Cullin 4B Ubiquitin Ligase Is Important for Cell Survival and Regulates TGF-β1 Expression in Pleural Mesothelioma

Our plain-English summary of Cullin 4B Ubiquitin Ligase Is Important for Cell Survival and Regulates TGF-β1 Expression in Pleural Mesothelioma, by J. Kreienbühl, S. Changkhong, V. Orlowski, et al (International Journal of Molecular Sciences, 2023).

The study

• Authors: J. Kreienbühl, S. Changkhong, V. Orlowski, M.B. Kirschner, I. Opitz, M. Meerang
• Journal: International Journal of Molecular Sciences
• Published: 2023;24(17)
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

We previously demonstrated that cullin 4B (CUL4B) upregulation was associated with worse outcomes of pleural mesothelioma (PM) patients, while the overexpression of its paralog CUL4A was not associated with clinical outcomes. Here, we aimed to identify the distinct roles of CUL4B and CUL4A in PM using an siRNA approach in PM cell lines (ACC Meso-1 and Mero82) and primary culture. The knockdown of CUL4B and CUL4A resulted in significantly reduced colony formation, increased cell death, and delayed cell proliferation. Furthermore, similar to the effect of CUL4A knockdown, downregulation of CUL4B led to reduced expression of Hippo pathway genes including YAP1, CTGF, and survivin. Interestingly, CUL4B and not CUL4A knockdown reduced TGF-β1 and MMP2 expression, suggesting a unique association of CUL4B with this pathway. However, the treatment of PM cells with exogenous TGF-β1 following CUL4B knockdown did not rescue PM cell growth. We further analyzed ACC Meso-1 xenograft tumor tissues treated with the cullin inhibitor, pevonedistat, which targets protein neddylation, and observed the downregulation of human TGF-β1 and MMP2. In summary, our data suggest that CUL4B overexpression is important for tumor cell growth and survival and may drive PM aggressiveness via the regulation of TGF-β1 expression and, furthermore, reveal a new mechanism of action of pevonedistat.

In plain English

This was a laboratory study of pleural mesothelioma cells by researchers at the University Hospital Zürich. It did not examine asbestos exposure. The authors used siRNA (small RNA molecules) to lower the level of a protein called cullin 4B (CUL4B), and of its close relative CUL4A. They did this in two established mesothelioma cell lines (ACC Meso-1 and Mero-82) and in one primary culture (M15.32) grown from a patient's pleural effusion.

Lowering CUL4B reduced colony formation in all three and increased the number of dead cells. A drop in cells entering DNA copying was seen in ACC Meso-1 and M15.32, but it was not statistically significant. In ACC Meso-1, the only cells tested, CUL4B knockdown did not change sensitivity to cisplatin.

Lowering either protein reduced YAP1 and CTGF. Only CUL4B knockdown reduced TGF-β1 and MMP2. Adding TGF-β1 did not restore colony formation. In xenograft tumor tissue from the authors' earlier work, treatment with pevonedistat was followed by significantly lower TGF-β1 and MMP2.

The authors concluded that high CUL4B may drive mesothelioma aggressiveness through regulation of TGF-β1.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study using siRNA knockdown in mesothelioma cell lines and a primary cell culture, plus gene-expression analysis of xenograft tumor tissue from the authors' earlier study
• Who was studied: Two established pleural mesothelioma cell lines (ACC Meso-1 and Mero-82) and one primary culture (M15.32) grown from the pleural effusion of a patient with pleural mesothelioma treated at the University Hospital Zurich
• Exposure: Asbestos exposure was not studied. The experiments were: siRNA knockdown of CUL4B (two siRNAs) and of CUL4A; treatment with TGF-β1 (4 ng/mL), cisplatin, the proteasome inhibitor Bortezomib, and the cullin inhibitor pevonedistat
• Main results:
  • Both CUL4B siRNAs reduced CUL4B mRNA by more than 80%
  • Colony formation was reduced after CUL4B knockdown in all cell lines
  • After CUL4B knockdown there were slightly more early apoptotic cells and, more significantly, more dead cells
  • Fewer cells entered S-phase after CUL4B knockdown in ACC Meso-1 and M15.32, but this was not statistically significant; Mero-82 showed no change
  • CUL4B knockdown did not change the sensitivity of ACC Meso-1 cells to cisplatin (only ACC Meso-1 was tested)
  • YAP1 and CTGF were significantly reduced after knockdown of either CUL4A or CUL4B; survivin was significantly reduced only in the primary cells (M15.32)
  • MMP2 was reduced after CUL4B knockdown but showed minimal or no change after CUL4A knockdown; TGF-β1 expression was also reduced after CUL4B knockdown
  • In ACC Meso-1 culture fluid, TGF-β1 protein measured by ELISA was lower after CUL4B depletion, but the difference was not statistically significant (2 independent experiments)
  • Adding TGF-β1 did not restore colony formation after CUL4B knockdown
  • In ACC Meso-1 xenograft tumor tissue from the earlier study, pevonedistat treatment was associated with significantly lower human TGF-β1 and MMP2
  • Knocking down CUL4A and CUL4B together did not further increase the reduction of TGF-β1 and YAP1 expression
• The authors' conclusion: The authors concluded that high CUL4B expression may drive pleural mesothelioma aggressiveness through regulation of TGF-β1 expression
• Limitations the authors state:
  • The fall in YAP1 and CTGF might have been a result of cell cycle changes rather than a direct effect
  • The ELISA results apply only to ACC Meso-1 (TGF-β1 was undetectable in M15.32, and Mero82 had too many dead cells)
  • One non-silencing control (siNeg) itself affected cell viability and was dropped
  • Differences between drug effects in animals and in cell culture may reflect factors absent in culture, such as hypoxia and extracellular matrix

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma.

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• Expression of FGFR1-4 in Malignant Pleural Mesothelioma Tissue and Corresponding Cell Lines and its Relationship to Patient Survival and FGFR Inhibitor Sensitivity — G. Vlacic, M.A. Hoda, et al. (2019)
• Cytosolic pH regulates proliferation and tumour growth by promoting expression of cyclin D1 — L.M. Koch, E.S. Birkeland, et al. (2020)

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