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Regulator of cullins-1 (ROC1) negatively regulates the Gli2 regulator SUFU to activate the hedgehog pathway in bladder cancer
Min Gu
Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9110-8054
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The regulator of cullins-1 (ROC1) is an essential subunit in the cullin-RING ligase (CRL) protein complex and has been shown to be critical in bladder cancer cell survival and progression. This study aimed to explore the molecular mechanism of ROC1 action in the malignant progression of bladder cancer.
Methods: This study utilized ex vivo, in vitro, and in vivo nude mouse experiments to assess the underlying mechanisms of ROC1 in bladder cancer cells. The expression of the components of the sonic hedgehog (SHH) pathway was determined by western blot analysis. ROC1 expression in human tumors was evaluated by immunohistochemistry.
Results: ROC1 overexpression promoted the growth of bladder cancer cells, whereas knockdown of ROC1 expression had the opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target suppressor of fused homolog (SUFU) for ubiquitin-dependent degradation, allowing Gli2 release from the SUFU complex to activate the SHH pathway. Furthermore, knockdown of SUFU expression partially rescued the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. In ex vivo experiments, tissue microarray analysis of human bladder cancer specimens revealed a positive association of ROC1 expression with the SHH pathway activity.
Conclusion: This study demonstrated that dysregulation of the ROC1–SUFU–GLI2 axis plays an important role in bladder cancer progression and that targeting ROC1 expression is warranted in further investigations as a novel strategy for the future control of bladder cancer.
Keywords
Bladder cancer, ROC1, sonic hedgehog signaling, SUFU, Gli2
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- S01.tif
Supplemental Figure 1. ROC expression in ROC1 cDNA- or siRNA-transfected bladder cancer 5637 (A) and T24 (B) cells. Tumor cells were grown and stably transfected with ROC1 cDNA or transiently transfected with ROC1 siRNA and then subjected to western blot analysis of ROC1 protein.
- S02.tif
Supplemental Figure 2. Knockdown of ROC1 led to cancer cell arrest at the G2/M phase. Bladder cancer 5637 (A) and T24 (B) cells were transfected with siROC1 and stained with propidium iodide for flow cytometric analysis. The representative images are shown in the left panel, and the quantified data are shown below. Representative results of three independent experiments are shown as means ± SEM; ** P < 0.01, *** P < 0.001.
- S03.tif
Supplemental Figure 3. Immunostaining of Gli2 protein in ROC1-knocked down or ROC1-overexpressed 5637 and T24 cells. Bladder cancer 5637 (A) and T24 (B) cells were grown and transfected with siROC1 or pROC1, stained with Gli2 (Red), and reviewed under a confocal microscope. The nuclear DNA was stained with 4,6-diamidino-2-phenylindole (blue). Scale bar, 10 µm.
- S04.tif
Supplemental Figure 4. Knockdown of SUFU expression rescued tumor cell growth upon ROC1 knockdown. A cell viability assay was performed to assess the cell viability of 5637 cells transfected with siRNA targeting SUFU, ROC1, or both. Representative results of three independent experiments are shown as means ± SEM; * P < 0.05, ** P < 0.01.
- S05.tif
Supplemental Figure 5. Detection of Gli1 or Gli2 binding to SUFU in 5637 and T24 cells. Immunoprecipitation of SUFU from 5637 cells (A) and T24 cells (B). Nonspecific rabbit immunoglobulin G (IgG) was used as a negative control. Cell lysates were subjected to western blot analysis.
- S06.tif
Supplemental Figure 6. MLN4924 regulation of bladder cancer cell growth. (A and B) Cell viability assay. Bladder cancer 5637 (A) and T24 (B) cells were grown and treated with different concentrations of the CRL inhibitor MLN4924 and then subjected to a cell viability assay. (C and D) Colony formation assay. Bladder cancer 5637 (C) and T24 (D) cells were grown and treated with different concentrations of the CRL inhibitor MLN4924 and then subjected to a colony formation assay. Representative results of three independent experiments are shown as means ± SEM; * P < 0.05, *** P < 0.001.
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This preprint is under consideration at Cancer Cell International. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Primary research
Regulator of cullins-1 (ROC1) negatively regulates the Gli2 regulator SUFU to activate the hedgehog pathway in bladder cancer
Min Gu
Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9110-8054
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 4
Posted 18 Dec, 2020
No community comments so far
Review #2 received
Received 07 Jan, 2021
Reviewer #3 agreed
On 03 Jan, 2021
Reviewer #2 agreed
On 31 Dec, 2020
Review #1 received
Received 23 Dec, 2020
Reviewer #1 agreed
On 13 Dec, 2020
Reviewers invited
Invitations sent on 10 Dec, 2020
Editor assigned
On 08 Dec, 2020
Submission checks complete
On 08 Dec, 2020
Editor invited
On 08 Dec, 2020
No comments provided
Editorial decision: Minor revision
On 11 Nov, 2020
Review #2 received
Received 09 Nov, 2020
Review #1 received
Received 25 Oct, 2020
Reviewer #2 agreed
On 20 Oct, 2020
Reviewer #1 agreed
On 18 Oct, 2020
Editor assigned
On 17 Oct, 2020
Reviewers invited
Invitations sent on 17 Oct, 2020
Submission checks complete
On 16 Oct, 2020
Editor invited
On 16 Oct, 2020
No comments provided
Editorial decision: Major revision
On 18 Sep, 2020
Review #2 received
Received 17 Sep, 2020
Reviewer #2 agreed
On 05 Sep, 2020
Review #1 received
Received 03 Sep, 2020
Reviewer #1 agreed
On 22 Aug, 2020
Reviewers invited
Invitations sent on 08 Aug, 2020
Editor assigned
On 24 Jun, 2020
Submission checks complete
On 23 Jun, 2020
Editor invited
On 23 Jun, 2020
No comments provided
Editorial decision: Revise Before Review
On 22 Jun, 2020
Editor assigned
On 11 Jun, 2020
Editor invited
On 10 Jun, 2020
First submitted
On 29 May, 2020
Submission checks complete
On 29 May, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cancer Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The regulator of cullins-1 (ROC1) is an essential subunit in the cullin-RING ligase (CRL) protein complex and has been shown to be critical in bladder cancer cell survival and progression. This study aimed to explore the molecular mechanism of ROC1 action in the malignant progression of bladder cancer.
Methods: This study utilized ex vivo, in vitro, and in vivo nude mouse experiments to assess the underlying mechanisms of ROC1 in bladder cancer cells. The expression of the components of the sonic hedgehog (SHH) pathway was determined by western blot analysis. ROC1 expression in human tumors was evaluated by immunohistochemistry.
Results: ROC1 overexpression promoted the growth of bladder cancer cells, whereas knockdown of ROC1 expression had the opposite effect in bladder cancer cells. Mechanistically, ROC1 was able to target suppressor of fused homolog (SUFU) for ubiquitin-dependent degradation, allowing Gli2 release from the SUFU complex to activate the SHH pathway. Furthermore, knockdown of SUFU expression partially rescued the ROC1 knockdown-suppressed SHH activity as well as cancer cell growth inhibition. In ex vivo experiments, tissue microarray analysis of human bladder cancer specimens revealed a positive association of ROC1 expression with the SHH pathway activity.
Conclusion: This study demonstrated that dysregulation of the ROC1–SUFU–GLI2 axis plays an important role in bladder cancer progression and that targeting ROC1 expression is warranted in further investigations as a novel strategy for the future control of bladder cancer.
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Figure 3
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Figure 5