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. 2019 Jun;18(6):1045-1056.
doi: 10.1158/1535-7163.MCT-18-0146. Epub 2019 Apr 8.

Inhibition of Ubiquitin-Specific Protease 14 Suppresses Cell Proliferation and Synergizes with Chemotherapeutic Agents in Neuroblastoma

Yang Yu  1   2 , Yanling Zhao  2 , Yihui Fan  2 , Zhenghu Chen  1 , Hui Li  2 , Jiaxiong Lu  2 , Kevin Guo  2 , Sarah E Woodfield  1 , Sanjeev A Vasudevan  1 , Jianhua Yang  3 , Jed G Nuchtern  4   2
Affiliations

Inhibition of Ubiquitin-Specific Protease 14 Suppresses Cell Proliferation and Synergizes with Chemotherapeutic Agents in Neuroblastoma

Yang Yu et al. Mol Cancer Ther. 2019 Jun.

Abstract

Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1:
Figure 1:. USP14 knockdown inhibits cell proliferation and induces cell apoptosis in NB.
A. Endogenous expression of USP14 in a panel of NB cell lines including IMR-32, NGP, NB-19, CHLA-255, SH-SY5Y, SK-N-AS, LAN-6, and SK-N-BE2. B, C. Immunoblotting assay showing USP14 knockdown in IMR-32 (B), NGP (B), SK-N-BE2 (B), SH-SY5Y (C), SK-N-AS (C), and LAN-6 (C) cells by two independent lentivirus shRNAs, sh-1 and sh-2. Cell growth curve assay shows a proliferation defect in six USP14 knockdown NB cells. The cell counting kit-8 (CCK-8) was used to determine cell viability. Error bars represent SDs of six samples. ***P<0.001. D, E. Six USP14-knockdown cells were harvested for the protein immunoblotting assay. PARP and Caspase-3 cleavages were detected by immunoblotting with the antibodies. β-actin was used as a loading control for whole cell extracts in all samples.
Figure 2:
Figure 2:. The effect of UCHL5 and PSMD14 knockdown on NB cell proliferation and USP14 knockdown inhibits anchorage-independent growth.
A, B. Immunoblotting assay showing UCHL5 and PSM14 knockdown in NGP and SH-SY5Y cells by lentivirus sh-UCHL5 and sh-PSMD14. Cell growth curve assay shows a proliferation defect in two UCHL5 knockdown NB cells. The cell counting kit-8 (CCK-8) was used to determine cell viability. Error bars represent SDs of six samples. **P<0.01 and ***P<0.001. C-H. Soft agar assay showing reduced colony formation in USP14-knockdown cells. Colony numbers were quantified. These experiments were performed in triplicate and reported as mean with SDs. ***P<0.001.
Figure 3:
Figure 3:. USP14 inhibitor b-AP15 suppresses cell proliferation, causes accumulation of ubiquitinated proteins and induces UPR in NB.
A. b-AP15 showing cytotoxic effect on NB cells. Six NB cell lines IMR-32, NGP, SK-N-BE2, SH-SY5Y, SK-N-AS, and LAN-6 were incubated with serial dilutions of b-AP15 or medium alone for 72 hours. Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability. B. IC50 values of six cell lines tested were measured based on CCK-8 assay. C. Morphological changes of six NB cell lines after incubation with serial dilutions of b-AP15 for 72 hours were shown. D. Two USP14-knockdown cells, NGP and SH-SY5Y, were harvested, and subjected to immunoblotting with anti-Lys48-linked specific antibody. Same amounts of lysates were performed Coomassie Blue staining as loading controls. E. NGP and SH-SY5Y cells were treated with b-AP15 (1 μM) for the indicated time points. Cell lysates were subjected to immunoblotting as the above. F, G. USP14 knockdown NGP and SH-SY5Y (F) cells or b-AP15 treated NB cells (G) were subjected to immunoblotting with anti-HSP40, HSP70, and USP14 antibodies.
Figure 4:
Figure 4:. USP14 inhibitor b-AP15 suppresses NB tumor growth in vivo.
A. Representative pictures of orthotopic tumors in each group. The nude mice bearing tumors were treated with b-AP15 (5 mg/kg) and DMSO respectively for two weeks. B. Tumor weights from the last day of treatment. Data was from one experiment. **P<0.01 and ***P<0.001. C. Representative light microscopy images of cleaved capspse-3 and Ki67 immunohistochemistry staining from NGP xenograft tumors treated with b-AP15 and DMSO control. Quantification of cleaved caspase-3 and Ki67 (positive cell per high-power field) demonstrates induction of apoptosis and inhibition of tumor growth in NGP xenografts treated with b-AP15. D. The nude mice bearing tumors were treated with 5 mg/kg of b-AP15 by intraperitoneal injection once daily for two days. The mice were then euthanized, and immunoblotting assay was used to detect b-AP15-induced apoptosis.
Figure 5.
Figure 5.. The mRNA level of USP14 in NB patient samples.
A. Correlation of USP14 mRNA levels of NB tumor samples and the overall survival of patients was analyzed using Seqc-498-cohort (n=498), Kocak-cohort (n=476), Versteeg-cohort (n=88), and Oberthurs-cohort (n=251). B. USP14 mRNA levels in patient samples at different Stages were analyzed using these four datasets.
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