Yasumichi Kuwahara

Fenretinide induces sustained‐activation of JNK/p38 MAPK and apoptosis in a reactive oxygen species‐dependent manner in neuroblastoma cells

Fenretinide, which mediates apoptosis in neuroblastoma cells, is being considered as a novel therapeutic for neuroblastoma. The cytotoxic mechanisms of fenretinide, however, have not been fully elucidated. Sustained‐activation of JNK and p38 MAPK signaling has been shown recently to have a pivotal role in stress‐induced apoptosis. Whether fenretinide activates the signaling in neuroblastoma cells is not known. In the present study, fenretinide induced sustained‐activation of both JNK and p38 MAPK in neuroblastoma cells. Pretreatment with the antioxidant L‐ascorbic acid almost completely inhibited the accumulation of fenretinide‐induced intracellular reactive oxygen species (ROS), activation of JNK and p38 MAPK and apoptosis. Intracellular ROS production and activation of stress signaling was not altered by fenretinide in resistant neuroblastoma cells. Our study demonstrates that in neuroblastoma cells, fenretinide induces sustained‐activation of JNK and p38 MAPK in an ROS‐dependent manner and indicates that JNK and p38 MAPK signaling might mediate fenretinide‐induced apoptosis. Our results also indicate that suppression of the fenretinide‐induced ROS productive system and the downstream JNK and p38 MAPK signaling pathways causes neuroblastoma cells to become resistant to fenretinide.

Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction

PURPOSE MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA. PATIENTS AND METHODS Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB. RESULTS The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to the normal range in the patients in remission (n = 3), whereas the ratios increased to high levels in the patients who relapsed (n = 2) or failed to achieve remission (n = 1). CONCLUSION Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.

Antitumor Activity of Gefitinib in Malignant Rhabdoid Tumor Cells In vitro and In vivo

Purpose: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. Current treatments have had only limited success. Epidermal growth factor receptor (EGFR) was found recently to be expressed on MRT cell lines. Gefitinib (trade name Iressa) is an oral and selective EGFR-tyrosine kinase inhibitor and has been demonstrated to be effective in inhibiting the proliferation of cancer cells in vivo as well as in clinical trials. This encouraged us to examine the antitumor effects of gefitinib on MRT cells in vitro and in vivo. Experimental Design: The expression of EGFR in two MRT tumors and two MRT cell lines (MP-MRT-AN and KP-MRT-NS), established from these two tumor tissues, was examined by immunohistochemistry, immunofluorescence, and immunoblot. The effect of gefitinib on EGFR phosphorylation was examined by immunoblot. The effects of gefitinib on cell growth and apoptosis were examined by cell growth assay and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The in vivo effect of gefitinib was assessed in athymic mice that had been xenografted with MRT cells. Results: The expression of EGFR was detected in both tumor tissues and cell lines. Gefitinib inhibited EGFR-phosphorylation (IC50 < 0.1 μmol/L) and in vitro cell growth (IC50 = approximately 10–12 μmol/L), and a high concentration of gefitinib (20 μmol/L) induced apoptosis in vitro (MP-MRT-AN, 42.9% and KP-MRT-NS, 47.2%). Furthermore, gefitinib at 150 mg/kg had a cytostatic effect on established MRT xenografts (MP-MRT-AN, P = 0.039 and 0.0014; and KP-MRT-NS, P = 0.048 and 0.0086). Conclusions: Our results demonstrate that gefitinib has antitumor effects in MRT cells in vitro and in vivo and, thus, has promise as a novel and therapeutic strategy for MRT.

SNF5 reexpression in malignant rhabdoid tumors regulates transcription of target genes by recruitment of SWI/SNF complexes and RNAPII to the transcription start site of their promoters.

Malignant rhabdoid tumor (MRT), a highly aggressive cancer of young children, displays inactivation or loss of the hSNF5/INI1/SMARCB1 gene, a core subunit of the SWI/SNF chromatin-remodeling complex, in primary tumors and cell lines. We have previously reported that reexpression of hSNF5 in some MRT cell lines causes a G1 arrest via p21CIP1/WAF1 (p21) mRNA induction in a p53-independent manner. However, the mechanism(s) by which hSNF5 reexpression activates gene transcription remains unclear. We initially searched for other hSNF5 target genes by asking whether hSNF5 loss altered regulation of other consensus p53 target genes. Our studies show that hSNF5 regulates only a subset of p53 target genes, including p21 and NOXA, in MRT cell lines. We also show that hSNF5 reexpression modulates SWI/SNF complex levels at the transcription start site (TSS) at both loci and leads to activation of transcription initiation through recruitment of RNA polymerase II (RNAPII) accompanied by H3K4 and H3K36 modifications. Furthermore, our results show lower NOXA expression in MRT cell lines compared with other human tumor cell lines, suggesting that hSNF5 loss may alter the expression of this important apoptotic gene. Thus, one mechanism for MRT development after hSNF5 loss may rely on reduced chromatin-remodeling activity of the SWI/SNF complex at the TSS of critical gene promoters. Furthermore, because we observe growth inhibition after NOXA expression in MRT cells, the NOXA pathway may provide a novel target with clinical relevancy for treatment of this aggressive disease. Visual Overview: http://mcr.aacrjournals.org/content/11/3/251/F1.large.jpg. Mol Cancer Res; 11(3); 251–60. ©2013 AACR. Visual Overview

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.

Trastuzumab Activates Allogeneic or Autologous Antibody-Dependent Cellular Cytotoxicity against Malignant Rhabdoid Tumor Cells and Interleukin-2 Augments the Cytotoxicity

Purpose: Malignant rhabdoid tumor (MRT) is an early childhood cancer with poor prognosis. Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER-2), has been shown to be effective against breast cancer and other cancers. We investigated the effect of trastuzumab on MRT cell lines. Experimental Design: We examined expression of HER-2 on four MRT cell lines and two tumor tissues by indirect immunofluorescence, flow cytometry, and immunohistochemistry. The effect of trastuzumab against MRT cells was examined by cell growth assay. To observe the antibody-dependent cellular cytotoxicity of effector cells, we examined the cytotoxicity of trastuzumab in combination with allogeneic or autologous human peripheral blood mononuclear cells with and without IL-2 using the chromium release assay. Results: All four MRT cell lines and both MRT tissues expressed HER-2 protein. Trastuzumab alone did not reduce the viability of the MRT cell lines. On the other hand, the cytotoxicity of trastuzumab against each of the MRT cell lines was significantly increased by the presence of allogeneic and autologous peripheral blood mononuclear cells (P < 0.01). There was a strong correlation coefficient (r = 0.825) between HER-2 expression and the cytotoxicity enhanced by trastuzumab. Moreover, trastuzumab in combination with peripheral blood mononuclear cells augmented by interleukin-2 (IL-2) was significantly more cytotoxic than trastuzumab alone or IL-2 alone (P < 0.01). Conclusions: Our results indicate that (1) trastuzumab can exert antitumor effects on MRT cells by using the antibody-dependent cellular cytotoxicity of effector cells and (2) IL-2 can enhance the cytotoxicity of trastuzumab against MRT cells.

PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells

We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

Therapeutic targeting of PGBD5-induced DNA repair dependency in pediatric solid tumors

PGBD5 DNA transposase confers therapeutically actionable dependency in solid tumors. Synthetic lethality, pediatric edition Although a variety of therapeutic regimens are available for pediatric solid tumors, they are often ineffective and typically nonspecific. Henssen et al. determined that expression of a DNA transposase called PGBD5 is common in these tumors and presents a therapeutic vulnerability. The authors demonstrated that cells expressing PGBD5 are dependent on DNA repair through nonhomologous end joining, then identified a drug that inhibits this DNA repair pathway and is therefore active against many pediatric tumor types, particularly when combined with chemotherapy, while sparing surrounding nontumor tissues. Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma, and Ewing sarcoma, express an active DNA transposase, PGBD5, that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we discovered that mouse and human cells deficient in nonhomologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity–deficient mutant, was sufficient to induce sensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment and underwent apoptosis in both dividing and G1-phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against most neuroblastoma, medulloblastoma, Ewing sarcoma, and rhabdoid tumor cells tested while sparing nontransformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including substantial antitumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

Diffuse Anterior Retinoblastoma with Sarcoidosis-Like Nodule.

Background: Retinoblastomas account for 4% of malignancies in children, 1-2% of which are diffuse infiltrating retinoblastomas. Diffuse anterior retinoblastoma is rare and does not involve the retina. Here, we report on a diffuse anterior retinoblastoma with large sarcoidosis-like nodules on the iris that were responsive to anti-inflammatory therapy. Case: We present a 6-year-old girl who had anterior uveitis with white nodules on the iris and posterior surface of the cornea in her right eye. The nodules initially responded well to anti-inflammatory treatment. However, anterior segment optical coherence tomography (AS-OCT) showed that the nodules gradually grew, shrinking the iris. We then collected the aqueous humor for diagnosis. A biopsy revealed clusters of small cells with a high nuclear-to-cytoplasm ratio with partial rosette formation. Therefore, we diagnosed diffuse anterior retinoblastoma without retinal involvement and performed enucleation of the right eye. The histopathology demonstrated undifferentiated cells similar to those seen on the biopsy, and tumor cells invaded the iris stroma, posterior surface of the cornea, ciliary body, and sclera. After the enucleation, she underwent chemotherapy and remains alive. Conclusion: A differential diagnosis of retinoblastoma should be considered when white nodules refractory to anti-inflammatory therapy occur in the eye, even in the absence of obvious retinal masses. AS-OCT findings are useful in assessing retinoblastoma.