Molecular Regulation of Autophagy and Asymmetric Cell Division by Cancer Stem Cell Marker CD133.

CD133, also called prominin-1, is widely known as a cancer stem cell marker, and its high expression correlates with a poor prognosis in many cancers. CD133 was originally discovered as a plasma membranous protein in stem/progenitor cells. It is now known that Src family kinases phosphorylate the C-terminal of CD133. However, when Src kinase activity is low, CD133 is not phosphorylated by Src and is preferentially downregulated into cells through endocytosis. Endosomal CD133 then associates with HDAC6, thereby recruiting it to the centrosome via dynein motors. Thus, CD133 protein is now known to localize to the centrosome as endosomes as well as to the plasma membrane. More recently, a mechanism to explain the involvement of CD133 endosomes in asymmetric cell division was reported. Here, we would like to introduce the relationship between autophagy regulation and asymmetric cell division mediated by CD133 endosomes.

Asymmetric Pericentrosomal CD133 Endosomes Induce the Unequal Autophagic Activity During Cytokinesis in CD133-Positive Human Neuroblastoma Cells.

CD133 is a transmembrane protein that mainly localizes to the plasma membrane in hematopoietic/neural stem cells and cancer stem cells. Although CD133 also localizes to the cytoplasm and is degraded through autophagy, the precise mechanisms responsible for the autophagic degradation of endosomal CD133 currently remain unknown. We demonstrated that endosomal CD133 has unique properties for cell homeostasis. Endosomal CD133 is degraded through p62/SQSTM1-mediated selective autophagy. However, in low basal autophagic cells, such as SK-N-DZ and SH-SY5Y cells, endosomal CD133 accumulates at the pericentrosomal region and conversely suppresses autophagy. Endosomal CD133 also asymmetrically distributes to the pericentrosomal region and induces unequal autophagic activity between 2 daughter cells during cytokinesis in SK-N-DZ and TGW cells. In addition, the asymmetric distribution of pericentrosomal CD133 endosomes and nuclear ß-catenin cooperatively suppresses autophagic activity against p62 in SK-N-DZ cells. Thus, the present study suggests that the asymmetric distribution of pericentrosomal CD133 endosomes induces the symmetry breaking of autophagic activity during cytokinesis in cooperation with nuclear ß-catenin.

Wilms tumour surveillance in at-risk children: Literature review and recommendations from the SIOP-Europe Host Genome Working Group and SIOP Renal Tumour Study Group.

Since previous consensus-based Wilms tumour (WT) surveillance guidelines were published, novel genes and syndromes associated with WT risk have been identified, and diagnostic molecular tests for previously known syndromes have improved. In view of this, the International Society of Pediatric Oncology (SIOP)-Europe Host Genome Working Group and SIOP Renal Tumour Study Group hereby present updated WT surveillance guidelines after an extensive literature review and international consensus meetings. These guidelines are for use by clinical geneticists, pediatricians, pediatric oncologists and radiologists involved in the care of children at risk of WT. Additionally, we emphasise the need to register all patients with a cancer predisposition syndrome in national or international databases, to enable the development of better tumour risk estimates and tumour surveillance programs in the future.

Frequent breakpoints of focal deletion and uniparental disomy in 22q11.1 or 11.2 segmental duplication region reveal distinct tumorigenesis in rhabdoid tumor of the kidney.

SMARCB1 is mutated in most rhabdoid tumors (RTs) developing in the kidney (RTK) and various other organs. Focal deletions found in patients with 22q11.2 deletion syndrome show breakpoints within clusters of segmental duplications (SDs), and those in some RTs show breakpoints in the 22q11-q12 region. SDs are known to cause focal deletion mediated by non-allelic homologous recombination. The present study identified SMARCB1 alterations in all 30 RTKs, using SNP array CGH, MLPA, and sequence analyses. Twenty-eight tumors had a total of 51 breakpoints forming focal 22q deletion and/or uniparental disomy (22qUPD), and the other two had compound mutation with no breakpoints in 22q. Twenty-four (47.1%) of the 51 breakpoints were within SDs, and occurred in 16 (53.3%) of the 30 tumors. The association of breakpoints with SDs was found not only in focal deletion, but also in 22qUPD, indicating that SDs mediate the first and second hits (focal deletion) and the second hit (22qUPD) of SMARCB1 alteration. Of the 51 breakpoints, 14 were recurrent, and 10 of the 14 were within SDs, suggesting the presence of hotspots in the 22q11.2 region. One recurrent breakpoint outside SDs resided in SMARCB1, suggesting inactivation of the gene by out-of-frame fusion. The association between SDs and focal deletion has been reported in two other types of cancer. RTKs may be the third example of SD-associated tumors. Thus, the present study indicated that RTKs exploit genomic instability in the 22q11.1-11.2 SDs region, and 22qUPD caused by mitotic recombination may also be mediated by SDs.

The Role of MYCN in Symmetric vs. Asymmetric Cell Division of Human Neuroblastoma Cells.

Asymmetric cell division (ACD) is an important physiological event in the development of various organisms and maintenance of tissue homeostasis. ACD produces two different cells in a single cell division: a stem/progenitor cell and differentiated cell. Although the balance between self-renewal and differentiation is precisely controlled, disruptions to ACD and/or enhancements in the self-renewal division (symmetric cell division: SCD) of stem cells resulted in the formation of tumors in Drosophila neuroblasts. ACD is now regarded as one of the characteristics of human cancer stem cells, and is a driving force for cancer cell heterogeneity. We recently reported that MYCN controls the balance between SCD and ACD in human neuroblastoma cells. In this mini-review, we discuss the mechanisms underlying MYCN-mediated cell division fate.

Kasumi leukemia cell lines: characterization of tumor genomes with ethnic origin and scales of genomic alterations.

Kasumi-1 has played an important role in an experimental model with t(8;21) translocation, which is a representative example of leukemia cell lines. However, previous studies using Kasumi-1 show discrepancies in the genome profile. The wide use of leukemia cell lines is limited to lines that are well-characterized. The use of additional cell lines extends research to various types of leukemia, and to further explore leukemia pathogenesis, which can be achieved by uncovering the fundamental features of each cell line with accurate data. In this study, ten Kasumi cell lines established in Japan, including five that were previously unknown, have been characterized by SNP microarray and targeted sequencing. SNP genotyping suggested that the genetic ancestry in four of the ten Kasumi cell lines was not classified as Japanese but covered several different east-Asian ethnicities, suggesting that patients in Japan are genetically diverse. TP53 mutations were detected in two cell lines with complex array profiles, indicating chromosomal instability (CIN). A quantitative assessment of tumor genomes at the chromosomal level was newly introduced to reveal total DNA sizes and Scales of Genomic Alterations (SGA) for each cell line. Kasumi-1 and 6 derived from relapsed phases demonstrated high levels of SGA, implying that the level of SGA would reflect on the tumor progression and could serve as an index of CIN. Our results extend the leukemia cellular resources with an additional five cell lines and provide reference genome data with ethnic identities for the ten Kasumi cell lines.

Blastemal predominant type Wilms tumor in Japan: Japan Children's Cancer Group.

BACKGROUND: Persistence of blastemal components after chemotherapy is a marker of poor outcome in Wilms tumor (WT). Recent reports from local Japanese areas have described pre-chemotherapy blastemal predominant type WT to also be a risk factor for relapse. The significance, however, of blastemal predominant WT remains to be evaluated in a larger study. This study retrospectively evaluated the prognostic significance of pre-chemotherapy blastemal predominant type WT in the Japan Wilms tumor Study (JWiTS) trials. METHODS: The JWiTS trial (1996-2013) was a prospective, single-arm study. The outcomes of blastemal predominant type WT were retrospectively evaluated compared with non-blastemal type WT excluding anaplasia between 1996 and 2013. Relapse-free survival (RFS) and overall survival (OS) were estimated. RESULTS: Of 319 primary renal tumors diagnosed by the central pathology review system, advanced stage of pre-chemotherapy blastemal predominant type WT (n = 53; 16.1%) occurred more frequently in older children than non-blastemal type WT (n = 225), and was especially frequent in female patients registered in the JWiTS trials. No significant difference in 10 years RFS and OS (78.8% vs 84.5; P = 0.201) or in 10 years RFS and OS (89.3% vs 93.5; P = 0.45) was seen between pre-chemotherapy blastemal predominant type and non-blastemal type WT. CONCLUSIONS: Relapse-free survival and OS are not significantly different between pre-chemotherapy blastemal predominant type and non-blastemal type WT.

Recycling endosomal CD133 functions as an inhibitor of autophagy at the pericentrosomal region.

CD133 is a transmembranous protein that mainly localises to the plasma membrane in haematopoietic and neural stem cells as well as cancer stem cells. Although CD133 also localises to the cytoplasm, the mechanism of action and function of cytoplasmic CD133 currently remain unknown. We herein demonstrated that when Src family kinase activity is weak, CD133 interacts with HDAC6 and is transported to the pericentrosomal region after internalization and endosome formation via the dynein-based traffic system. Pericentrosomal CD133 is then recycled to the plasma membrane via recycling endosomes. At the pericentrosomal region, endosomal CD133 captures GABARAP, an initiator of autophagy, and inhibits GABARAP-mediated ULK1 activation and the subsequent initiation of autophagy. Furthermore, pericentrosomal CD133 suppresses cell differentiation, such as primary cilium formation and neurite outgrowth, by inhibiting autophagy. Thus, the present results provide evidence to suggest that pericentrosomal CD133 has the unique property of maintaining the undifferentiated status of cells by inhibiting autophagy.

Combined Genetic and Chromosomal Characterization of Wilms Tumors Identifies Chromosome 12 Gain as a Potential New Marker Predicting a Favorable Outcome.

To identify prognostic factors, array CGH (aCGH) patterns and mutations in WT1 and 9 other genes were analyzed in 128 unilateral Wilms tumors (WTs). Twenty patients had no aCGH aberrations, and 31 had WT1 alterations [silent and WT1 types: relapse-free survival (RFS), 95% and 83%, respectively]. Seventy-seven patients had aCGH changes without WT1 alterations (nonsilent/non-WT1 type) and were subtyped into those with or without +12, 11q-, 16q-, or HACE1 loss. RFS was better for those with than those without +12 (P = .010) and worse for those with than those without 11q-, 16q-, or HACE1 loss (P = .001, .025, or 1.2E-04, respectively). Silent and WT1 type and 8 subtype tumors were integrated and classified into 3 risk groups: low risk for the silent type and +12 subgroup; high risk for the no +12 plus 11q-, 16q-, or HACE1 loss subgroup; intermediate risk for the WT1 type and no +12 plus no 11q-, 16q-, or HACE1 loss subgroup. Among the 27 WTs examined, the expression of 146 genes on chromosome 12 was stronger in +12 tumors than in no +12 tumors, while that of 10 genes on 16q was weaker in 16q- tumors than in no 16q- tumors. Overexpression in 75 out of 146 upregulated genes and underexpression in 7 out of 10 downregulated genes correlated with better and worse overall survival, respectively, based on the public database. +12 was identified as a potential new marker predicting a favorable outcome, and chromosome abnormalities may be related to altered gene expression associated with these abnormalities.

Outcome of renal tumors registered in Japan Wilms Tumor Study-2 (JWiTS-2): A report from the Japan Children's Cancer Group (JCCG).

BACKGROUND: Japan Wilms Tumor Study-2 (JWiTS-2) mandated central pathology review for all case registrations. The study aimed to compare the outcomes of patients with unilateral Wilms tumor enrolled on the JWiTS-1 and JWiTS-2 trials. PROCEDURE: The JWiTS-2 trial (2006-2014), a prospective, single-arm study, required compulsory submission of histologic slides to central pathology, while in the JWiTS-1 trial, such submission was not compulsory. Relapse-free survival (RFS) and overall survival (OS) versus cases in the JWiTS-1 trial (1996-2005) were statistically evaluated. RESULTS: Of 277 enrolled patients with primary renal tumors diagnosed by the central pathology review system, 225 patients with unilateral renal tumors were followed up over 9 years. The RFS and OS of Wilms tumor (n = 178) were 90.4% (P = 0.0003) and 96.8% (P = 0.054), respectively, as compared to 74.9% and 89.4% in JWiTS-1. RFS rates of stages I-III Wilms tumor in JWiTS-2 were more than 90%, although the outcome of stage IV Wilms tumor was significantly poorer (RFS: 66.2%) (P = 0.0094). RFS and OS of clear cell sarcoma of the kidney (CCSK; n = 31) were 82.4% (P = 0.30) and 91.3% (P = 0.42), respectively, as compared to 68.8% and 81.3% in JWiTS-1, and those of rhabdoid tumor of the kidney (RTK; n = 16) were 18.8% (P = 0.88) and 25.0% (P = 0.80), respectively, as compared to 23.5% and 23.5% in JWiTS-1. CONCLUSIONS: RFS and OS for stages I-III Wilms tumor were improved in JWiTS-2 compared to JWiTS-1, whereas outcomes for stage IV Wilms tumor, CCSK, and RTK did not improve.

NM23 downregulation and lysophosphatidic acid receptor EDG2/lpa1 upregulation during myeloid differentiation of human leukemia cells.

The NM23 gene is overexpressed in many hematological malignancies and its overexpression predicts poor treatment outcomes. NM23 overexpression is thought to suppress myeloid differentiation of leukemia cells, but the molecular mechanism is unknown. In breast cancer cells, the lysophosphatidic acid (LPA) receptor EDG2/lpa1 was downregulated by NM23-H1 overexpression, and this reciprocal expression pattern was associated with suppressed or induced cell motility/metastasis. Here, we examined the relationship between EDG2 and NM23 expression during myeloid differentiation of leukemia cells. NM23 expression decreased and EDG2 expression increased during all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60, NB4, and THP-1 leukemia cells. Moreover, this inverse correlation was more evident when myeloid differentiation was enhanced by ellagic acid, an inhibitor of NM23 activity. In contrast, there was no inverse correlation between EDG2 and NM23 expression during erythroid differentiation of HEL and K562 cells. ATRA plus LPA enhanced the motility of leukemia cells as well as breast cancer cells in an EDG2-dependent manner. These results suggest a common molecular mechanism between myeloid differentiation of leukemia cells and migration of breast cancer cells depending on NM23 and EDG2 expression levels.

Genomic analysis-integrated whole-exome sequencing of neuroblastomas identifies genetic mutations in axon guidance pathway.

Neuroblastoma (NB) is a childhood solid malignant tumor originating from precursor cells of the peripheral nervous system. We have previously established a risk classification system based on DNA copy number profiles. To further explore the pathogenesis of NBs in distinct risk groups, we performed whole-exome sequencing analysis of 57 primary and 7 recurrent/metastatic tumors with unique chromosomal aberration profiles as categorized by our genomic sub-grouping system. Overall, a low frequency of somatic mutations was found. Besides ALK (4/64, 6.3%), SEMA6C, SLIT1 and NRAS, genes involved in the axon guidance pathway, were identified as recurrently mutated in 6 of 64 tumors (9.4%). Pathway enrichment analysis revealed enrichment of 25 mutated genes in the mitogen-activated protein kinase (MAPK) pathway, 13 genes in the Wnt pathway, and 12 genes in the axon guidance pathway. Genomic analyses demonstrated that primary and matched recurrent or metastatic tumors obtained from sporadic and monozygotic twin NBs were clonally related with variable extents of genetic heterogeneity. Monozygotic twin NBs displayed different evolutionary trajectories. These results indicate the involvement of the axon guidance, MAPK and Wnt pathways in NB and demonstrate genomic diversity with NB progression.

BRCA1 alterations with additional defects in DNA damage response genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy.

The BRCA-like phenotype is a feature that some sporadic breast cancers share with those occurring in BRCA1 or BRCA2 mutation carriers. As tumors with the phenotype have defects in the DNA damage response pathway, which may increase sensitivity to drugs such as DNA cross-linking agents and PARP inhibitors, a method to identify this phenotype is important. The prediction of chemoresistance, which frequently develops in these tumors, is also crucial for improving therapy. We examined genomic aberrations and BRCA1 promoter methylation in tumors of 73 breast cancer (20 HR-/HER2- and 53 HR+/HER2-) patients, who received neoadjuvant chemotherapy with anthracycline, cyclophosphamide, and taxane, using SNP array CGH and quantitative PCR. The methylation and/or loss or uniparental disomy (UPD) of BRCA1 (BRCA1 alterations) and the loss or UPD of BRCA2 (BRCA2 alterations) were detected in 27 (37%) and 21 (29%), respectively, of the 73 tumors. Tumors with BRCA1 or BRCA2 alterations were associated with a higher number of genomic aberrations (P < 0.001 and P < 0.001) and higher percentage of TP53 alterations (P < 0.001 and P < 0.001) than those without. Overall survival (OS) rates were similar between patients with or without BRCA1 or BRCA2 alterations. However, when 27 patients with BRCA1-altered tumors were classified into those with or without the loss or UPD of PALB2, PAGR1, RAD51B, FANCM, MLL4, or ERCC1/2 in tumors, patients with additional defects in DNA damage response genes had worse OS (P = 0.037, 0.045, 0.038, 0.044, 0.041, or 0.019) than those without. These defects may confer chemoresistance and predict poor outcomes in patients with BRCA1-altered breast cancer.

Molecular cytogenetic characterization of two established ESFT cell lines.

Ewing's sarcoma/primitive neuroectodermal tumor/Askin's tumor (Ewing`s sarcoma family of tumors: ESFT) is the most common type of malignant tumor of bone and soft tissue in children and young adults, and morphologically is a member of a group of small round cell tumors. We report, here, on the establishment of two human ESFT cell lines, FU-PNET-3 and FU-PNET-4, from the iliac and the chest wall, respectively, the cells of both cell lines were tumorigenic in immunodeficient mice. Histologically, both original and xenograft tumors and cultured cells were composed of small round cells with positive immunoreactivity for CD99 and Nkx2.2. Molecular biological examination demonstrated chimeric transcripts of EWSR1 exon 7 to FLI1 exon 6 in FU-PNET-3 cells, and EWSR1 exon 10 to FLI1 exon 6 in FU-PNET-4 cells. Cytogenetic analysis revealed chromosome translocation t(11;22)(q24;q12) and some secondary changes in both cultured cells. These histological, molecular biological, and cytogenetical findings indicate ESFT in both cell lines. ESFT is well studied, but its recurrent fusion genes are heterogeneous and its biological behaviors are unclear. The FU-PNET-3 and FU-PNET-4 cell lines have been well examined and may become useful tools for studying the genetic and biological behavioral properties of ESFT.

Anaplastic histology Wilms' tumors registered to the Japan Wilms' Tumor Study Group are less aggressive than that in the National Wilms' Tumor Study 5.

PURPOSE: To evaluate the clinical features and treatment results of anaplastic histology (AH) Wilms' tumor (WT) patients registered in the Japan Wilms' Tumor Study (JWiTS) group to elucidate the clinical characteristics of AH in the Japanese population. PATIENTS AND METHODS: Of 344 WT patients who were enrolled in JWiTS between 1995 and 2013, 17 had AH. Treatment using the JWiTS protocols was similar to the fifth National Wilms' Tumor Study 5 (NWTS-5) protocols. Clinical characteristics and mutation status of TP53 gene were evaluated and compared with those in NWST-5 study. RESULTS: AH incidences in JWiTS were 4.9 %, lower than that in NWTS-5. Seven tumors had focal AH and 10 had diffuse AH. Clinical stages of AH patients were stage I in seven, stage II in three, stage III in five, stage IV in one and unknown in one. Four-year event-free survival and overall survival rates were 90.9 and 86.7 %, respectively. Two patients with diffuse AH and none with focal AH had TP53 mutation. CONCLUSION: Japanese patients presented with higher incidence, earlier stages and may have better outcomes than American patients, indicating a possible biological heterogeneity of AH WT. Further analysis is necessary to elucidate the different characteristic of AH WT between Japanese and American populations.

Clinical prognostic value of DNA methylation in hepatoblastoma: Four novel tumor suppressor candidates.

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

Management of pediatric renal tumor: Past and future trials of the Japan Wilms Tumor Study Group.

The Japan Wilms Tumor Study group (JWiTS) was founded in 1996 to improve outcomes for children with renal tumor in Japan, and a nationwide multicenter cooperative study was initiated thereafter. JWiTS-1 (1996-2005) was analyzed, and JWiTS-2 (2005-2014) is now under analysis; the following problems have been identified and used to decide future study protocol: (i) there has been a decline in survival rate for patients with rhabdoid tumor of the kidney (RTK) and new treatment strategies are required; (ii) the survival rate for bilateral Wilms tumors (BWT) has improved, but results for renal preservation are unsatisfactory; (iii) the prognosis of stage IV favorable nephroblastoma is very good, suggesting that the current protocols provide overtreatment, particularly for patients with lung metastasis; and (iv) no effective biological risk factors exist for predicting the outcome of Wilms tumor, and a study of the genetic changes of these tumors is necessary to determine biological markers for use in risk classification. To solve these issues, the development of a new risk classification of pediatric renal tumors is required. In addition, different study protocols should be developed according to the risk-based classification of the patients. Further, a new study protocol for BWT began in 2015, and new study protocols are being prepared for RTK, and for Wilms tumor with lung metastasis. In addition, an analysis of biological markers with regard to risk classification is to be performed. Furthermore, to create new protocols for patients with rare renal tumors, international collaboration with Children's Oncology Group and International Society of Pediatric Oncology is necessary.

NUAK2 Amplification Coupled with PTEN Deficiency Promotes Melanoma Development via CDK Activation.

The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted.

Trim32 facilitates degradation of MYCN on spindle poles and induces asymmetric cell division in human neuroblastoma cells.

Asymmetric cell division (ACD) is a physiologic process during development and tissue homeostasis. ACD produces two unequal daughter cells: one has stem/progenitor cell activity and the other has potential for differentiation. Recent studies showed that misregulation of the balance between self-renewal and differentiation by ACD may lead to tumorigenesis in Drosophila neuroblasts. However, it is still largely unknown whether human cancer stem-like cells exhibit ACD or not. Here, using human neuroblastoma cells as an ACD model, we found that MYCN accumulates at spindle poles by GSK-3ß phosphorylation during mitosis. In parallel, the ACD-related ubiquitin ligase Trim32 was recruited to spindle poles by CDK1/cyclin B-mediated phosphorylation. Trim32 interacted with MYCN at spindle poles during mitosis, facilitating proteasomal degradation of MYCN at spindle poles and inducing ACD. Trim32 also suppressed sphere formation of neuroblastoma-initiating cells, suggesting that the mechanisms of ACD produce differentiated neuroblastoma cells that will eventually die. Thus, Trim32 is a positive regulator of ACD that acts against MYCN and should be considered as a tumor-suppressor candidate. Our findings offer novel insights into the mechanisms of ACD and clarify its contributions to human tumorigenesis.