OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via deubiquitination activity

Oncogenic processes exert their greatest effect by targeting regulators of cell proliferation. Studying the mechanism underlying growth augmentation is expected to improve clinical therapies. The ovarian tumor (OTU) subfamily deubiquitinases have been implicated in the regulation of critical cell-signaling cascades, but most OTUs functions remain to be investigated. Through an unbiased RNAi screen, knockdown of OTUD5 is shown to significantly accelerate cell growth. Further investigation reveals that OTUD5 depletion leads to the enhanced transcriptional activity of TRIM25 and the inhibited expression of PML by altering the ubiquitination level of TRIM25. Importantly, OTUD5 knockdown accelerates tumor growth in a nude mouse model. OTUD5 expression is markedly downregulated in tumor tissues. The reduced OTUD5 level is associated with an aggressive phenotype and a poor clinical outcome for cancers patients. Our findings reveal a mechanism whereby OTUD5 regulates gene transcription and suppresses tumorigenesis by deubiquitinating TRIM25, providing a potential target for oncotherapy.


Statistics
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Software and code
Policy information about availability of computer code Data collection BD bioscience FACSCalibur was used to collect flow cytometric data. QuantStudio Dx Instrument (Applied Biosystems) was used to collect Real-time PCR data.

Data analysis
The data analysis was performed by Image Lab software (Version 3.0 beta), BD FACStation software version 6.0, Microsoft Excel (Version 11.0) and GraphPad Instat software (Version 5.01).
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Data
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Life sciences study design
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Sample size
All sample size was revealed in the sections of Materials and Methods or Figure Legends. For no sample size calculation was performed, we have chosen several large cohorts to perform statistical analysis, the sample size of which is sufficient for correlation analysis, Chi-square or Fisher's test. We also used publicly available TCGA database from large cohorts of tumor patients to perform survival analysis. For student's t test, at least 3 biological repeats was used to calculate p value. The sample size is commonly acceptable in biological experiments.
Data exclusions A total of 110 tumor samples were immunostained in the tissue microarray for primary liver cancer. The staining for E7 was missing. The staining for J1 was excluded from the statistics because of mixed hepatocellular carcinoma and normal live tissue. For OTUD5 expression correlation with demographic and clinical features in LUAD patients, the cutoff value for OTUD5 mRNA expression is set at 10 and two patients were excluded from the analysis. All the inclusion and exclusion criteria were stated in details in the main text. These exclusion criteria were pre-established because the expression level of OTUD5 in 2 patients out of 522 LUAD patients was extremely and abnormally higher in comparison with the others. We considered that the experimental or sample issues affect the data accuracy and therefore excluded the data of 2 patients from the final statistical analysis.

Replication
Each experiment was performed in three biological replicates with consistent results.
Randomization The research subjects were randomly assigned to the treatment or control group. Nude mice were chosen as xenograft models, and randomly allocated into experimental groups.

Blinding
For in vitro cell-based experiments, the investigators were not blinded during data acquisition and analysis. The application of treatments and processing procedures made it difficult for blinding but there was no human bias given all the data were collected independently using instrumentation. For the animal experiments the investigators were not blinded to the group allocation. For in vitro cell-based experiments, the investigators were not blinded during data acquisition and analysis. The application of treatments and processing procedures made it difficult for blinding but there was no human bias given all the data were collected independently using instrumentation. For the animal experiments the investigators were not blinded to the group allocation. During this process for cell and mouse experiments, only one researcher performed an experiment at a time and therefore it is difficult to blind him to the experiment. However, we ensure that the outcomes being measured are as objective as possible. We used duplicate assessment of outcomes and reported the level of agreement achieved by the investigators. At least two observers measured xenograft tumor volumes/weights or the latency of tumor formation/survival time to alleviate human bias in these data. All immunohistochemistry sections were blindly assessed by two pathologists independently. The third pathologist's assessment is performed if controversial results are acquired.

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Cell line source(s) Hep3B, Huh7 and 293T cells were purchased from National Infrastructure of cell line resource (Beijing, China).

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All the cell lines were obtained from National Infrastructure of cell line resource and have been proven to be negative for mycoplasma contamination.
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Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals 4-to 6-week-old male nude mice (NU/NU; 4-6 weeks old; male; strain 403; Charles River) were licensed by Beijing Municipal Committee of Science and Technology. All mice were housed in a temperature-controlled room (22 ± 2 °C) with 40-60% humidity, with a light/dark cycle of 12h/12h. All animal experiments were done according animal protocol (No. LA2018013) to Wenhui Zhao approved by the Animal Ethics Committee of the Peking University Health Science Center, China.

Wild animals
No wild animals were used in this study.

Field-collected samples
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Ethics oversight
The study is compliant with all relevant ethical regulations for animal experiments. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Peking University.
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Human research participants
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Population characteristics
The demographical and clinical features of all liver cancer and NSCLC patients are shown in supplementary Table 3 and 4.

Recruitment
We also assessed OTUD5 expression in 21 non-small-cell lung carcinoma (NSCLC) patients. The patients who underwent curative surgery at The Affiliated Zhangjiagang Hospital of Soochow University from March 2019 to July 2019 were enrolled in this study. Histological confirmation of primary NSCLC was obtained from the Department of Pathology at the hospital. None of the patients received preoperative adjuvant chemotherapy.
We cannot recruit the NSCLC patients with late-stage diseases in this study, especially for those patients with distant metastasis, who are not fit for curative surgery. Therefore, most of the patients included in the current study were from early-stage NSCLC patients. Further study was required to determine OTUD5 expression and its correlation with PML level in more progressive NSCLC patients.

Ethics oversight
The written permission was requested and received from all NSCLC patients in the study. The use of the human specimens was approved by The Zhangjiagang Hospital Institutional Review Board (No.2019001). To determine OTUD5 expression in primary liver cancer, we purchased commercial available human liver tissue microarray (ALENA biotechnology; Xi'an; China).
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Flow Cytometry
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All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

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October 2018

Methodology Sample preparation
The cells were plated at 50-70% of confluency with 500 nM TSA dissolved in DMSO or not. After 24 hours, the cells were collected and stained with propidium iodide (PI) for flow cytometric analysis.

Instrument
FACSCalibur, BD bioscience Software FACADiva software Cell population abundance 10,000 cells were analyzed for each sample Gating strategy Staining is with a nucleic acid dye like PI and is analyzed according to the standard procedure.
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