Tumor and Stem Cell Biology Trim 32 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 thatmisregulation of the balance between self-renewal and differentiation by ACDmay lead to tumorigenesis inDrosophila neuroblasts. However, it is still largely unknownwhether 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-3b 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 againstMYCN 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. Cancer Res; 74(19); 1–11. 2014 AACR. Introduction Asymmetric cell division (ACD) is a physiologic process that occurs during development and tissue homeostasis in a large variety of organisms. ACD produces two unequal daughter cells; one has multipotent stem and/or progenitor cell activity and the other has potential for differentiation. Recent ACD studies using model organism systems revealed that the balance between self-renewal and differentiation by ACD is robustly controlled and that misregulation of this balance may lead to tumorigenesis in Drosophila neuroblasts (1–3). Neuroblastoma is one of the major childhood tumors (4–6) and is derived from normal neural crest cells, which serve as multipotent stem cells that differentiate into mature tissues, including peripheral neurons (7). Of the many genetic and biochemical features of neuroblastoma, MYCN oncogene amplification has been shown to correlate with an aggressive phenotype and a poor outcome (4–6). Recent studies have shown that MYCN shows not only oncogenic activity but also plays a central role in self-renewal growth of normal neural stem and precursor cells (8–12). Although the precise role of MYCN in control of the balance between cell self-renewal and differentiation is still unknown, it is now suspected that neuroblastoma has a cancer stem cell–like property due to aberrant MYCN expression in multipotent neural crest cells (7). Human neuroblastoma cell lines have a unique property in that, although they show unlimited cell proliferation, they are easily induced to become mature neuronal cells by drugs such as retinoic acids (7). Thus, we considered that a human neuroblastoma cell line is the most suitable system for understanding the mechanism of ACD in human cells because it has both self-renewal and differentiation abilities. In fact, we previously reported that NuMA, one of the conserved ACDrelated polarity cues, distributed to one side of the cell cortex during cell division, was detected inmanyMYCN-nonamplified human neuroblastoma cell lines, and that MYCN powerfully induced self-renewal division against ACD inMYCN-amplified neuroblastoma cells (13). In this study, we attempted to identify the cellular components that abolish the self-renewal proliferation function by MYCN. Materials and Methods Cell lines and transfections All cell lines except TGW were obtained from the American Type Culture Collection (ATCC). TGW was obtained from Japan Health Science Research Resources Bank, Osaka, Japan. All cell lines have been validated by short tandem repeat analysis. These cell lines were maintained in complete medium [Dulbecco's Modified Eagle Medium, supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and Division of Cancer Therapeutics, Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan Corresponding Authors: Hideki Izumi, Division of Cancer Therapeutics, Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan. Phone: 81-48-722-1111; Fax: 81-48-722-1739; E-mail: hideki@cancer-c.pref. saitama.jp; and Yasuhiko Kaneko, kaneko@cancer-c.pref. saitama.jp doi: 10.1158/0008-5472.CAN-14-0169 2014 American Association for Cancer Research. Cancer Research www.aacrjournals. org OF1 Research. on June 9, 2017. © 2014 American Association for Cancer cancerres.aacrjournals. org Downloaded from Published OnlineFirst August 6, 2014; DOI: 10.1158/0008-5472.CAN-14-0169