WIP1 Phosphatase as a Potential Therapeutic Target in Neuroblastoma

The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.野生型p53诱导磷酸酶1（WIP1，wild-type p53-induced phosphatase 1）是一种丝氨酸/苏氨酸磷酸酶，可负向调控多种参与DNA损伤应答的蛋白，包括p53、CHK2、组蛋白H2AX及ATM，且在乳腺癌、卵巢癌等人类癌症中存在过表达或扩增现象。我们对多种肿瘤组织中的WIP1 mRNA表达水平进行了检测，发现其在乳腺癌、白血病、髓母细胞瘤和神经母细胞瘤中表达水平最高。神经母细胞瘤在确诊时均为TP53野生型肿瘤，且p53抑制是肿瘤发生所必需的过程。既往研究表明，神经母细胞瘤存在17号染色体长臂（17q）扩增，该区域包含WIP1（PPM1D）基因，且与不良临床结局相关。因此，本研究旨在探究采用选择性拮抗剂GSK2830371抑制WIP1，是否能够通过重新激活p53介导的肿瘤抑制通路，从而抑制神经母细胞瘤细胞增殖。携带野生型TP53等位基因的神经母细胞瘤细胞系对GSK2830371处理高度敏感，而携带突变型TP53的细胞系则对GSK2830371产生耐药。在本研究检测的多数PPM1D基因座拷贝数增加的神经母细胞瘤细胞系中，其TP53亦为野生型且对GSK2830371A敏感；与之相反，未发生PPM1D拷贝数增加的细胞系对WIP1抑制的敏感性参差不齐，其敏感性的主要决定因素为TP53的突变状态。鉴于WIP1参与细胞DNA损伤应答，且神经母细胞瘤治疗所用药物可通过诱导DNA损伤引发细胞凋亡，本研究进一步探究了GSK2830371是否能够与临床标准化疗药物产生协同作用。与单独使用任一药物相比，联合使用GSK2830371与多柔比星或卡铂处理野生型TP53神经母细胞瘤细胞系，可通过激活半胱天冬氨酸蛋白酶3/7（caspase 3/7）通路增强细胞死亡效应。本研究数据表明，WIP1抑制有望成为一种全新的神经母细胞瘤治疗策略，并可与现有化疗方案联合应用。