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Inhibition of PP2A by LB100 sensitizes bladder cancer cells to chemotherapy by inducing p21 degradation

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Abstract

Purpose

Bladder carcinoma (BLCA) is the most common urinary tract malignancy and exhibits a poor response to chemotherapy. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in a wide variety of regulatory cellular processes, including apoptosis and the DNA-damage response (DDR). LB100, a small molecule inhibitor of PP2A, has been shown to act as a chemo-sensitizer in multiple types of cancer. However, the anti-tumor effect and mode of action of LB100 in BLCA have yet to be identified.

Methods

In vitro and in vivo experiments were performed to assess the anti-tumor effect of LB100 alone or in combination with gemcitabine. Mass spectrometry (MS)-based phosphoproteomics analysis was used to identify the downstream substrates of PP2A and to explore the mechanism underlying LB100-induced DNA damage and apoptosis. In addition, we established a chemo-resistant BLCA cell line (RT-112-R) by prolonged drug exposure and determined the effect of LB100 in enhancing genotoxicity in BLCA cell lines and xenograft mouse models.

Results

We found that LB100 is sufficient to induce an anti-tumor response in BLCA cells by inducing DNA damage and apoptosis both in vitro and in vivo. Furthermore, we found that PP2A potentially dephosphorylates p-p21-ser130 to stabilize p21. Inhibition of PP2A by LB100 increased the level of p-p21-ser130, subsequently leading to a reduction in p21 level in a dose-dependent manner. In addition, we found that treatment of LB100 abrogated the G1/S cell cycle checkpoint, resulting in increased phosphorylation of γH2AX in BLCA cells. Moreover, LB100 enhanced genotoxicity in chemo-resistant BLCA cells by inducing DNA damage and apoptosis in vitro and in vivo.

Conclusion

Our findings indicate that PP2A may serve as a potential therapeutic target in BLCA through regulating p21 stability.

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Data availability

All data sets generated during the study are available in this article and its supplementary files.

Abbreviations

PP2A:

Protein phosphatase 2A

DDR:

DNA-damage response

BLCA:

Bladder carcinoma

MS:

Mass spectrometry

FA:

Formic acid

HCD:

Higher-energy collisional dissociation

FDR:

False-discovery rate

Parp1:

Poly ADP-ribose polymerase 1

shRNA:

Short hairpin RNA

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Acknowledgements

We thank the technical staff of the animal facility for their assistance.

Funding

The study was supported by the National Natural Science Foundation of China (#81872446), the National Key Research and Development Program of China (2017YFC1308604), the Youth talent project of Xingliao Yingcai program (XLYC1807071), the Liaoning Livelihood Science and Technology Plan Joint Project (No.2021JH2/10300130) and “345” Talent Project of Shengjing Hospital of China Medical University (No.M0397).

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Author notes

  1. Song Gao, Liping Shan and Mo Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, 110004, China

    Xinyi Tao, Xinyu Li & Yang Liu

  2. Innovative Research Center for Integrated Cancer Omics, Shengjing Hospital of China Medical University, Shenyang, 110004, China

    Xinyi Tao, Xinyu Li & Yang Liu

  3. (CAS) Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, China Academy of Sciences, Dalian, 116023, China

    Yan Wang, Xi Zhan, Yalei Yin & Mingliang Ye

  4. Department of Oncology, Shengjing Hospital of China Medical University, Shenyang, 110004, China

    Song Gao

  5. Department of Urology, Shengjing Hospital of China Medical University, Shenyang, 110004, China

    Liping Shan & Mo Zhang

  6. Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital of China Medical University, Shenyang, 110004, China

    Zhonghao Jiang

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  1. Song Gao
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Contributions

Study concept and design: Song Gao, Xinyu Li, Mingliang Ye & Yang Liu.

Acquisition of data: Song Gao, Liping Shan, Zhonghao Jiang & Xinyi Tao.

Analysis and interpretation of data: Song Gao, Liping Shan, Mo Zhang, Yan Wang, Xi Zhan & Yalei Yin.

Drafting of the manuscript: Xinyu Li & Yang Liu.

Statistical analysis: Song Gao, Liping Shan & Mo Zhang.

Acquisition of funding: Song Gao & Yang Liu.

Study supervision: Xinyu Li, Mingliang Ye & Yang Liu.

Corresponding authors

Correspondence to Xinyu Li, Mingliang Ye or Yang Liu.

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All animal procedures were performed in accordance with the National Institutes of Health Guide on the Care and Use of Laboratory Animals. This study was performed with approval of the China Medical University.

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Supplementary Information

ESM 1

Figure S1. LB100 induced cell death in bladder cancer cells. (a). RT-4, T-24, 639V and MGHU1 were treated with LB100 for 24h. Typical images were captured using a microscopic imaging system and shown. (b). RT112, MGHU1, 97-1,639-V, RT-4 and T-24 were treated with 10μM LB100 for 24h. Cells viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (c&d). RT-4, T-24, MGHU1 and 639-V were treated with 10μM LB100 for 24h. The extracted protein was analyzed by immunoblotting with the indicated antibodies. (e). 639V cells were injected into BALB/c nude mice. Mice were randomly subjected to vehicle (NT) or LB100 (2 mg/kg). Body weights of mice in different groups are shown. All data are quantified as the mean±SEM. Figure S2. Detection of PP2A phosphorylated substrate. (a). RT-4 cells were treated with 5μM LB100 for 4h. Protein lysates were subjected to a phosphoproteomics assay using mass spectrometer. Relative phosphorylated protein was screened out by Venn assay. Figure S3. Quantitative results of western blot. (a&b). p21 and p-γH2AX expression in T24 and RT-4 cells were examined by protein intensity assay using Image J. (c-e). CDC25C, p21 and p-γH2AX expression in T24 and RT-4 cells were examined by protein intensity assay using Image J. (f-h). p21, p-p21-s130 and PP2A expression in T24 and RT-4 cells were examined by protein intensity assay using Image J. Figure S4. Correlation analysis between PP2A and p21 in BLCA cells. (a). RT-4, T-24, TCCSUP, 5637, UMUC3, J82 and RT-112 cells (1×106) were collected. The extracted protein was analyzed by immunoblotting with the indicated antibodies. (b). The correlation between PP2A and p21 was shown. Figure S5. LB100 promoted gemcitabine-induced cytotoxicity in BLCA cells. (a&b). T-24, T-24-shPP2A, RT-4 and RT-4-shPP2A cells (5×103) were treated with LB100 and gemcitabine for 24h. Cell counts were assessed by CellTiter-Glo® Luminescent Cell Viability Assay. (c-f). PP2A and p-γH2AX expression in RT-112 and RT-112R cells were examined by protein intensity assay using Image J. All data are quantified as the mean±SEM. Asterisks indicate a significant difference, *** indicates p < 0.001. Figure S6. LB100 sensitized chemo-resistant cells to gemcitabine. (a). RT-112 and RT-112-R cells (1×106)were treated with LB100, gemcitabine, and AZD7762 or in combination for 24h. The extracted protein was analyzed by immunoblotting with the indicated antibodies. (b-e). PP2A, p21, p-p21-S130 and p-γH2AX expression in RT-112 and RT-112R cells were examined by protein intensity assay using Image J. Figure S7. Tumor weight in RT-4 and RT-112 xenograft models. (a). RT-4 cells were planted into BALB/c nude mice. Mice were randomly subjected to vehicle (NT), LB100 (1.5 mg/kg, n = 5), gemcitabine (Gem, 2.5 mg/kg, n = 5), and the combination (n = 5). Tumor weights of mice in different groups were evaluated and shown. (b). RT-112-R cells were planted into BALB/c nude mice. Mice were randomly subjected to 4 groups including vehicle (NT), LB100 (1.5 mg/kg, n = 5), gemcitabine (Gem, 2.5 mg/kg, n = 5), and the combination group (n = 5). Tumor weight of mice was evaluated after treatment in different groups and shown. All data are quantified as the mean±SEM. Figure S8. LB100 combined with gemcitabine inhibited tumor growth by inducing p21 degradation in mice. (a). PP2A, p21 and p-p21-S130 proteins were assessed in different groups by immunoblotting with the indicated antibodies. (b&c). The expression levels of PP2A, p21 and p-p21-S130 expression were examined by protein intensity assay using Image J. All data are quantified as the mean±SEM. Asterisks indicate a significant difference, *** indicates p < 0.001. (PPT 3629 kb)

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Gao, S., Shan, L., Zhang, M. et al. Inhibition of PP2A by LB100 sensitizes bladder cancer cells to chemotherapy by inducing p21 degradation. Cell Oncol. 45, 1203–1215 (2022). https://doi.org/10.1007/s13402-022-00710-8

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