Tadayuki Koda

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.

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.

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.

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.

ATM-dependent nuclear accumulation of IKK- α plays an important role in the regulation of p73-mediated apoptosis in response to cisplatin

I kappa B kinase (IKK) complex plays an important role in the regulation of signaling pathway that activates nuclear factor–kappa-B (NF-κB). Recently, we reported that cisplatin (CDDP) treatment causes a remarkable nuclear accumulation of IKK-α in association with stabilization and activation of p73. However, underlying mechanisms of CDDP-induced nuclear accumulation of IKK-α are elusive. Here, we found that ataxia–telangiectasia mutated (ATM) is one of upstream mediators of IKK-α during CDDP-induced apoptosis. In response to CDDP, ATM was phosphorylated at Ser-1981, which was accompanied with nuclear accumulation of IKK-α in HepG2 cells, whereas CDDP treatment had undetectable effects on IKK-α in ATM-deficient cells. Indirect immunofluorescence experiments demonstrated that phosphorylated form of ATM colocalizes with nuclear IKK-α in response to CDDP. In vitro kinase assay indicated that ATM phosphorylates IKK-α at Ser-473. Moreover, IKK-α-deficient MEFs displayed CDDP-resistant phenotype as compared with wild-type MEFs. Taken together, our present results suggest that ATM-mediated phosphorylation of nuclear IKK-α, which stabilizes p73, is one of the main apoptotic pathways in response to CDDP.

ΔNp63/BMP-7-dependent expression of matrilin-2 is involved in keratinocyte migration in response to wounding

p63 is expressed as multiple variants including TA and DeltaN forms. Since p63-deficient mice displayed profound defects of stratified epithelia, p63 is an essential transcription factor required for epidermal morphogenesis. However, precise molecular mechanisms behind contribution of p63 to normal skin formation and healing skin wounds remained unclear. In this study, we demonstrated that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of extracellular matrix protein matrilin-2. Knocking down of DeltaNp63 in human keratinocyte HaCaT cells led to a significant reduction of matrilin-2. Intriguingly, BMP-7 which is one of DeltaNp63-target gene products, induced matrilin-2 and attenuated inhibitory effect of siRNA against DeltaNp63 on matrilin-2. Furthermore, a remarkable cell migration in response to wounding took place in DeltaNp63- or matrilin-2-knocked down cells. Taken together, our present findings indicate that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of matrilin-2.

TAp63-dependent induction of growth differentiation factor 15 (GDF15) plays a critical role in the regulation of keratinocyte differentiation

Since p63-deficient mice display severe defects in formation of epidermis, p63 has been considered to be a multi-isoform p53 family member essential for epidermal development. However, it is still unclear how p63 could contribute to keratinocyte differentiation. In the present study, we have found that TAp63α is induced in association with the upregulation and a secretion of growth differentiation factor 15 (GDF15) during the keratinocyte differentiation of HaCaT cells bearing p53 mutation. Short interference RNA-mediated knockdown of the endogenous TAp63 resulted in a remarkable reduction of GDF15. Luciferase reporter assay and reverse transcription–PCR analysis demonstrated that enforced expression of TAp63α significantly increases the luciferase activity driven by GDF15 promoter and the expression of GDF15. Consistent with these results, the proximal p53/p63-binding site within the GDF15 promoter region was required for the TAp63α-mediated transcriptional activation of GDF15, and TAp63α was recruited onto this site. Furthermore, siRNA-mediated knockdown of the endogenous GDF15 permitted cell growth and inhibited the expression of the differentiation markers such as keratin 10 and involucrin in response to differentiation stimuli. Taken together, our present results provide a novel insight into understanding the molecular mechanisms behind TAp63α-mediated keratinocyte differentiation.

Muscle Atrophy and Motor Neuron Degeneration in Human NEDL1 Transgenic Mice

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease. Approximately 20% cases of familial ALS show the mutation in the superoxide dismutase-1 (SOD1) gene. We previously demonstrated that homologue to E6AP carboxyl terminus- (HECT-) type ubiquitin protein E3 ligase (NEDL1) physically bind to mutated SOD1 protein but not wild-type SOD1 and promote the degradation of mutated SOD1 protein through ubiquitin-mediated proteasome pathway. To further understand the role of NEDL1 involved in the pathogenesis of familial ALS, we generated transgenic mice with human NEDL1 cDNA. The transgenic mice with human NEDL1 expression showed motor dysfunctions in rotarod, hanging wire, and footprint pattern examination. Histological studies indicated degeneration of neurons in the lumbar spinal cord and muscle atrophy. The number of activated microglia in the spinal cord of transgenic mice was significantly higher than that of wild-type mice, suggesting that inflammation might be observed in the spinal cord of transgenic mice. In conclusion, these findings suggest that the human NEDL1 transgenic mice might develop ALS-like symptoms, showing signs of motor abnormalities, accompanied with significant reduction in muscle strength.

Sp1-mediated transcriptional regulation of NFBD1/MDC1 plays a critical role in DNA damage response pathway.

NFBD1/MDC1 is a large nuclear protein with an anti‐apoptotic potential which participates in DNA damage response. Recently, we have demonstrated that NFBD1 has an inhibitory effect on pro‐apoptotic p53 and DNA damage‐induced transcriptional repression of NFBD1 plays an important role in p53‐dependent apoptotic response. In this study, we have found that NFBD1 promoter region contains canonical Sp1‐, STAT‐1‐ and NF‐Y‐binding sites and finally we have identified Sp1 as a transcriptional activator for NFBD1. The 5′‐RACE and bioinformatic analyses revealed that NFBD1 encodes at least four transcriptional variants arising from distinct transcriptional start sites. Luciferase reporter assays using a series of NFBD1 promoter deletion mutants demonstrated that the proximal Sp1‐binding site is required for the transcriptional activation of NFBD1. Indeed, the endogenous Sp1 was recruited onto the proximal Sp1‐binding site as examined by chromatin immunoprecipitation (ChIP) assay and siRNA‐mediated knockdown of the endogenous Sp1 in HeLa cells reduced the expression levels of NFBD1, which renders cells sensitive to adriamycin (ADR). In support of this notion, mithramycin A (MA, Sp1 inhibitor) treatment resulted in a significant down‐regulation of NFBD1. Taken together, our present findings suggest that Sp1‐mediated transcriptional regulation of NFBD1 plays an important role in the regulation of DNA damage response.

A novel potent tumour promoter aberrantly overexpressed in most human cancers

The complexity and heterogeneity of tumours have hindered efforts to identify commonalities among different cancers. Furthermore, because we have limited information on the prevalence and nature of ubiquitous molecular events that occur in neoplasms, it is unfeasible to implement molecular-targeted cancer screening and prevention. Here, we found that the FEAT protein is overexpressed in most human cancers, but weakly expressed in normal tissues including the testis, brain, and liver. Transgenic mice that ectopically expressed FEAT in the thymus, spleen, liver, and lung spontaneously developed invasive malignant lymphoma (48%, 19/40) and lung-metastasizing liver cancer (hepatocellular carcinoma) (35%, 14/40) that models human hepatocarcinogenesis, indicating the FEAT protein potently drives tumorigenesis in vivo. Gene expression profiling suggested that FEAT drives receptor tyrosine kinase and hedgehog signalling pathways. These findings demonstrate that integrated efforts to identify FEAT-like ubiquitous oncoproteins are useful and may provide promising approaches for cost-effective cancer screening and prevention.