Russo, Eleonora (2013) "Signaling and functions of the calcium/calmodulin-dependent kinase II (CaMKII) in medullary thyroid carcinoma". [Tesi di dottorato]

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Germline point mutations of the RET gene (REarranged during Transfection) are present in about 70% of sporadic medullary thyroid carcinoma (MTC), a malignant tumor that arises from calcitonin-secreting C cells of thyroid gland, and in almost 100% of the dominantly inherited multiple endocrine neoplasia (MEN) type 2A and 2B and the familial medullary thyroid carcinoma (FMTC). The calcium-calmodulin dependent kinase II (CaMKII) is an ubiquitous serine/threonine protein kinase involved in multiple signalings and biological functions. In epithelial and mesenchimal cells, CaMKII participates with Ras to Raf-1 activation, phosphorylating Raf-1 at S338, a phosphorylation necessary for ERK activation upon different physiologic and pathologic stimuli in the mitogen activated protein kinase (MAPK) cascade. In papillary thyroid carcinoma, CaMKII is activated by BRafV600E, oncogenic Ras and by RET rearrangements (RET/PTC) and participates to the activation of the ERK pathway by oncogenic Ras and RET/PTC, thus modulating tumor cell proliferation. The main aim of this thesis is to determine whether CaMKII is involved also in MTC harboring activating point mutation of RET. Recently it has been shown that an endogenous inhibitor of CaMKII (hCaMKIIN) is expressed in several cell types. Its expression is negatively correlated with the severity of human colon adenocarcinoma, suggesting a pivotal role of CaMKII in the development and progression of carcinomas with oncogenic activation of the MAPK pathway. To determine the role of CaMKII in the RET signaling and in MTC, two activated RET mutants (C634W and M918T) have been expressed in NIH-3T3 cells, observing the following CaMKII activation. MTC cell lines (TT and MZ-CRC1) harboring the most frequent MEN2A and MEN2B mutations (C634W and M918T respectively), have been treated with the CaMKII selective inhibitor KN93 and the following effects on the MAPK pathway and cell cycle have been monitored. Accordingly with results of the RET mutant expression in NIH-3T3, CaMKII was activated in MTC cell lines. Inhibition of CaMKII in these cells induced a decrease of Raf-1 phosphorylation at ser338. Accordingly, also the dephosphorylation of MEK and ERK was observed. CaMKII inhibition was followed by a reduced cyclin D and p27 accumulation, and by a reduction of cell proliferation. These results demonstrate that CaMKII is involved in cell cycle and proliferation in MTC cell lines harboring the RET oncogene. To confirm the actual role of CaMKII in the development and progression of MTC, I determined the relative hCaMKIINα mRNA expression in primary MTC tumors and its correlation with some clinicopathological parameters at surgery. In MTC affected patients hCaMKIINα mRNA expression was inversely correlated with serum calcitonin, tumor extension, tumor staging and presence of metastatic lymph nodes. The results of this thesis indicate that CaMKII has a role in cancers harboring oncogenic point mutation of RET and could represent a new therapeutic target for pharmacological intervention in these tumors.

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