Dissertation committee:
Σοφία Τσελένη-Μπαλαφούτα, Ομότιμη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Δημήτριος Στραβοπόδης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Ευανθία Αναστασιάδου, Ερευνήτρια Γ', ΙΙΒΕΑΑ
Νικόλαος Καβαντζάς, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Αγρογιάννης, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Δημήτριος Κλέτσας, Διευθυντής, ΙΒΕ "Δημόκριτος",
Γεώργιος Παυλόπουλος, Ερευνητής Β', ΕΚΒΕ "Αλέξανδρος Φλέμιγκ"
Summary:
Malignancies of thyroid gland are the most common malignancies of the endocrine system as they account for 1-5% of all cancer cases worldwide. Papillary carcinomas (PTC) are the most common type of thyroid cancer, accounting for approximately 80% of cases, usually, with good prognosis. However, specific variants such as tall cell variants (TCV) demonstrate aggressive characteristics and poor outcome. Deep understanding of the molecular correlations and gene pathways that dictate the cell programming and therefore, the fate of the disease and the outcome of the patient is crucial for a better stratification and the development of potential treatments.
Towards that direction, we conducted a population study and analyzed the distribution of specific clinicopathological features. We revealed a strong association between TCVs and the presence of nuclear and tissue alterations such as pseudoinclusions and fibrogenesis, respectively. This was associated with deregulation of specific processes such as protein processing in ER, NFKβ, cell-cell adhesion and regulation of cytoskeleton, crucial pathways involved in Epithelial to Mesenchymal Transition (EMT). EMT is a key mechanism in cell differentiation and migration (metastasis) rearranging the location and function of structural proteins (such as molecules of the cytoskeleton) as well as molecules that ensure cell junctions (tight, focal and adherens junction). The nuclear structure and the presence of grooves, invaginations, creases and pseudoinclusions were analyzed through Advanced microscopy techniques. RNA-induced silencing complex (RISC) components such as Drosha, DGCR8, Dicer and Argonaute proteins (AGOs) were intensely present inside these structures in diffused or specific-located punctuate patterns. Strikingly, AGOs, and specifically AGO2, follow these structures demonstrating an intense signal inside and along them. AGOs "load" miRNAs and direct them to the mRNA targets for post-transcriptional gene regulation. The presence of AGO2, the foremost member of the AGO family, most likely reflects the great cellular need for tight gene regulation leading and management of local homeostasis. This fact is not surprising since local protein synthesis in response to specific molecular signals dictates the morphology, function and fate of the cells and therefore, the outcome of the disease. Conclusively, we propose that AGO2 creates a niche, the “AGO2 locasomes”, for proper local homeostasis. Anyhow, AGO2 is known to be compartmentalized into structures such as GW- and P-bodies, stress granules, adherens junctions and midbody, serving the "local" needs for post-transcriptional gene regulation. Using immunofluorescence, image analysis, bioinformatics and cytogenetics, it was demonstrated that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The open-ended are most likely intercellular channels while close-ended are cytokinetic bridges. In the latter AGO2 colocalizes at the midbody-arms with cytoskeletal components such as α-Τubulin and Aurora B and various kinases. AGO2, phosphorylated on serine 387 colocalizes with Dicer at the midbody ring on a p38 MAPK dependent manner. Further, the stress sensitivity of AGO2 was revealed and its importance for the ensurement and fidelity in chromosome segregation and cytokinetis was demonstrated. We suggested that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress, aiming to generate the appropriate micro-environment for local homeostasis. In conclusion, AGO2 seems to accumulate at specific cellular structures such as pseudoinclusions, and tubular protrusions, together with other RISC components, serving the idea of AGO2 locasomes, for the restoration of the local post-transcriptional homeostasis.
To better understand the mechanisms involved, we attempted to determine the coding and non-coding genes (such as miRNAs), expressed in normal and malignant thyroid substrates. We used High-Sequencing of RNAs isolated by Cross-Linking Immuno-Precipitation (HITS-CLIP) methodology to record the tripartite complex AGO2::miRNA::mRNA and identify the "active" miRNome, ie the set of miRNAs that bind to the RISC complex and their mRNAs targets. Informatics analysis revealed that a group of 31 deregulated miRNAs with high statistical significance, between normal and cancer cell lines. Comparative analysis based on their differential expression and their mRNA targets revealed implicated molecular pathways such as regulation of actin cytoskeleton pathway, transcriptional dysregulation in cancer, ΝFK-β signaling pathway and miRNAs in cancer which support the EMT.
Subsequently, we focused on specific miRNAs, such as miR26. miR26 was a promising target as (i) it has the same transcriptional behavior across different cancer cell lines, (ii) it appears to be downregulated in cancer cell lines and patient tissue samples, (iii) all the members of miR26 family have the same transcriptional profile and (iv) our results are in accordance with the literature. In in vitro assays, using cell lines, overexpression of miR26 leads to restriction of the oncogenic potential since its expression has a reverse correlation with the ability of cells to proliferate. In mouse xenografts, generated by FTC133 cells transfected with miR26a-5p mimicking sequences, retardation in thyroid tumor development was observed and a promotion of a peripheral and not a central -as expected- tumor necrosis. RNA-seq differential expression analysis between cells carrying mimic miR26 or scrambled sequences revealed 1,201 statistically significant deregulated genes correlated with cell cycle, apoptosis, cell division, cell adhesion and transcriptional deregulation in cancer. 133 were experimentally verified as direct targets of miR26. The 100 top-expressed genes were used for pathway analysis via PathWalks algorithm. The cluster of interest contained fundamental pathways such as cell cycle, miRNAs in cancer and aging processes. Finally, a comparative informatics analysis of HITS-CLIP and RNA-seq data uncovered 50 deregulated molecules including PAWR and STRADB. The latter are correlated with cell proliferation and fate, crucial conditions for EMT. Conclusively, miR26 and its direct targets may constitute crucial molecules for oncogenesis and promising, potential agents for diagnosis and therapy of thyroid malignancies.
Keywords:
Thyroid cancer, EMT, Population study, Micro-staging, Pseudoinclusions, AGOs, RISC Complex, MiR26, Mimic Sequences MiR26, HITS-CLIP
