expression or impairment of its degradation systems. To date, much evidence has shown that MCL1 is important for cancer cell survival and drug resistance in lung cancers. In this review, we discuss the role and mechanism of high MCL1 expression in lung cancer.