Characterization of KLHL14 anti-oncogenic action in malignant mesothelioma

Our plain-English summary of Characterization of KLHL14 anti-oncogenic action in malignant mesothelioma, by A. Canciello, R.B. Domínguez, B. Barboni, et al (Heliyon, 2024).

The study

• Authors: A. Canciello, R.B. Domínguez, B. Barboni, A. Giordano, A. Morrione
• Journal: Heliyon
• Published: 2024;10(6):e27731
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant mesothelioma (MM) is a very aggressive neoplasia with a short life expectancy and limited therapeutic options. Thus, the identification of novel molecular targets is a matter of great urgency. Kelch-like (KLHL) proteins play an important role in a number of physiological and pathological cell-regulatory processes. Among this family, the function of KLHL14 is still very poorly characterized. KLHL14 was originally identified as a gene involved in regulating the epithelial-mesenchymal transition (EMT) process. Here, we demonstrate that KLHL14 not only prevents EMT but also plays an anti-oncogenic role in MM. Indeed, KLHL14 depletion enhanced proliferation, motility, invasion and colony formation in MM cells. Importantly, we also demonstrated that KLHL14 mechanism of action is dependent on Transforming Growth Factor β (TGF-β). In fact, TGF-β promotes de novo synthesis, increases protein stability and induces nuclear-cytoplasmic shuttling of KLHL14. Collectively, this research is an important step further to decipher KLHLs mechanism of action and further contributes to the understanding of the molecular mechanisms regulating MM.

© 2024 The Authors.

In plain English

This was a laboratory study of malignant mesothelioma (MM) cell lines examining the protein KLHL14, whose function the authors describe as poorly characterized; asbestos is mentioned only as background. Using NCI-H28 (epithelioid), MSTO-211H (biphasic), NCI-H2052 (sarcomatoid), NCI-H2452 and immortalized mesothelial MeT-5A cells, the authors measured KLHL14 levels and location, depleted KLHL14 with siRNA, and treated cells with TGF-beta1 and with inhibitors of nuclear export, protein synthesis and the proteasome. In the cBioPortal database, KLHL14 mRNA was mostly expressed in the epithelioid subtype (71.3%), then biphasic (26.4%) and sarcomatoid (2.3%). KLHL14 showed nuclear expression, with no canonical nuclear localization signal identified. Depleting KLHL14 raised EMT transcription factors (ZEB, SNAI, Twist) and N-cadherin, lowered E-cadherin and ZO-1, enhanced TGF-beta-induced wound closure, and increased proliferation, transwell migration, Matrigel invasion and colony number and size. TGF-beta raised KLHL14 levels over time. It reduced nuclear KLHL14 in some experiments but increased it in others, and the authors said its effect on nucleus-cytoplasm movement may depend on histological subtype. The authors concluded KLHL14 may act as a tumor suppressor in MM.

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: in vitro laboratory study using human mesothelioma cell lines (siRNA depletion, drug treatments, immunoblot, immunofluorescence, functional assays), plus a database query
• Who was studied: Human MM cell lines representing epithelioid (NCI-H28), sarcomatoid (NCI-H2052) and biphasic (MSTO-211H) subtypes, plus NCI-H2452, with immortalized mesothelial MeT-5A as control. Also studied: A549, Caco-2 and HEK-293T cells, and cBioPortal data on the three pleural mesothelioma subtypes
• Exposure: Not an asbestos exposure study. Cells were exposed to TGF-beta1 at 10 ng/mL, Leptomycin B at 20 nM, cycloheximide at 100 microM, MG-132 at 10 microM, and siRNA depletion of KLHL14 at 25 nM
• Main results:
  • In the cBioPortal database, KLHL14 mRNA was predominantly expressed in the epithelioid subtype (71.3%), then biphasic (26.4%), then sarcomatoid (2.3%)
  • KLHL14 protein was highest in MSTO-211H cells. Nuclear localization predominated in NCI-H2052 and MSTO-211H cells, while NCI-H28 and Met-5A cells showed mostly cytoplasmic KLHL14
  • No classical nuclear localization or nuclear export signal was identified in the KLHL14 sequence by the bioinformatic tools used
  • In MSTO-211H and NCI-H2052 cells, siRNA depletion of KLHL14 increased mRNA of ZEB, SNAI, Twist and CDH2 (N-cadherin) and reduced CDH1 (E-cadherin)
  • At protein level, KLHL14 depletion raised SNAIL, SLUG and ZEB1 and lowered ZO-1. There was no significant difference in vimentin between control and depleted cells
  • In MSTO-211H cells, TGF-beta increased KLHL14 mRNA and protein over time, reduced nuclear KLHL14 and slightly increased cytoplasmic KLHL14
  • In fractionation experiments, TGF-beta significantly increased both nuclear and cytoplasmic KLHL14 in NCI-H28 cells. The authors stated that the effect on nuclear-cytoplasmic movement may depend on histological subtype
  • In NCI-H2052 and NCI-H2452 cells, KLHL14 depletion significantly enhanced TGF-beta-induced wound closure compared with non-targeting control cells. It also significantly increased proliferation in both lines
  • Colonies were more numerous and larger in KLHL14-depleted NCI-H2052 and NCI-H2452 cells than in controls. Depletion also further increased transwell migration and Matrigel invasion in both unstimulated and TGF-beta-stimulated cells
• The authors' conclusion: The authors concluded that KLHL14 has an anti-oncogenic role in MM and that TGF-beta signaling regulates its expression, nuclear translocation and stability. They said it may function as a tumor suppressor, and that further work is needed
• Limitations the authors state:
  • The authors stated the database survival finding could not be tested for statistical significance because there were too few reported cases
  • The authors stated that the specific mechanism of action of KLHL14 in MM, including its protein substrates, requires further experiments
  • The authors stated that the study mainly explored TGF-beta signaling and EMT-related factors, and that other possible mechanisms are still under investigation
  • The authors noted that in vivo experiments and clinical trials are still needed

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Life Expectancy, De Novo.

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