Hajime Hosoi

Amino Acid-dependent Control of p70s6k INVOLVEMENT OF tRNA AMINOACYLATION IN THE REGULATION

In human T-lymphoblastoid cells, downstream signaling events of mammalian target of rapamycin (mTOR), including the activity of p70s6k and phosphorylation of eukaryotic initiation factor 4E-binding protein 1, were dependent on amino acid concentration in the culture media, whereas other growth-related protein kinases were not. Amino acid-induced p70s6kactivation was completely inhibited by rapamycin but only partially inhibited by wortmannin. Moreover, amino acid concentration similarly affected the p70s6k activity, which was dependent on a rapamycin-resistant mutant (S2035I) of mTOR. These data indicate that mTOR is required for amino acid-dependent activation of p70s6k. The mechanism by which amino acids regulate p70s6k activity was further explored: 1) amino acid alcohols, which inhibit aminoacylation of tRNA by their competitive binding to tRNA synthetases, suppressed p70s6k activity; 2) suppression of p70s6k by amino acid depletion was blocked by cycloheximide or puromycin, which inhibit utilization of aminoacylated tRNA in cells; and 3) in cells having a temperature-sensitive mutant of histidyl tRNA synthetase, p70s6k was suppressed by a transition of cells to a nonpermissible temperature, which was partially restored by addition of high concentrations of histidine. These results indicate that suppression of tRNA aminoacylation is able to inhibit p70s6k activity. Deacylated tRNA may be a factor negatively regulating p70s6k.

Predominant Nuclear Localization of Mammalian Target of Rapamycin in Normal and Malignant Cells in Culture

Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells.

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.

Restoration of p53 Pathway by Nutlin-3 Induces Cell Cycle Arrest and Apoptosis in Human Rhabdomyosarcoma Cells

Purpose: Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53. The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines. Experimental Design: Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed. Results: Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21 expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53. Conclusions: Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.

Epigenetic silencing of prostaglandin E receptor 2 (PTGER2) is associated with progression of neuroblastomas

We previously identified a cluster of prostanoid receptor genes, prostaglandin D2 receptor (PTGDR) and prostaglandin E receptor 2 (PTGER2), as possible targets for DNA methylation in advanced types of neuroblastoma (NB) using bacterial artificial chromosome array-based methylated CpG island amplification method. Among them, in this study, we found that PTGER2 was frequently silenced in NB cell lines, especially in those with MYCN amplification, through epigenetic mechanisms. In NB cell lines, DNA methylation pattern within a part of CpG island was inversely correlated with PTGER2 expression, and histone H3 and H4 deacetylation and histone H3 lysine 9 methylation within the putative promoter region were more directly correlated with silencing of this gene. Methylation of PTGER2 was observed more frequently in advanced-type of primary NBs compared with early-stage tumors. Growth of NB cells lacking endogenous PTGER2 expression was inhibited by restoration of the gene product by transient and stable transfection. A PTGER2-selective agonist, butaprost, increased intracellular cyclic adenosine monophosphate (cAMP) level, inhibited cell growth and induced apoptosis of NB cells stably expressing exogenous PTGER2. 8-Bromo-cAMP also inhibited growth of NB cells lacking PTGER2 expression, but not cells expressing this gene. Taken together, it is suggested that NB cells may lose responsiveness to PTGER2-mediated growth inhibition/apoptosis through epigenetic silencing of PTGER2 and/or disruption of downstream cAMP-dependent pathway during the neuroblastomagenesis.

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age ⩾12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8–30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6–9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome.

Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.

A review of 331 rhabdomyosarcoma cases in patients treated between 1991 and 2002 in Japan

BackgroundTo prepare for a Japanese nationwide group study of patients with rhabdomyosarcoma (RMS), we examined the characteristics and outcomes of RMS patients treated recently in Japan.MethodsWe classified 331 RMS patients treated between 1991 and 2002 at 63 institutions according to the Intergroup Rhabdomyosarcoma Study V (IRS-V) risk-group classification.ResultsTen-year survival rates were 86.3% for patients in low-risk subgroup A, 80.7% for low-risk subgroup B, 62.7% for intermediate-risk subgroup A, 61.7% for intermediate-risk subgroup B, and 38.1% for the high-risk group. The outcomes of the patients in the former three groups were 8%, 12%, and 21% worse than the outcomes of the respective patients in the IRS-III and early IRS-IV data. The frequency of the alveolar histological subtype was 21.8%. Chimera genes, which are useful markers for the alveolar subtype, had been examined in only 10% of the patients treated in the period of this investigation. The survival rates of our patients with embryonal and alveolar histological subtypes (65.9% and 63.4%, respectively) were not significantly different. Among the patients in the high-risk group, the 5-year survival of patients who received high-dose chemotherapy (HDC; 58.2%) was significantly better than that of patients who did not receive HDC (18.4%).ConclusionPatients in the lower-risk groups with embryonal-type tumors had poorer outcomes in this retrospective study. The better outcome of patients in the high-risk group is apparently due to the outstanding results obtained with an HDC regimen in a single institution. These results suggest that there is a need for: (1) a standard therapy, (2) a rapid central pathology review including a chimera gene analysis for the lower-risk group, and (3) evaluation of the efficacy of the high-dose regimen for the high-risk group in Japan.

Circulating Methylated-DCR2 Gene in Serum as an Indicator of Prognosis and Therapeutic Efficacy in Patients with MYCN Nonamplified Neuroblastoma

Background:MYCN amplification (MNA) in neuroblastoma is a strong indicator of poor prognosis. However, some MYCN nonamplified (non-MNA) cases show poor outcomes, and examining the status of the gene requires an operation, which may have surgical complications. Therefore, a new marker is needed to identify cases of non-MNA neuroblastomas with poor prognoses using less risky procedures. Aberrant hypermethylation of the DCR2 promoter has recently been associated with rapidly progressing neuroblastoma. We aimed to develop a noninvasive DCR2 methylation assay for patients with neuroblastoma using serum DNA, which predominantly originates from tumor-released DNA. Methods: Using DNA-based real-time PCR, we simultaneously quantified a methylated-DCR2 specific sequence (M) and a reference sequence (R) located in the promoter region in serum DNA, and evaluated DCR2 methylation status as M/R ratios in 86 patients with neuroblastoma. Results: Serum DCR2 M/R ratios were strongly correlated with those in the tumor (r = 0.67; P = 0.002). DCR2 methylation was associated with stage both in the whole neuroblastoma group and in the non-MNA group (P < 0.001), and DCR2-methylated patients showed significantly poorer 5-year event-free survival in the whole neuroblastoma group (43% versus 84%; P < 0.001), especially in the non-MNA group (12% versus 96%;P < 0.001). Among five DCR2-methylated patients whose clinical courses were followed, serum M/R ratios were close to 0 in the patients in remission, whereas the ratios increased in patients who relapsed. Conclusions: Detection of methylated-DCR2 in serum DNA has promise as a noninvasive assay for predicting prognosis and therapeutic efficacy in neuroblastoma, especially in non-MNA cases. Furthermore, it might be a sensitive marker of tumor recurrence in DCR2-methylated cases.

Sensitivity of Malignant Rhabdoid Tumor cell lines to PD 0332991 is inversely correlated with p16 expression

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC(50) values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r=0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p<0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.