Kiyohiro Ando

Activation of AMP-activated Protein Kinase Induces p53-dependent Apoptotic Cell Death in Response to Energetic Stress

Tumor suppressor p53-dependent stress response pathways play an important role in cell fate determination. In this study, we have found that glucose depletion promotes the phosphorylation of AMP-activated protein kinase catalytic subunit α (AMPKα) in association with a significant up-regulation of p53, thereby inducing p53-dependent apoptosis in vivo and in vitro. Thymocytes prepared from glucose-depleted wild-type mice but not from p53-deficient mice underwent apoptosis, which was accompanied by a remarkable phosphorylation of AMPKα and a significant induction of p53 as well as pro-apoptotic Bax. Similar results were also obtained in human osteosarcoma-derived U2OS cells bearing wild-type p53 following glucose starvation. Of note, glucose deprivation led to a significant accumulation of p53 phosphorylated at Ser-46, but not at Ser-15 and Ser-20, and a transcriptional induction of p53 as well as proapoptotic p53 AIP1. Small interference RNA-mediated knockdown of p53 caused an inhibition of apoptosis following glucose depletion. Additionally, apoptosis triggered by glucose deprivation was markedly impaired by small interference RNA-mediated depletion of AMPKα. Under our experimental conditions, down-regulation of AMPKα caused an attenuation of p53 accumulation and its phosphorylation at Ser-46. In support of these observations, enforced expression of AMPKα led to apoptosis and resulted in an induction of p53 at protein and mRNA levels. Furthermore, p53 promoter region responded to AMPKα and glucose deprivation as judged by luciferase reporter assay. Taken together, our present findings suggest that AMPK-dependent transcriptional induction and phosphorylation of p53 at Ser-46 play a crucial role in the induction of apoptosis under carbon source depletion.

Expression profiling and differential screening between hepatoblastomas and the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas

Hepatoblastoma is one of the most common malignant liver tumors in young children. Recent evidences have suggested that the abnormalities in Wnt signaling pathway, as seen in frequent mutation of the β-catenin gene, may play a role in the genesis of hepatoblastoma. However, the precise mechanism to cause the tumor has been elusive. To identify novel hepatoblastoma-related genes for unveiling the molecular mechanism of the tumorigenesis, a large-scale cloning of cDNAs and differential screening of their expression between hepatoblastomas and the corresponding normal livers were performed. We constructed four full-length-enriched cDNA libraries using an oligo-capping method from the primary tissues which included two hepatoblastomas with high levels of alpha-fetoprotein (AFP), a hepatoblastoma without production of AFP, and a normal liver tissue corresponded to the tumor. Among the 10 431 cDNAs randomly picked up and successfully sequenced, 847 (8.1%) were the genes with unknown function. Of interest, the expression profile among the two subsets of hepatoblastoma and a normal liver was extremely different. A semiquantitative RT–PCR analysis showed that 86 out of 1188 genes tested were differentially expressed between hepatoblastomas and the corresponding normal livers, but that only 11 of those were expressed at high levels in the tumors. Notably, PLK1 oncogene was expressed at very high levels in hepatoblastomas as compared to the normal infants livers. Quantitative real-time RT–PCR analysis for the PLK1 mRNA levels in 74 primary hepatoblastomas and 29 corresponding nontumorous livers indicated that the patients with hepatoblastoma with high expression of PLK1 represented significantly poorer outcome than those with its low expression (5-year survival rate: 55.9 vs 87.0%, respectively, p=0.042), suggesting that the level of PLK1 expression is a novel marker to predict the prognosis of hepatoblastoma. Thus, the differentially expressed genes we have identified may become a useful tool to develop new diagnostic as well as therapeutic strategies of hepatoblastoma.

KIF1Bβ Functions as a Haploinsufficient Tumor Suppressor Gene Mapped to Chromosome 1p36.2 by Inducing Apoptotic Cell Death

Deletion of the distal region of chromosome 1 frequently occurs in a variety of human cancers, including aggressive neuroblastoma. Previously, we have identified a 500-kb homozygously deleted region at chromosome 1p36.2 harboring at least six genes in a neuroblastoma-derived cell line NB1/C201. Among them, only KIF1Bβ, a member of the kinesin superfamily proteins, induced apoptotic cell death. These results prompted us to address whether KIF1Bβ could be a tumor suppressor gene mapped to chromosome 1p36 in neuroblastoma. Hemizygous deletion of KIF1Bβ in primary neuroblastomas was significantly correlated with advanced stages (p = 0.0013) and MYCN amplification (p < 0.001), whereas the mutation rate of the KIF1Bβ gene was infrequent. Although KIF1Bβ allelic loss was significantly associated with a decrease in KIF1Bβ mRNA levels, its promoter region was not hypermethylated. Additionally, expression of KIF1Bβ was markedly down-regulated in advanced stages of tumors (p < 0.001). Enforced expression of KIF1Bβ resulted in an induction of apoptotic cell death in association with an increase in the number of cells entered into the G2/M phase of the cell cycle, whereas its knockdown by either short interfering RNA or by a genetic suppressor element led to an accelerated cell proliferation or enhanced tumor formation in nude mice, respectively. Furthermore, we demonstrated that the rod region unique to KIF1Bβ is critical for the induction of apoptotic cell death in a p53-independent manner. Thus, KIF1Bβ may act as a haploinsufficient tumor suppressor, and its allelic loss may be involved in the pathogenesis of neuroblastoma and other cancers.

SQSTM1 Is a Pathogenic Target of 5q Copy Number Gains in Kidney Cancer

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and is often linked to loss of chromosome 3p, which harbors the VHL tumor suppressor gene, loss of chromosome 14q, which includes HIF1A, and gain of chromosome 5q. The relevant target(s) on chromosome 5q is not known. Here, we show that 5q amplification leads to overexpression of the SQSTM1 oncogene in ccRCC lines and tumors. Overexpression of SQSTM1 in ccRCC lines promoted resistance to redox stress and increased soft agar growth, while downregulation of SQSTM1 decreased resistance to redox stress, impaired cellular fitness, and decreased tumor formation. Therefore, the selection pressure to amplify 5q in ccRCC is driven, at least partly, by SQSTM1.

Expression of TSLC1, a candidate tumor suppressor gene mapped to chromosome 11q23, is downregulated in unfavorable neuroblastoma without promoter hypermethylation

Although it has been well documented that loss of human chromosome 11q is frequently observed in primary neuroblastomas, the smallest region of overlap (SRO) has not yet been precisely identified. Previously, we performed array‐comparative genomic hybridization (array‐CGH) analysis for 236 primary neuroblastomas to search for genomic aberrations with high‐resolution. In our study, we have identified the SRO of deletion (10‐Mb or less) at 11q23. Within this region, there exists a TSLC1/IGSF4/CADM1 gene (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1), which has been identified as a putative tumor suppressor gene for lung and some other cancers. Consistent with previous observations, we have found that 35% of primary neuroblastomas harbor loss of heterozygosity (LOH) on TSLC1 locus. In contrast to other cancers, we could not detect the hypermethylation in its promoter region in primary neuroblastomas as well as neuroblastoma‐derived cell lines. The clinicopathological analysis demonstrated that TSLC1 expression levels significantly correlate with stage, Shimadas pathological classification, MYCN amplification status, TrkA expression levels and DNA index in primary neuroblastomas. The immunohistochemical analysis showed that TSLC1 is remarkably reduced in unfavorable neuroblastomas. Furthermore, decreased expression levels of TSLC1 were significantly associated with a poor prognosis in 108 patients with neuroblastoma. Additionally, TSLC1 reduced cell proliferation in human neuroblastoma SH‐SY5Y cells. Collectively, our present findings suggest that TSLC1 acts as a candidate tumor suppressor gene for neuroblastoma.

RUNX3 Modulates DNA Damage-mediated Phosphorylation of Tumor Suppressor p53 at Ser-15 and Acts as a Co-activator for p53

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation

In response to DNA damage, p73 plays a critical role in cell fate determination. In this study, we have found that Plk1 (polo-like kinase 1) associates with p73, phosphorylates p73 at Thr-27, and thereby inhibits its pro-apoptotic activity. During cisplatin-mediated apoptosis in COS7 cells in which the endogenous p53 is inactivated by SV40 large T antigen, p73 was induced to accumulate in association with a significant down-regulation of Plk1. Consistent with these observations, Plk1 reduced the stability of the endogenous p73. Immunoprecipitation and in vitro pulldown assay demonstrated that p73 binds to the kinase domain of Plk1 through its NH2-terminal region. Luciferase reporter assay and reverse transcription-PCR analysis revealed that Plk1 is able to block the p73-mediated transcriptional activation. Of note, kinase-deficient Plk1 mutant (Plk1(K82M)) retained an ability to interact with p73; however, it failed to inactivate the p73-mediated transcriptional activation, suggesting that kinase activity of Plk1 is required for the inhibition of p73. Indeed, in vitro kinase assay indicated that p73 is phosphorylated at Thr-27 by Plk1. Furthermore, small interference RNA-mediated knockdown of the endogenous Plk1 in p53-deficient H1299 cells resulted in a significant increase in the number of cells with sub-G1 DNA content accompanied by the up-regulation of p73 and pro-apoptotic p53AIP1 as well as the proteolytic cleavage of poly(ADP-ribose) polymerase. Thus, our present results suggest that Plk1-mediated dysfunction of p73 is one of the novel molecular mechanisms to inhibit the p53-independent apoptosis, and the inhibition of Plk1 might provide an attractive therapeutic strategy for cancer treatment.

Plk1 regulates liver tumor cell death by phosphorylation of TAp63

We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21Cip1/WAF1 and 14-3-3σ, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus

The PIDDosome (PIDD–RAIDD–caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2–dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function.

Transcriptional regulation of tumor suppressor p53 by cAMP-responsive element-binding protein/AMP-activated protein kinase complex in response to glucose deprivation

Tumor suppressor p53 plays a pivotal role in the regulation of cell fate determination in response to a variety of cellular stress including carbon source depletion. In this study, we found that cAMP‐responsive element‐binding protein (CREB) collaborates with AMP‐activated protein kinase α (AMPKα) to regulate the transcription of p53. Luciferase reporter assays showed that the genomic fragment spanning from −531 to −239 of human p53 gene is required for the transactivation of p53 in response to glucose deprivation. Within this region, we found out a putative CREB‐binding site. siRNA‐mediated knockdown of CREB resulted in a significant inhibition of the up‐regulation of p53 and apoptosis under glucose deprivation. Consistent with these observations, glucose deprivation induced the transcription of p53 and CREB. Additionally, glucose deprivation led to an efficient recruitment of CREB onto the promoter region of p53 gene carrying the canonical CREB‐binding site, indicating that CREB has an ability to bind to the promoter region of p53 gene and transactivate p53. Furthermore, the amounts of CREB/phospo‐AMPKα complex increased in response to glucose deprivation. Taken together, our present findings suggest that p53 is transcriptionally regulated by CREB/phospho‐AMPKα complex and thereby contributing to the induction of apoptosis under carbon source depletion.