RET: A Multi-Faceted Gene in Human Cancer

Receptor tyrosine kinases (RTK) are transmembrane (TM) proteins featuring an intracellular domain containing the tyrosine kinase (TK) enzyme. RTKs are often involved in cancer formation [13]. Notable examples are epidermal growth factor receptor (EGFR/ HER1) and anaplastic lymphoma kinase (ALK) in non-small cell lung carcinoma (NSCLC) [4], KIT in gastrointestinal stromal tumors (GIST) [5], FLT3 in acute myeloid leukemia (AML) [6], and HER2/ ERBB2/neu in breast cancer [7]. In some cases, cancer cells up-regulate expression of the RTK (as an example HER2 in breast cancer), its cognate growth factor or both, in other cases, structural alterations such as chromosomal rearrangements leading to the RTK recombination to heterologous genes (as an example EML4-ALK in lung adenocarcinoma) or point mutations (as EGFR, KIT or FLT3 mutations in NSCLC, GIST, or AML, respectively), lead to unchecked kinase and oncogenic activity [1-3]. This notion has stimulated the search for agents, such as monoclonal antibodies against the RTK extracellular domain (like trastuzumab for HER2 or cetuximab for EGFR) or ATP-competitive small molecule protein kinase inhibitors (PKIs) (like gefitinib and erlotinib for EGFR or crizotinib for ALK), to combat cancers driven by oncogenic RTKs [1-3]. The RET RTK was originally identified as an oncogene activated by a rearrangement occurred in vitro during transfection of NIH3T3 cells with human lymphoma DNA [8]. RET protein belongs to a cell-surface complex able to bind glial-derived neurotrophic factor (GDNF) ligands (GDNF, neurturin, artemin, and persephin) in conjunction with co-receptors of the GDNF receptor α family, designated GFRα 1-4 [9]. Binding to the ligand-co-receptor complex leads to RET dimerization and kinase activation. RET expression is tightly regulated during development and in the adulthood is limited to specific tissues, including neural crest-derived cells. RET is essential for the development of the enteric neurvous system and kidney, and germline loss-of-function mutations in RET cause Hirschsprung disease (aganglionic megacolon) and congenital anomalies of the kidney or lower urinary tract [10,11]. RET gene maps to chromosome 10q11.2. Fig. 1 shows that it is splitted in 21 coding exons. Exons 1-10 code for the extracellular region; exon 11 codes for the COOH-terminal part of the extracellular region, the TM domain, and the intracellular juxtamembrane domain. Finally, exons 12-21 code for the intracellular domain. An alternative splicing at exon 19 determine the synthesis of three RET protein isoforms with different C-terminal tails. In RET9 (1072 aa), exon 19 is unspliced; in RET51 (1114 aa), exon 19 is spliced to exon 20; in RET43 (1106 aa), exon 19 is spliced to to exon 21 [12-15]. RET9 and RET51 are the most abundant and well characterized isoforms (Fig. 1). RET protein features an extracellular portion (RET-EC) tha

Differential diagnosis of thyroid nodules using fine-needle aspiration cytology and oncogene mutation screening: are we ready?

Thyroid nodules are a very common clinical finding, and although the majority of them are benign, thyroid carcinoma accounts for about 5-15% of nodules. Fine-needle aspiration cytology (FNAC) is actually used for the differential diagnosis of these lesions. Although in most cases this examination clearly distinguishes benign from malignant lesions, some fine-needle aspiration (FNA) samples fall into undetermined thyroid cytology categories, which according to the most recent classification of thyroid FNAC consist of ‘suspicious for malignancy’, ‘suspicious for follicular or Hurtle cell neoplasm’, and ‘follicular lesion of undetermined significance/atypia of undetermined significance’. Moreover, some samples are insufficient for diagnosis. Taken together, these categories account for almost 20-30% of nodules. Owing to the high risk of papillary thyroid carcinoma, patients with lesions that are ‘suspicious for malignancy’ are currently subjected to lobectomy or total thyroidectomy. On the other hand, patients with ‘atypia of undetermined significance’ undergo repeated FNAs, and patients with ‘suspicious for follicular or Hurtle cell neoplasm’ are subjected to diagnostic lobectomy and subsequently, in the case of histological diagnosis of carcinoma, total thyroidectomy. Recent studies clearly indicate that molecular analysis of thyroid nodules can significantly improve the diagnostic power of cytology and drive the appropriate clinical management of these patients

The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.

Abstract Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation. (Cancer Res 2006; 66(12): 6280-7

Activation of the Erk8 Mitogen-activated Protein (MAP) Kinase by RET/PTC3, a Constitutively Active Form of the RET Proto-oncogene

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demon- strate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we charac- terized Tyr981, a known binding site for c-Src, as a major determi- nant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr981-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions

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

Glial cell line-derived neurotrophic factor induces proliferative inhibition of NT2/D1 cells through RET-mediated up-regulation of the cyclin-dependent kinase inhibitor p27kip 1

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The disruption of the CCDC6 - PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma

Treatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours

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

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein Kinases (JNKs): evidence for a divergence of the ERKs and JNKs pathways induced by Ret

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprung's disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 ®broblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-de®cient RET mutant, associated with Hirschsprung's disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42