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Neuroblastoma (NB) is the most common extracranial tumor in children. Unlike in most adult tumors, tumor suppressor protein 53 (p53) mutations occur with a relatively low frequency in NB and the downstream function of p53 is intact in NB cell lines. Wip1 is a negative regulator of p53 and hindrance of Wip1 activity by novel inhibitor GSK2830371 is a potential strategy to activate p53's tumor suppressing function in NB. Yet, the in vivo efficacy and the possible mechanisms of GSK2830371 in NB have not yet been elucidated. Here we report that novel Wip1 inhibitor GSK2830371 induced Chk2/p53-mediated apoptosis in NB cells in a p53-dependent manner. In addition, GSK2830371 suppressed the colony-formation potential of p53 wild-type NB cell lines. Furthermore, GSK2830371 enhanced doxorubicin- (Dox) and etoposide- (VP-16) induced cytotoxicity in a subset of NB cell lines, including the chemoresistant LA-N-6 cell line. More importantly, GSK2830371 significantly inhibited tumor growth in an orthotopic xenograft NB mouse model by inducing Chk2/p53-mediated apoptosis in vivo. Taken together, this study suggests that GSK2830371 induces Chk2/p53-mediated apoptosis both in vitro and in vivo in a p53 dependent manner.

Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common childhood extracranial malignant tumor. In NB, somatic mutations of the tumor suppressor, p53, are exceedingly rare. Unlike in adult tumors, the majority of p53 downstream functions are still intact in NB cells with wild-type p53. Thus, restoring p53 function by blocking its interaction with p53 suppressors such as MDM2 is a viable therapeutic strategy for NB treatment. Herein, we show that MDM2 inhibitor SAR405838 is a potent therapeutic drug for NB. SAR405838 caused significantly decreased cell viability of p53 wild-type NB cells and induced p53-mediated apoptosis, as well as augmenting the cytotoxic effects of doxorubicin (Dox). In an in vivo orthotopic NB mouse model, SAR405838 induced apoptosis in NB tumor cells. In summary, our data strongly suggest that MDM2-specific inhibitors like SAR405838 may serve not only as a stand-alone therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact MDM2-p53 axis.

Neuroblastoma (NB) is the most common extracranial neoplasm in children. In NB, loss of p53 function is largely due to cytoplasmic sequestration rather than mutation. Ubiquitin-conjugating enzyme E2 N (UBE2N), also known as Ubc13, is an E2 ubiquitin-conjugating enzyme that promotes formation of monomeric p53 that results in its cytoplasmic translocation and subsequent loss of function. Therefore, inhibition of UBE2N may reactivate p53 by promoting its nuclear accumulation. Here, we show that NSC697923, a novel UBE2N inhibitor, exhibits potent cytotoxicity in a panel of NB cell lines evidenced by its ability to induce apoptosis. In p53 wild-type NB cells, NSC697923 induced nuclear accumulation of p53, which led to its increased transcriptional activity and tumor suppressor function. Interestingly, in p53 mutant NB cells, NSC697923 induced cell death by activating JNK pathway. This effect was reversible by blocking JNK activity with its selective inhibitor, SP600125. More importantly, NSC697923 impeded cell growth of chemoresistant LA-N-6 NB cell line in a manner greater than conventional chemotherapy drugs doxorubicin and etoposide. NSC697923 also revealed in vivo antitumor efficacy in NB orthotopic xenografts. Taken together, our results suggest that UBE2N is a potential therapeutic target in NB and provide a basis for the rational use of UBE2N inhibitors like NSC697923 as a novel treatment option for NB patients.

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy.