Regulation of p27Kip1 Protein Levels Contributes to Mitogenic Effects of the RET/PTC Kinase in Thyroid Carcinoma Cells

Abstract We show that treatment of a panel of thyroid carcinoma cell lines naturally harboring the RET/PTC1 oncogene, with the RET kinase inhibitors PP1 and ZD6474, results in reversible G1 arrest. This is accompanied by interruption of Shc and mitogen-activated protein kinase (MAPK) phosphorylation, reduced levels of G1 cyclins, and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 because of a reduced protein turnover. MAP/extracellular signal-regulated kinase 1/2 inhibition by U0126 caused G1 cyclins down-regulation and p27Kip1 up-regulation as well. Forced expression of RET/PTC in normal thyroid follicular cells caused a MAPK- and proteasome-dependent down-regulation of p27Kip1. Reduction of p27Kip1 protein levels by antisense oligonucleotides abrogated the G1 arrest induced by RET/PTC blockade. Therefore, in thyroid cancer, RET/PTC-mediated MAPK activation contributes to p27Kip1 deregulation. This pathway is implicated in cell cycle progression and in response to small molecule kinase inhibitors

AXL is an oncotarget in human colorectal cancer

AXL is a tyrosine kinase receptor activated by GAS6 and regulates cancer cell proliferation migration and angiogenesis. We studied AXL as new therapeutic target in colorectal cancer (CRC). Expression and activation of AXL and GAS6 were evaluated in a panel of human CRC cell lines. AXL gene silencing or pharmacologic inhibition with foretinib suppressed proliferation, migration and survival in CRC cells. In an orthotopic colon model of human HCT116 CRC cells overexpressing AXL, foretinib treatment caused significant inhibition of tumour growth and peritoneal metastatic spreading. AXL and GAS6 overexpression by immunohistochemistry (IHC) were found in 76,7% and 73.5%, respectively, of 223 human CRC specimens, correlating with less differentiated histological grading. GAS6 overexpression was associated with nodes involvement and tumour stage. AXL gene was found amplified by Fluorescence in situ hybridization (FISH) in 8/146 cases (5,4%) of CRC samples. Taken together, AXL inhibition could represent a novel therapeutic approach in CRC

The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes

Oncogenic activation of the RET receptor tyrosine kinase is common in different human cancers. We found that the pyrazolo-pyrimidine PP1 inhibited RET-derived oncoproteins with a half maximal inhibitor concentration of 80 nM. Furthermore, RET/PTC3-transformed cells treated with 5 microM of PP1 lost proliferative autonomy and showed morphological reversion. PP1 prevented the growth of two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene. These findings suggest targeting RET oncogenes with PP1 or related compounds as a novel treatment strategy for RET-associated neoplasms

The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells.

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is a ATP-competitive inhibitor of RET, EGFR and VEGFRs kinases. Here, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying the cysteine 634 to tryptophan (C634W) and the methionine 918 to threonine (M918T) RET mutation, respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 MAPK phosphorylation. Single receptor knock-down by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells escape from RET blockade through compensatory over-activation of EGFR

Association of Very Rare NOTCH2 Variants with Clinical Features of Alagille Syndrome

Background. Alagille syndrome (ALGS) is a rare autosomal dominant genetic disease caused by pathogenic variants in two genes: Jagged Canonical Notch Ligand 1 (JAG1) and Notch Receptor 2 (NOTCH2). It is characterized by phenotypic variability and incomplete penetrance with multiorgan clinical signs. Methods. Using Next Generation Sequencing (NGS), we analyzed a panel of liver-disease-related genes in a population of 230 patients with cholestasis and hepatopathies. For the rare variants, bioinformatics predictions and pathogenicity classification were performed. Results. We identified eleven rare NOTCH2 variants in 10 patients, two variants being present in the same patient. Ten variants had never been described before in the literature. It was possible to classify only two null variants as pathogenic, whereas the most of variants were missense (8 out of 11) and were classified as uncertain significance variants (USVs). Among patients with ALGS suspicion, two carried null variants, two carried variants predicted to be pathogenic by bioinformatics, one carried a synonymous variant and variants in glycosylation-related genes, and two carried variants predicted as benign in the PEST domain. Conclusions. Our results increased the knowledge about NOTCH2 variants and the related phenotype, allowing us to improve the genetic diagnosis of ALGS