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

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

Constitutive and AP20187-induced Ret activation in photoreceptors does not protect from light-induced damage.

Purpose Delivery of glial cell-derived neurotrophic factor (GDNF), either as a recombinant protein or by retinal gene transfer results in photoreceptor (PR) neuroprotection in genetic models of retinitis pigmentosa (RP). The mechanism of GDNF action and its direct targets in the retina remain unknown. The goal of the present study was to test the neuroprotective effect of GDNF from light-induced damage, a commonly used stimulus of PR degeneration, and to determine whether protection occurs directly on PRs. Methods Adeno-associated viral vectors (AAV) were developed that expressed either GDNF or a constitutively (RetMen2A) or pharmacologically activated chimeric GDNF receptor (Fv2Ret). Fv2Ret homodimerization and activation are induced by the administration of the small dimerizer drug AP20187. AAV2/2 vectors and the cytomegalovirus (CMV) promoter were used to transduce GDNF in the retina, whereas RetMen2A and Fv2Ret were transduced by AAV2/5 vectors and their expression restricted to PRs by the rhodopsin promoter. In vivo GDNF levels were measured by ELISA, RetMen2A and Fv2Ret expression and activation in vitro and/or in vivo were assessed by Western blot and immunofluorescence analyses. ERG measurements and histologic analyses were performed to assess morphologic and functional rescue, respectively. Results GDNF gene transfer resulted in sustained protein expression in the eye. In addition, the results confirmed in vivo that PR-restricted activation of Ret signaling occurred after either AAV-mediated expression of RetMen2A or AP20187-dependent Fv2Ret activation. However, this or AAV-mediated GDNF retinal gene transfer did not result in functional or morphologic PR protection from light-induced damage. Conclusions The results suggest that the apoptotic pathways responsible for light-induced PR degeneration are not inhibited by GDNF. However, GDNF signaling was shown to be regulated in time and levels in the retina by the AP20187/Fv2Ret system which is therefore available to be tested as gene-based therapeutic strategy in models of PR degeneration responsive to GDNF

Structure Prediction and Validation of the ERK8 Kinase Domain

Extracellular signal-regulated kinase 8 (ERK8) has been already implicated in cell transformation and in the protection of genomic integrity and, therefore, proposed as a novel potential therapeutic target for cancer. In the absence of a crystal structure, we developed a three-dimensional model for its kinase domain. To validate our model we applied a structure- based virtual screening protocol consisting of pharmacophore screening and molecular docking. Experimental characterization of the hit compounds confirmed that a high percentage of the identified scaffolds was able to inhibit ERK8. We also confirmed an ATP competitive mechanism of action for the two best-performing molecules. Ultimately, we identified an ERK8 drug-resistant \u27\u27gatekeeper\u27\u27 mutant that corroborated the predicted molecular binding mode, confirming the reliability of the generated structure. We expect that our model will be a valuable tool for the development of specific ERK8 kinase inhibitors

The Use of Music and Brain Stimulation in Clinical Settings: Frontiers and Novel Approaches for Rehabilitation in Pathological Aging

Aging is a phase of life characterized by the increasing risk of occurring neurodegenerative pathologies, as well as stroke and physical decline. Patients in such clinical conditions are known to benefit from programs able to promote the improvement of associated cognitive, functional, and behavioral disorders. In recent times, growing empirical evidence showed the efficacy of active and passive music-based interventions to be the highest when used for healing these diseases. Additionally, very latest research found the combination of electrical neurostimulation with music to have potential utility for clinical older adult populations, as it may amplify the impulse to neuroplasticity and, by consequence, the rehabilitation gains. Reiterating of active music making induces changes in multiple brain regions bringing to the enhancement of cognitive and sensorimotor skills, while merely listening to pleasurable music stimulates dopaminergic regions of the brain improving cognition, motivation, and mood in a variety of neurological diseases. The versatility of music-based interventions in combination with new technologies allows an effective application of innovative therapeutic techniques. Moreover, their easy implementation in healthcare settings and their positive effects on both recovery and patients’ quality of life makes the integration of music-based interventions with conventional rehabilitation approaches highly desirable

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

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

R0 surgical resection of giant dedifferentiated retroperitoneal liposarcomas in the COVID era with and without nephrectomy: A case report

: Retroperitoneal sarcomas (RPSs) are rare findings that can grow into large masses without eliciting severe symptoms. At present, surgical resection is the only radical therapy, whenever it can be performed with the aim to achieve a complete removal of the tumor. The present report describes two consecutive cases of RPSs that resulted in dedifferentiated liposarcomas (DDLPSs) and these patients underwent R0 surgical resection with and without a nephron-sparing procedure. The diagnostic workup, the surgical approach, the impact of late surgical management due to the COVID pandemic and the latest literature on the topic are discussed and analyzed. The patients, who refused to undergo any medical examination during the prior 2 years due to the COVID pandemic, were admitted to Federico II University Hospital (Naples, Italy) complaining about weight loss and general abdominal discomfort. In the first case, a primitive giant abdominal right neoplasm of retroperitoneal origin enveloping and medializing the right kidney was observed. The second patient had a similar primitive retroperitoneal giant left neoplasm, which did not affect the kidney. Given the characteristics of the masses and the absence of distant metastases, after a multidisciplinary discussion, radical surgical removal was carried out for both patients. The lesions appeared well-defined from the surrounding tissues, and markedly compressed all the adjacent organs, without signs of infiltration. In the first patient, the right kidney was surrounded and undetachable from the tumor and it was removed en bloc with the mass. The second patient benefited from a nephron-sparing resection, due to the existence of a clear cleavage plane. The postoperative courses were uneventful. Both the histological examinations were oriented towards a DDLPS and both patients benefited from adjuvant chemotherapy. In conclusion, the treatment of giant RPS is still challenging and requires multidisciplinary treatment as well as, when possible, radical surgical removal. The lack of tissue infiltration and the avoidance of excision or reconstruction of major organs (including the kidney) could lead to an easier postoperative course and an improved prognosis. When possible, surgical management of recurrences or incompletely resected masses must be pursued. Since the COVID pandemic caused limited medicalization of a number of population groups and delayed diagnosis of other oncologic diseases, an increased number of DDLPSs could be expected in the near future