Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of Tumour Suppressor TMEM127 Drives RET-mediated Transformation Through Disrupted Membrane Dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability, and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

Selpercatinib: First approved selective RET inhibitor.

Selpercatinib is a small molecule that binds at the RET kinase active site. It inhibits activity of constitutively dimerized RET fusion proteins and activated point mutants, thereby blocking downstream signals for proliferation and survival. It is the first selective RET inhibitor to be FDA approved for tumor agnostic targeting of oncogenic RET fusion proteins. To view this Bench to Bedside, open or download the PDF.

Evaluating Cell Membrane Localization and Intracellular Transport of Proteins by Biotinylation.

Protein translocation to the cell membrane and transport through intracellular compartments are dynamic processes frequently altered in cancer cells. Abnormal protein localization can affect key cell functions, including transduction of extracellular signals and organization of the cytoskeleton, significantly affecting oncogenicity and therapeutic responses. In this chapter, we describe a surface protein biotinylation method that allows the study of membrane localization and endosomal transport of membrane-associated proteins. Surface biotinylation can be used to evaluate baseline protein levels at the membrane, and other processes such as internalization, recycling, and degradation of proteins in response to different treatments or as a consequence of oncogenic mutations. Further, the combination of this technique with other strategies, such as treatments with transport inhibitors, allows investigation of specific steps of protein trafficking through the cell.

Comprehensive immunohistochemical analysis of RET, BCAR1, and BCAR3 expression in patients with Luminal A and B breast cancer subtypes.

PURPOSE: Breast cancer (BC) is considered a heterogeneous disease composed of distinct subtypes with diverse clinical outcomes. Luminal subtype tumors have the best prognosis, and patients benefit from endocrine therapy. However, resistance to endocrine therapies in BC is an obstacle to successful treatment, and novel biomarkers are needed to understand and overcome this mechanism. The RET, BCAR1, and BCAR3 genes may be associated with BC progression and endocrine resistance. METHODS: Aiming to evaluate the expression profile and prognostic value of RET, BCAR1, and BCAR3, we performed immunohistochemistry on tissue microarrays (TMAs) containing a cohort of 361 Luminal subtype BC. RESULTS: Low expression levels of these three proteins were predominantly observed. BCAR1 expression was correlated with nuclear grade (p = 0.057), and BCAR3 expression was correlated with lymph node status (p = 0.011) and response to hormonal therapy (p = 0.021). Further, low expression of either BCAR1 or BCAR3 was significantly associated with poor prognosis (p = 0.005; p = 0.042). Pairwise analysis showed that patients with tumors with low BCAR1/low BCAR3 expression had a poorer overall survival (p = 0.013), and the low BCAR3 expression had the worst prognosis with RET high expression stratifying these patients into two different groups. Regarding the response to hormonal therapy, non-responder patients presented lower expression of RET in comparison to the responder group (p = 0.035). Additionally, the low BCAR1 expression patients had poorer outcomes than BCAR1 high (p = 0.015). CONCLUSION: Our findings suggest RET, BCAR1, and BCAR3 as potential candidate markers for endocrine therapy resistance in Luminal BC.

RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.

RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinoma.

OBJECTIVES: Increased expression of REarranged during Transfection (RET) kinase is reported in 10-20 % of lung adenocarcinomas (LUAD) and is associated with metastasis and reduced survival. Ezrin is a scaffold protein that promotes protein interactions with the actin cytoskeleton to regulate cell migration and is also associated with invasion and metastasis in cancers. RET isoforms interact with unique combinations of scaffold proteins to promote distinct signaling pathways. We hypothesized that RET isoforms associate distinctly with Ezrin for cytoskeletal reorganization and LUAD cell migration processes. METHODS: HCC1833 and A549 LUAD, SH-SY5Y neuroblastoma or HEK-293 cells expressing RET and Ezrin were stimulated with the RET ligand glial cell line-derived neurotrophic factor (GDNF) and treated with RET, Ezrin or Src inhibitors. Co-immunoprecipitation or pull-down assays coupled to immunoblotting were used to investigate protein activation and interactions. Immunofluorescence confocal microscopy assessed LUAD cytoskeletal reorganization and colocalization of RET and Ezrin. Live-cell fluorescence imaging was used to measure cell migration and chemotaxis. RESULTS: GDNF promoted activation, interaction and colocalization of RET51 isoform and Ezrin. Inhibition of RET or Src impaired Ezrin interactions with RET and Src. GDNF stimulation enhanced the formation of actin-rich filopodia, in which both RET and Ezrin were enriched, and promoted chemotaxis in LUAD cells. However, inhibition of RET, Src or Ezrin suppressed filopodia formation, reduced colocalization of Ezrin with RET, and impaired cell migration and/ or chemotaxis. We further showed that GDNF-mediated activation of RET and Ezrin promoted RhoA-GTPase activity and signaling of ROCK1 and ROCK2 in LUAD cells. CONCLUSIONS: Expression and activation of RET51 mediates unique protein interactions with Ezrin to promote LUAD cell chemotaxis for cancer cell dissemination, which may have implications in LUAD metastatic progression.

A gain-of-functional screen identifies the Hippo pathway as a central mediator of receptor tyrosine kinases during tumorigenesis.

The Hippo pathway has emerged as a key signaling pathway that regulates various biological functions. Dysregulation of the Hippo pathway has been implicated in a broad range of human cancer types. While a number of stimuli affecting the Hippo pathway have been reported, its upstream kinase and extracellular regulators remain largely unknown. Here we performed the first comprehensive gain-of-functional screen for receptor tyrosine kinases (RTKs) regulating the Hippo pathway using an RTK overexpression library and a Hippo signaling activity biosensor. Surprisingly, we found that the majority of RTKs could regulate the Hippo signaling activity. We further characterized several of these novel relationships [TAM family members (TYRO3, AXL, METRK), RET, and FGFR family members (FGFR1 and FGFR2)] and found that the Hippo effectors YAP/TAZ are central mediators of the tumorigenic phenotypes (e.g., increased cell proliferation, transformation, increased cell motility, and angiogenesis) induced by these RTKs and their extracellular ligands (Gas6, GDNF, and FGF) through either PI3K or MAPK signaling pathway. Significantly, we identify FGFR, RET, and MERTK as the first RTKs that can directly interact with and phosphorylate YAP/TAZ at multiple tyrosine residues independent of upstream Hippo signaling, thereby activating their functions in tumorigenesis. In conclusion, we have identified several novel kinases and extracellular stimuli regulating the Hippo pathway. Our findings also highlight the pivotal role of the Hippo pathway in mediating Gas6/GDNF/FGF-TAM/RET/FGFR-MAPK/PI3K signaling during tumorigenesis and provide a compelling rationale for targeting YAP/TAZ in RTK-driven cancers.

GGA3-mediated recycling of the RET receptor tyrosine kinase contributes to cell migration and invasion.

The RET receptor tyrosine kinase plays important roles in regulating cellular proliferation, migration, and survival in the normal development of neural crest derived tissues. However, aberrant activation of RET, through oncogenic mutations or overexpression, can contribute to tumourigenesis, regional invasion, and metastasis of several human cancers. RET is expressed as two main isoforms with unique C-terminal sequences that differ in protein interactions and subcellular trafficking in response to RET activation, and which also have distinct oncogenic potentials. The long isoform, termed RET51, is internalized from the membrane in response to stimulation by its ligand, GDNF, but is known to recycle back to the surface via RAB11 endosomes. However, the mechanisms regulating this process and its cellular effects have not been defined. Here, we show that recycling of RET51 requires a multicomponent complex that includes the endosomal-sorting protein GGA3, which mediates GDNF-dependent slow recycling of RET51 receptors to the plasma membrane. Our data show that the GRB2 adapter associates with RET51 through interactions with its C-terminal sequences, facilitating recruitment of active ARF6 and GGA3 interaction, and that depletion of GGA3 or ARF6 reduced RET51 recycling. Further, GGA3 knockdown accelerated RET51 degradation and also attenuated RET-mediated AKT activation. Finally, we showed that recycling of RET51 to the cell surface through association with GGA3 and ARF6 contributes to RET51-dependent cell motility, migration, and invasion. Our data establish RET recycling as a mechanism coordinating location and duration of RET signals in order to direct cell movement and invasion.

65 YEARS OF THE DOUBLE HELIX: Exploiting insights on the RET receptor for personalized cancer medicine.

The focus of precision cancer medicine is the use of patient genetic signatures to predict disease occurrence and course and tailor approaches to individualized treatment to improve patient outcomes. The rearranged during transfection (RET) receptor tyrosine kinase represents a paradigm for the power of personalized cancer management to change cancer impact and improve quality of life. Oncogenic activation of RET occurs through several mechanisms including activating mutations and increased or aberrant expression. Activating RET mutations found in the inherited cancer syndrome multiple endocrine neoplasia 2 permit early diagnosis, predict disease course and guide disease management to optimize patient survival. Rearrangements of RET found in thyroid and lung tumors provide insights on potential disease aggressiveness and offer opportunities for RET-targeted therapy. Aberrant RET expression in a subset of cases is associated with tumor dissemination, resistance to therapies and/or poorer prognosis in multiple cancers. The potential of RET targeting through repurposing of small-molecule multikinase inhibitors, selective RET inhibitors or other novel approaches provides exciting opportunities to individualize therapies across multiple pathologies where RET oncogenicity contributes to cancer outcomes.

GDNF and the RET Receptor in Cancer: New Insights and Therapeutic Potential.

The Glial cell line-derived neurotrophic Family Ligands (GFL) are soluble neurotrophic factors that are required for development of multiple human tissues, but which are also important contributors to human cancers. GFL signaling occurs through the transmembrane RET receptor tyrosine kinase, a well-characterized oncogene. GFL-independent RET activation, through rearrangement or point mutations occurs in thyroid and lung cancers. However, GFL-mediated activation of wildtype RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET and GFL expression have been implicated in metastasis or invasion in diverse human cancers including breast, pancreatic, and prostate tumors, where they are linked to poorer patient prognosis. In addition to directly inducing tumor growth in these diseases, GFL-RET signaling promotes changes in the tumor microenvironment that alter the surrounding stroma and cellular composition to enhance tumor invasion and metastasis. As such, GFL RET signaling is an important target for novel therapeutic approaches to limit tumor growth and spread and improve disease outcomes.

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Differential roles of RET isoforms in medullary and papillary thyroid carcinomas.

The RET receptor tyrosine kinase mediates cell proliferation, survival and migration in embryogenesis and is implicated in the transformation and tumour progression in multiple cancers. RET is frequently mutated and constitutively activated in familial and sporadic thyroid carcinomas. As a result of alternative splicing, RET is expressed as two protein isoforms, RET9 and RET51, which differ in their unique C-terminal amino acids. These isoforms have distinct intracellular trafficking and associated signalling complexes, but functional differences are not well defined. We used shRNA-mediated knockdown (KD) of individual RET isoforms or of total RET to evaluate their functional contributions in thyroid carcinoma cells. We showed that RET is required for cell survival in medullary (MTC) but not papillary thyroid carcinoma (PTC) cells. In PTC cells, RET depletion reduced cell migration and induced a flattened epithelial-like morphology. RET KD decreased the expression of mesenchymal markers and matrix metalloproteinases and reduced anoikis resistance and invasive potential. Further, we showed that RET51 depletion had significantly greater effects on each of these processes than RET9 depletion in both MTC and PTC cells. Finally, we showed that expression of RET, particularly RET51, was correlated with malignancy in a panel of human thyroid tumour tissues. Together, our data show that RET expression promotes a more mesenchymal phenotype with reduced cell-cell adhesion and increased invasiveness in PTC cell models, but is more important for tumour cell survival, proliferation and anoikis resistance in MTC models. Our data suggest that the RET51 isoform plays a more prominent role in mediating these processes compared to RET9.

Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 µ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

ATM gene mutations in sporadic breast cancer patients from Brazil.

PURPOSE: The Ataxia-telangiectasia mutated (ATM) gene encodes a multifunctional kinase, which is linked to important cellular functions. Women heterozygous for ATM mutations have an estimated relative risk of developing breast cancer of 3.8. However, the pattern of ATM mutations and their role in breast cancer etiology has been controversial and remains unclear. In the present study, we investigated the frequency and spectrum of ATM mutations in a series of sporadic breast cancers and controls from the Brazilian population. METHODS: Using PCR-Single Strand Conformation Polymorphism (SSCP) analysis and direct DNA sequencing, we screened a panel of 100 consecutive, unselected sporadic breast tumors and 100 matched controls for all 62 coding exons and flanking introns of the ATM gene. RESULTS: Several polymorphisms were detected in 12 of the 62 coding exons of the ATM gene. These polymorphisms were observed in both breast cancer patients and the control population. In addition, evidence of potential ATM mutations was observed in 7 of the 100 breast cancer cases analyzed. These potential mutations included six missense variants found in exon 13 (p.L546V), exon 14 (p.P604S), exon 20 (p.T935R), exon 42 (p.G2023R), exon 49 (p.L2307F), and exon 50 (p.L2332P) and one nonsense mutation in exon 39 (p.R1882X), which was predicted to generate a truncated protein. CONCLUSIONS: Our results corroborate the hypothesis that sporadic breast tumors may occur in carriers of low penetrance ATM mutant alleles and these mutations confer different levels of breast cancer risk.