LIMD2, is a LIM domain protein, which is defined by the presence of one segment containing two adjacent cysteine-histidine-rich zinc fingers separated by a hydrophobic linker that functions as a protein-binding interface, as previously revisited. However, the signaling pathway through which LIMD2 promotes morphological changes to trigger the metastatic behavior still remains unknown. The authors showed that LIMD2 controlled the acquisition of multiple hallmarks of tumor progression as anchorage-independent growth and increased migration of different cancer types and cell lines. LIMD2 overexpression has been correlated with a higher degree of invasiveness in breast and melanoma cancer cell lines. Using the Cancer Genome Atlas (TCGA) dataset, we provided further evidence that LIMD2 expression was higher in BRAF-like than in RAS-like PTC. Remarkably, its expression was higher in PTC samples and papillary thyroid cell lines harboring the BRAF V600E mutation than its expression in PTC harboring RET/PTC fusion. The expression of LIMD2 was subsequently confirmed in over 80% of the metastatic PTC and in nearly 95% of matched lymph node metastases. Several transcripts were highly expressed in lymph node metastasis, comprising LIMD2. To learn how PTC spreads to the lymph nodes, we performed gene expression profiling on matched normal thyroid, primary PTC, and its lymph node metastasis to identify genes associated with the metastatic process. Although metastasis is the main cause of recurrence, failure of cancer therapy, and mortality, it remains poorly understood. Moreover, the American Thyroid Association (ATA) thyroid cancer guidelines proposed a dynamic risk stratification scheme that included additional prognostic variables such as the extent of lymph node involvement (i.e., number and size) and mutational status of the primary tumor to determine the risk of disease recurrence or persistence. In addition, 1.0–13% of patients present distant metastasis at time of diagnosis, which negatively impacts the survival rate. Through cooperation with different kinases, it contributes to the increased genomic instability that ultimately promotes PTC progression.Īlthough most patients with papillary thyroid carcinoma (PTC) have a favorable prognosis, 30–90% of patients exhibit clinical or occult cervical lymph nodes. Collectively, our findings show that LIMD2 enhances phosphorylation of kinases associated with EMT and invasion. Flow cytometry and HCS validation analyses further corroborated the phospho-protein array data. 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. Several members of the MAPK superfamily showed robust reduction in phosphorylation.
To unveil the signals supervising these phenotypic changes, we employed phospho-protein array. Ultrastructural analyses showed that cell polarity and mitochondria function and morphology were restored in LIMD2 KO cells. LIMD2 KO reduced in vitro invasion and migration. Western blot and high-content screening (HCS) analysis confirmed functional KO of LIMD2. 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). We previously showed that LIMD2 was a novel metastasis-associated gene. Metastatic disease represents the main clinical challenge that impacts survival rate. 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.
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