Although papillary thyroid carcinoma (PTC) has a good prognosis, 20–90% of patients show metastasis to regional lymph nodes and 10–15% of patients show metastasis to distant sites. Metastatic disease represents the main clinical challenge that impacts survival rate. We previously showed that LIMD2 was a novel metastasis-associated gene. In this study, to interrogate the role of LIMD2 in cancer invasion and metastasis, we used CRISPR-mediated knockout (KO) of LIMD2 in PTC cells (BCPAP and TPC1). Western blot and high-content screening (HCS) analysis confirmed functional KO of LIMD2. LIMD2 KO reduced in vitro invasion and migration. Ultrastructural analyses showed that cell polarity and mitochondria function and morphology were restored in LIMD2 KO cells. To unveil the signals supervising these phenotypic changes, we employed phospho-protein array. Several members of the MAPK superfamily showed robust reduction in phosphorylation. A Venn diagram displayed the overlap of kinases with reduced phosphorylation in both cell lines and showed that they were able to initiate or sustain the epithelial-mesenchymal transition (EMT) and DNA damage checkpoint. Flow cytometry and HCS validation analyses further corroborated the phospho-protein array data. Collectively, our findings show that LIMD2 enhances phosphorylation of kinases associated with EMT and invasion. Through cooperation with different kinases, it contributes to the increased genomic instability that ultimately promotes PTC progression.
Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.
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