Data from Functional Analysis of <i>MET</i> Exon 14 Skipping Alteration in Cancer Invasion and Metastatic Dissemination
Feng WangYang LiuWanglong QiuElaine ShumMonica FengDejian ZhaoDeyou ZhengAlain BorczukHaiying ChengBalázs Halmos
0
Citation
47
Reference
10
Related Paper
Abstract:
<div>Abstract<p><i>MET</i> exon 14 skipping alteration (<i>METΔ14Ex</i>) is an actionable oncogenic driver that occurs in 2% to 4% of non–small cell lung cancer (NSCLC) cases. The precise role of <i>METΔ14Ex</i> in tumor progression of NSCLC is poorly understood. Using multiple isogenic <i>METΔ14Ex</i> cell models established with CRISPR editing, we demonstrate that METΔ14Ex expression increases receptor kinase activity and downstream signaling by impairing receptor internalization and endocytic degradation, significantly boosting cell scatter, migration, and invasion capacity <i>in vitro</i> as well as metastasis <i>in vivo</i>. RNA sequencing analysis revealed that <i>METΔ14Ex</i> preferentially activates biological processes associated with cell movement, providing novel insights into its unique molecular mechanism of action. Activation of PI3K/Akt/Rac1 signaling and upregulation of multiple matrix metallopeptidases (MMP) by METΔ14Ex induced cytoskeleton remodeling and extracellular matrix disassembly, which are critical functional pathways that facilitate cell invasion and metastasis. Therapeutically, MET inhibitors dramatically repressed <i>METΔ14Ex</i>-mediated tumor growth and metastasis <i>in vivo</i>, indicating potential therapeutic options for <i>METΔ14Ex</i>-altered NSCLC patients. These mechanistic insights into <i>METΔ14Ex</i>-mediated invasion and metastasis provide a deeper understanding of the role of <i>METΔ14Ex</i> in NSCLC.</p>Significance:<p>These findings reveal the mechanistic function of <i>METΔ14Ex</i> alteration in driving metastasis and define novel metastasis-related pathways that could be targeted for more effective treatment of lung cancer with <i>METΔ14Ex</i> alterations.</p></div>Keywords:
Internalization
Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.
Internalization
Horseradish peroxidase
Cite
Citations (12)
Abstract While the physiological function and mechanisms of agonist‐dependent G protein‐coupled receptor (GPCR) internalization have been extensively studied, the functional characterization of constitutive internalization of these critically important receptors has received less attention. Here we relate the constitutive internalization of more than 30 therapeutically targeted GPCRs to their agonist‐induced internalization. The constitutive internalization ranges from levels of bulk membrane endocytosis in some cases to levels of agonist‐induced internalization for other receptors. Moreover, for receptors with high constitutive internalization this occludes further agonist‐induced internalization. Additionally, Gq‐coupled GPCRs show a significantly higher rate of constitutive internalization than Gs‐ and Gi‐coupled receptors. Finally, we consolidate the proposed link between the constitutive internalization, as assessed by a cytometry‐based assay, and the constitutive activity of these receptors, as previously reported by a β‐arrestin recruitment assay across the range of pharmacologically relevant receptors. In summary, we provide a quantitative comparison of GPCR internalization across a range of pharmacologically relevant receptors providing generalized insight into the relations between constitutive internalization, constitutive activity and agonist‐induced internalization, which has so far relied on mutational studies in individual receptors.
Internalization
Cite
Citations (9)
Expression of truncated G-CSFR forms in patients with SCN/AML induces hyperproliferation and prolonged cell survival. Previously, we showed that ligand internalization is delayed and degradation of truncated G-CSFR forms is defective in patients with SCN/AML.In this study, we investigated the potential roles of dileucine and tyrosine-based motifs within the cytoplasmic domain of the G-CSFR in modulating ligand/receptor internalization. Using standard binding assays with radiolabeled ligand and COS-7 cells, substitutions in the dileucine motif or deletion of tyrosine residues in the G-CSFR did not alter internalization. Attachment of the transferrin receptor YTRF internalization motif to a truncated G-CSFR form from a patient with SCN/AML corrected defective internalization, but not receptor degradation suggesting that receptor internalization and degradation occur independently via distinct domains and/or processes.Our data suggest that distinct domains within the G-CSFR mediate separate processes for receptor internalization and degradation. Our findings using standard binding assays differ from recently published data utilizing flow cytometry.
Internalization
Immunoprecipitation
Transferrin receptor
Cite
Citations (4)
Our data demonstrate that the uptake of surface Ia into an intracellular compartment of B lymphoma or normal spleen cells is limited to about 20% after 2 to 3 h. The extent of internalization does not vary with several types of stimulation, including LPS, phorbol esters, anti-Ig-plus phorbol ester-stimulated EL-4 T cell supernatant, and Con A supernatant. Resting and activated B cells had similar rates of internalization. The rate and extent of uptake of surface Ia molecules into an intracellular compartment was monitored quantitatively through the use of a mAb radiolabeled with 125I. The internalization of Ia molecules was compared to that of transferrin receptor, a receptor that undergoes rapid internalization and recycling and accumulates in a intracellular pool that can be trapped by monensin. The internalization of Ia was not affected by monensin, although its synthetic pathway is disturbed by this drug. The potential use of internalized Ia for formation of T cell-triggering complexes of Ia and Ag fragments is not ruled out by these data, but it appears unlikely that internalization provides the major mechanism permitting Ia interaction with Ag.
Internalization
Transferrin receptor
Compartment (ship)
Cite
Citations (8)
The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.
Internalization
Alanine
Mannose 6-phosphate
Cite
Citations (42)
Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here, we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.
Internalization
Cite
Citations (7)
Internalization
Cite
Citations (21)
Internalization
Cite
Citations (31)
Internalization of monoclonal antibody (MAb) conjugates is an important feature of tumour targeting, both with respect to the therapeutic action of substances coupled to the antibody and to retention of radionuclides. Problems of analysing internalization in vitro and in vivo, of manipulating internalization, and of evaluating the involvement of normal tissues are illustrated by recent experimental data and are discussed in the light of published evidence.
Internalization
Cite
Citations (14)
Antibody-drug conjugates (ADCs) have promising potential as an effective therapeutic agent for various cancer therapies. Antigen-mediated internalization induces the delivery of ADCs into cancer cells, resulting in activation of the attached cytotoxic agents. The internalization and internalization efficiency of ADC are critical for its anti-cancer efficacy. How to verify the internalization and the internalization efficiency of ADCs could be very useful to identify appropriate antibody candidates with internalization characteristics favorable for ADCs. This chapter describes the experiments for evaluating internalization and strategies to improve internalization efficiency of ADCs, which would benefit for the identification and development of next generation ADCs in the future.
Internalization
Conjugate
Cite
Citations (0)