Maturi, Rufina (2025) Unraveling Klhl14 role in the thyroid: linking differentiation, EMT and senescence. [Tesi di dottorato]

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Abstract

Kelch-like family member 14 (Klhl14) is part of the Kelch-like protein family, functioning as a substrate recognition component of an E3-ubiquitin ligase complex. It has emerged as a pivotal regulator implicated in both tumorigenesis and protein homeostasis. Klhl14 displayed dichotomous functions in cancer biology: on one hand, serving as an oncogene in ovarian and endometrial cancers, on the other hand, acting as a tumor suppressor in malignancies such as diffuse large B-cell lymphoma, malignant mesothelioma and thyroid cancer. While its tumor-suppressive effects have been observed in multiple malignancies, Klhl14 remains poorly understood in terms of its mechanistic actions within the various biological contexts. Our study aims to investigate Klhl14’s role in thyroid physiology using an advanced mouse-derived thyroid organoid model. We observed increased Klhl14 expression in mature thyroid cells, aligning with master regulators of differentiation. Moreover, Klhl14 knockdown (Klhl14-KD) organoids displayed impaired growth and viability, linking Klhl14 to cellular proliferation and survival. Whole proteomic analysis of Klhl14-KD organoids identified key alterations in proteins associated with inflammation, cell migration, cytokine regulation, apoptosis and senescence, suggesting a dual role of Klhl14 in senescence and epithelial-mesenchymal transition (EMT). EMT activation was supported by the increased expression of mesenchymal master regulators and effector molecules, whereas senescence was indicated by increased levels of senescence-associated factors, such as p16, p21, Ccl5, Mcp1. Intriguingly, these cellular changes were reversible with transforming growth factor-beta (TGF-β) type I receptor ALK5 kinase inhibition, underscoring TGF-β as an important player in observed Klhl14-KD phenotypes. Indeed, reducing TGF-β activity resulted in mitigated senescence and EMT signatures, revealing a regulatory axis between Klhl14 and TGF-β, with potential feedback between these molecules. Our findings highlight Klhl14 as a critical regulator in thyroid organoid development, influencing differentiation, proliferation, EMT, and senescence, and positioning Klhl14 as an upstream modulator of TGF-β activity. This study not only elucidates Klhl14’s role in thyroid physiology but also suggests its potential as a molecular marker or therapeutic target in thyroid malignancies and possibly in a broader cancer context.

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