Clinical and genetic studies of papillary thyroid carcinoma : with focus on telomere maintenance and the microRNA machinery

<p dir="ltr">Papillary thyroid carcinoma (PTC) is generally indolent, but a subset exhibits aggressive behavior driven by molecular alterations. Identifying reliable biomarkers and molecular mechanisms to predict and manage this aggressive subset remains an important unmet clinical need, as current risk stratification tools are insufficient to guide personalized therapy. Reactivation of telomerase reverse transcriptase (TERT), most often via promoter mutations, is a key feature of tumor aggressiveness. Follicular-patterned tumors, including follicular thyroid carcinoma (FTC), pose diagnostic challenges. While epigenetic alterations such as global loss of 5-hydroxymethylcytosine (5hmC) have been proposed as surrogate markers for TERT promoter mutations, their predictive value is limited. Moreover, the regulation of TERT through transcriptional and epigenetic mechanisms, and its interaction with other oncogenic pathways in PTC, remain incompletely understood. In parallel, dysregulation of microRNAs (miRNAs) has been implicated in tumor progression, invasion, and metastasis in multiple cancers, including thyroid carcinoma, highlighting their potential as biomarkers and therapeutic targets.</p><p dir="ltr">In Paper I, immunohistochemical (IHC) detection of 5hmC was assessed as a potential predictive marker for TERT promoter mutations in FTC. Using two antibody clones across 29 tumors with known TERT status, 5hmC IHC showed low sensitivity for predicting TERT promoter mutations, despite prior associations in PTC. These results indicate that global 5hmC loss is not a reliable predictor in follicular thyroid tumors, highlighting the need for more direct molecular assessments of TERT activation.</p><p dir="ltr">Paper II identified the transcription factor, ETS homologous factor (EHF), as a critical mediator linking BRAFV600E signaling to TERT transcription. In analyses of PTC cohorts from public databases and Karolinska, high EHF expression was associated with BRAFV600E mutation, TERT promoter mutations, worse clinical behaviors, and shorter survival. Functional assays in vitro demonstrated that EHF overexpression increased TERT expression in cells with BRAFV600E and TERT promoter mutations, whereas BRAFV600E knockdown reduced both EHF and TERT expression levels. Chromatin immunoprecipitation suggested direct EHF binding to the mutant TERT promoter, providing mechanistic insight into transcriptional TERT activation in PTC.</p><p dir="ltr">Paper III explored alternative mechanisms of TERT activation, including promoter hypermethylation and copy number gains. Hypermethylation at promoter specific CpG sites and copy number gains were associated with elevated TERT expression, advanced disease, and poor survival, even in tumors lacking TERT promoter mutations. Integrative analyses indicate that promoter mutations, promoter hypermethylation, and copy number gains represent complementary mechanisms that all are associated with TERT activation, contributing to PTC aggressiveness.</p><p dir="ltr">In Paper IV, post-transcriptional regulation by miRNAs was examined in relation to PTC aggressiveness. DICER1 expression was downregulated in PTC, particularly in advanced stages, and associated with widespread miRNA dysregulation. Among these, miR-1179, miR-126-5p, and miR-139-5p were consistently associated with aggressive clinical features and poor survivals in two PTC cohorts. Notably, reduced miR-139-5p expression retained prognostic value in TERT promoter wild- type tumors, suggesting its potential as an independent prognostic biomarker and therapeutic target. Functional enrichment analyses of predicted targets implicated these miRNAs in key pathways regulating proliferation, survival, and cell motility.</p><p dir="ltr">Collectively, these studies provide a comprehensive view of TERT regulation in PTC, highlighting the multifaceted mechanisms driving tumor aggressiveness. While global 5hmC loss is not a reliable marker for TERT promoter mutations in follicular-patterned tumors, transcriptional regulation via the ETS factor EHF, TERT promoter hypermethylation, and copy number gains represent complementary pathways contributing to TERT activation and poor clinical outcomes in PTC. Furthermore, dysregulation of specific miRNAs offers insight into post- transcriptional control of PTC progression and identifies potential prognostic biomarkers. Together, these findings advance our understanding of molecular determinants of aggressiveness in thyroid cancer and underscore the importance of integrated molecular and epigenetic profiling for risk stratification and precision treatment.</p><h3>List of scientific papers</h3><p dir="ltr">I. Martin Hysek#, Samuel L Hellgren, Vincenzo Condello, <b>YIYI XU</b> , Catharina Larsson, Jan Zedenius, C Christofer Juhlin. 5hmC immunohistochemistry: A predictor of TERT promoter mutational status in follicular thyroid carcinoma? J Histochem Cytochem. 2023 Aug;71(8):451-458. <a href="https://doi.org/10.1369/00221554231190437" rel="noreferrer" target="_blank">https://doi.org/10.1369/00221554231190437</a></p><p dir="ltr">II. <b>YIYI XU</b>, Jiwei Gao, Na Wang, Jan Zedenius, Inga-Lena Nilsson, Weng- Onn Lui, Dawei Xu, C Christofer Juhlin, Catharina Larsson", Ninni Mu#. BRAF-induced EHF expression affects TERT in aggressive papillary thyroid cancer. J Clin Endocrinol Metab. 2025 Feb 18;110(3):693-705. <a href="https://doi.org/10.1210/clinem/dgae589" rel="noreferrer" target="_blank">https://doi.org/10.1210/clinem/dgae589</a></p><p dir="ltr">III. <b>YIYI XU</b>#, Jiwei Gao, Vincenzo Condello, Weng-Onn Lui, C Christofer Juhlin, Ninni Mu, Catharina Larsson#. Epigenetic and genetic mechanisms of TERT activation in papillary thyroid carcinoma. [Manuscript]</p><p dir="ltr">IV. <b>YIYI XU</b>#, Ninni Mu, Vincenzo Condello, C Christofer Juhlin, Weng- Onn Lui, Catharina Larsson#. Identification of clinically relevant microRNAs in papillary thyroid carcinoma. [Manuscript]</p><p dir="ltr"># Corresponding author</p>