FOXK2 amplification promotes breast cancer development and chemoresistance.

Oncogene activation through DNA amplification or overexpression is a crucial driver of cancer initiation and progression. The FOXK2 gene, located on chromosome 17q25, encodes a transcription factor with a forkhead DNA-binding domain. Analysis of genomic datasets reveals that FOXK2 is frequently amplified and overexpressed in breast cancer, correlating with poor patient survival. Knockdown of FOXK2 significantly inhibited breast cancer cell proliferation, migration, anchorage-independent growth, and delayed tumor growth in a xenograft mouse model. Additionally, inhibiting FOXK2 sensitized breast cancer cells to chemotherapy. Co-overexpression of FOXK2 and mutant PI3KCA transformed non-tumorigenic MCF-10A cells, suggesting a role for FOXK2 in PI3KCA-driven tumorigenesis. CCNE2, PDK1, and ESR1 were identified as transcriptional targets of FOXK2 in MCF-7 cells. Small-molecule inhibitors of CCNE2/CDK2 (dinaciclib) and PDK1 (dichloroacetate) exhibited synergistic anti-tumor effects with PI3KCA inhibitor (alpelisib) in vitro. Inhibition of FOXK2 by dinaciclib synergistically enhanced the anti-tumor effects of alpelisib in a xenograft mouse model. Collectively, these findings highlight the oncogenic function of FOXK2 and suggest that FOXK2 and its downstream genes represent potential therapeutic targets in breast cancer.

Diagnosis Framework for Probable Alzheimer's Disease and Mild Cognitive Impairment Based on Multi-Dimensional Emotion Features.

BACKGROUND: Emotion and cognition are intercorrelated. Impaired emotion is common in populations with Alzheimer's disease (AD) and mild cognitive impairment (MCI), showing promises as an early detection approach. OBJECTIVE: We aim to develop a novel automatic classification tool based on emotion features and machine learning. METHODS: Older adults aged 60 years or over were recruited among residents in the long-term care facilities and the community. Participants included healthy control participants with normal cognition (HC, n = 26), patients with MCI (n = 23), and patients with probable AD (n = 30). Participants watched emotional film clips while multi-dimensional emotion data were collected, including mental features of Self-Assessment Manikin (SAM), physiological features of electrodermal activity (EDA), and facial expressions. Emotional features of EDA and facial expression were abstracted by using continuous decomposition analysis and EomNet, respectively. Bidirectional long short-term memory (Bi-LSTM) was used to train classification model. Hybrid fusion was used, including early feature fusion and late decision fusion. Data from 79 participants were utilized into deep machine learning analysis and hybrid fusion method. RESULTS: By combining multiple emotion features, the model's performance of AUC value was highest in classification between HC and probable AD (AUC = 0.92), intermediate between MCI and probable AD (AUC = 0.88), and lowest between HC and MCI (AUC = 0.82). CONCLUSIONS: Our method demonstrated an excellent predictive power to differentiate HC/MCI/AD by fusion of multiple emotion features. The proposed model provides a cost-effective and automated method that can assist in detecting probable AD and MCI from normal aging.

Influence of Geometric Characteristics on Water Flow and Solute Transport at Fracture Intersections.

Laboratory experiments and numerical simulations were performed to explore the influence of intersection geometry on fluid flow and solute transport in fractures. Fractures were engraved and sealed into an acrylic plate and two orthogonal intersections with different geometry were constructed. The effects of curvature and relative shear displacement at intersections on preferential flow and solute transport were investigated. By solving the Navier-Stokes (NS) equation, the fluid mixing and solute distribution were predicted. The results showed that the geometric characteristics at the intersection have a significant effect on the preferential flow and solute distribution. The results agreed well with the experimental results, in terms of flow direction, preferential flow rate, and heterogeneous solute distribution. With an increase in curvature, the flow difference between the two outlets increases gradually. Increasing curvature can reduce the preferential flow and weaken the inhomogeneity of solute distribution. An increase of relative shear displacement decreases the pressure gradient and flow rate at the entrance of the two branch fractures, and thereby increases preferential flow and inhomogeneity of solute distribution. The results provide a basis and reference for further exploring the relationship between the geometric characteristics of fracture intersections and flow behaviors.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

FOXK2 amplification and overexpression promotes breast cancer development and chemoresistance.

Activation of oncogenes through DNA amplification/overexpression plays an important role in cancer initiation and progression. Chromosome 17 has many cancer-associated genetic anomalies. This cytogenetic anomaly is strongly associated with poor prognosis of breast cancer. FOXK2 gene is located on 17q25 and encodes a transcriptional factor with a forkhead DNA binding domain. By integrative analysis of public genomic datasets of breast cancers, we found that FOXK2 is frequently amplified and overexpressed in breast cancers. FOXK2 overexpression in breast cancer patients is associated with poor overall survival. FOXK2 knockdown significantly inhibits cell proliferation, invasion and metastasis, and anchorage-independent growth, as well as causes G0/G1 cell cycle arrest in breast cancer cells. Moreover, inhibition of FOXK2 expression sensitizes breast cancer cells to frontline anti-tumor chemotherapies. More importantly, co-overexpression of FOXK2 and PI3KCA with oncogenic mutations (E545K or H1047R) induces cellular transformation in non-tumorigenic MCF10A cells, suggesting that FOXK2 is an oncogene in breast cancer and is involved in PI3KCA-driven tumorigenesis. Our study identified CCNE2, PDK1, and Estrogen receptor alpha (ESR1) as direct transcriptional targets of FOXK2 in MCF-7 cells. Blocking CCNE2- and PDK1-mediated signaling by using small molecule inhibitors has synergistic anti-tumor effects in breast cancer cells. Furthermore, FOXK2 inhibition by gene knockdown or inhibitors for its transcriptional targets (CCNE2 and PDK1) in combination with PI3KCA inhibitor, Alpelisib, showed synergistic anti-tumor effects on breast cancer cells with PI3KCA oncogenic mutations. In summary, we provide compelling evidence that FOXK2 plays an oncogenic role in breast tumorigenesis and targeting FOXK2-mediated pathways may be a potential therapeutic strategy in breast cancer.

Water optical properties over or around coral reefs in the coastal region: implications for coral reef remote sensing.

Coral reefs inhabit clear oceanic-type waters to turbid coastal regimes. For shallow coastal water environments, the coral reefs from remote sensing will be substantially influenced by the complexity and heterogeneity of the optical properties of water. Through a set of bio-optical and chemical measurements in July 2019 around Xidao Island, Sanya, China, we explored spatial variations of water components' concentrations (including suspended solids, phytoplankton, and dissolved organic carbon) and the optical absorptions (i.e., particulate pigments, detritus, and colored dissolved organic matter) in waters over or around coral reefs; further analysis of their influences upon the remote sensing reflectance (Rrs(λ)) of water was performed. It was observed that the bio-optical and chemical properties of the waters over or around coral reefs were spatially and vertically heterogeneous for different sampling sites. As expected, the suspended solids dominated the optical properties of coral reefs waters in areas of Xidao Island, which evidently influenced the Rrs(λ), especially for the surface waters (Pearson r > 0.60, p < 0.01). In addition, the dissolved organic carbon concentrations exhibited significant relations to the Rrs(λ) both in surface and bottom water layers, whereas the colored dissolved organic matter showed a weak negative correlation with the Rrs(λ). These findings will support the mapping and monitoring benthic habitats with remote sensing imagery in coastal regions, especially when a removal of the influence by particulate sediments was available.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

A robust performance evaluation method based on interval evidential reasoning approach under uncertainty.

Performance evaluation (PE) is an important part of equipment health management. If the monitoring information of the equipment is interfered, the evaluation results may be erroneous. A robust performance evaluation (RPE) method is proposed to solve this problem. The performance evaluation results are obtained by distinguishing the cases of single evidence with interference and two evidence with interference, and a robustness measurement based on interval similarity is proposed. To improve the accuracy of the evaluation results, the referential values in the IER evaluation model are optimized. The robustness thresholds of the input indexes are obtained under the satisfaction of the robustness constraints. If the interference value of the input index is within the thresholds, the deviation between the evaluation results using monitoring information with interference and those using monitoring information without interference is small. Finally, the proposed method is applied to a type of electric servo mechanism performance evaluation, and the case shows the validity of the RPE method.

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

A New Evidential Reasoning Rule Considering Interval Uncertainty and Perturbation.

The evidential reasoning (ER) rule has been widely applied in the multiple attribute decision making (MADM), which makes the decision-making process transparent and credible by using a belief structure. To improve the ability of the ER rule in dealing with the interval uncertainty, a new interval ER (IER) rule is proposed in this article. The interval uncertainty is described as the interval grade in the new frame of discernment (FoD) to model the local ignorance. It is proved that the IER rule is a generalization of the ER rule. To study the influence of perturbation on the IER rule, the perturbation is first introduced to the belief structure, and the perturbation analysis (PA) is conducted for the IER rule. An optimization model is established to estimate the perturbation threshold, which can measure the effectiveness of the inference result under perturbation. Two numerical examples and a case study are carried out, respectively, to show the implementation process of the proposed IER rule and validate its effectiveness in different decision-making scenarios.

LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

The Association between Spicy Food Consumption and Psychological Health in Chinese College Students: A Cross-Sectional Study.

BACKGROUND: Capsaicin is the main active ingredient in chili peppers and spicy food. Animal studies provide contradictory results on the role of capsaicin in psychiatric disorders. There are no epidemiological studies to investigate the relationship between spicy food consumption and psychological health. METHODS: A cross-sectional online survey was conducted. Psychological health was assessed with the Depression Anxiety Stress Scale, and spicy food consumption was assessed as frequency, strength, and duration of consumption. Multivariable logistic regression was conducted to determine the associations between spicy food consumption and psychological symptoms. RESULTS: Our sample comprised 1771 participants (male = 674, mean age = 21.97 years). The odds of having depressive, anxiety, and stress symptoms were 34.0%, 46.5%, and 19.1% in Chinese college students, respectively. After adjusting for a series of covariates, compared with non-consumers, the odds ratios (ORs) of depressive symptoms across spicy food consumption were 1.13 (95% CI: 0.87-1.46) for 1-2 days/week and 1.38 (95% CI: 1.02-1.86) for ≥3 days/week. With regard to anxiety symptoms, the ORs were 0.99 (95% CI: 0.78, 1.27) for 1-2 days/week and 1.50 (95% CI: 1.13-1.99) for ≥3 days/week. For stress symptoms, the ORs were 0.90 (95% CI: 0.66-1.23) for 1-2 days/week and 1.27 (95% CI: 0.89-1.80) for ≥3 days/week. The ORs for the depressive symptoms associated with different intensities of spicy food consumption were 1.00 (reference) for the reference group (non-consumers), 1.17 (95% CI: 0.90-1.52) for eating weakly spicy food, and 1.34 (95% CI: 1.01-1.78) for moderately to strongly spicy food. CONCLUSION: The findings suggested a positive association between frequently spicy food consumption and depressive/anxiety symptoms in adolescents, and no such association was found for stress symptoms.

Identification of origin and runoff of karst groundwater in the glacial lake area of the Jinsha River fault zone, China.

Karst groundwater plays important roles as a water supply and in sustaining the biodiversity and ecosystems of the eastern Qinghai-Xizang Plateau. Owing to the stratigraphic structure, high tectonic activity, and changeable climate of the region, the recharge source, runoff path, and dynamic characteristics of karst groundwater are highly complex, which poses challenges with regard to the protection of water resources and ecology. This study identified the origin and flow processes of karst groundwater in the glacial lake area of the Jinsha River fault zone using satellite remote sensing, hydrochemical and isotope analyses, and flow measurements. Results showed that active faults control the distribution of glacial lakes and the recharge, runoff, and discharge of karst groundwater. Glacial lake water is an important source of karst groundwater in the Jinsha River fault zone area. Specifically, glacial lake water continuously recharges the karst system via faults, fractures, and karst conduits, thereby maintaining the relative stability of karst spring flows. Through hierarchical cluster analysis, two main runoff conduits of karst water were distinguished: one along the Dingqu Fault and the other along the Eastern Zhairulong Fault, which together account for 59% of the total regional karst groundwater flow. The elevation difference between the recharge and discharge areas of the main karst springs is > 1000 m. Groundwater runoff is fast and residence time in the aquifer is short. The dissolution of calcite and dolomite mainly occurs during transit through the groundwater system, and cation exchange is weak. Therefore, the regional karst springs are predominantly HCO3-Ca·Mg type. To protect regional karst water resources and ecology, the monitoring and protection of glacial lakes should be strengthened.

Integrative Proteomic Characterization of Human Lung Adenocarcinoma.

Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90ß as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.

ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.

Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.

A time-resolved multi-omic atlas of the developing mouse liver.

Liver organogenesis and development are composed of a series of complex, well-orchestrated events. Identifying key factors and pathways governing liver development will help elucidate the physiological and pathological processes including those of cancer. We conducted multidimensional omics measurements including protein, mRNA, and transcription factor (TF) DNA-binding activity for mouse liver tissues collected from embryonic day 12.5 (E12.5) to postnatal week 8 (W8), encompassing major developmental stages. These data sets reveal dynamic changes of core liver functions and canonical signaling pathways governing development at both mRNA and protein levels. The TF DNA-binding activity data set highlights the importance of TF activity in early embryonic development. A comparison between mouse liver development and human hepatocellular carcinoma (HCC) proteomic profiles reveal that more aggressive tumors are characterized with the activation of early embryonic development pathways, whereas less aggressive ones maintain liver function-related pathways that are elevated in the mature liver. This work offers a panoramic view of mouse liver development and provides a rich resource to explore in-depth functional characterization.

CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions.

DNA damage response (DDR) is critically important for cell survival, genome maintenance, and its defect has been exploited therapeutically in cancer treatment. Many DDR-targeting agents have been generated and have entered the clinic and/or clinical trials. In order to provide a global and unbiased view of DDR network, we designed a focused CRISPR library targeting 365 DDR genes and performed CRISPR screens on the responses to several DDR inhibitors and DNA-damaging agents in 293A cells. With these screens, we determined responsive pathways enriched under treatment with different types of small-molecule agents. Additionally, we showed that POLE3/4-deficient cells displayed enhanced sensitivity to an ATR inhibitor, a PARP inhibitor, and camptothecin. Moreover, by performing DDR screens in isogenic TP53 wild-type and TP53 knock-out cell lines, our results suggest that the performance of our CRISPR DDR dropout screens is independent of TP53 status. Collectively, our findings indicate that CRISPR DDR screens can be used to identify potential targets of small-molecule drugs and reveal that TP53 status does not affect the outcome of these screens.

A Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor Receptor Proteome.

Ligand binding to the cell surface receptors initiates signaling cascades that are commonly transduced through a protein-protein interaction (PPI) network to activate a plethora of response pathways. However, tools to capture the membrane PPI network are lacking. Here, we describe a cross-linking-aided mass spectrometry workflow for isolation and identification of signal-dependent epidermal growth factor receptor (EGFR) proteome. We performed protein cross-linking in cell culture at various time points following EGF treatment, followed by immunoprecipitation of endogenous EGFR and analysis of the associated proteins by quantitative mass spectrometry. We identified 140 proteins with high confidence during a 2 h time course by data-dependent acquisition and further validated the results by parallel reaction monitoring. A large proportion of proteins in the EGFR proteome function in endocytosis and intracellular protein transport. The EGFR proteome was highly dynamic with distinct temporal behavior; 10 proteins that appeared in all time points constitute the core proteome. Functional characterization showed that loss of the FYVE domain-containing proteins altered the EGFR intracellular distribution but had a minor effect on EGFR proteome or signaling. Thus, our results suggest that the EGFR proteome include functional regulators that influence EGFR signaling and bystanders that are captured as the components of endocytic vesicles. The high-resolution spatiotemporal information of these molecules facilitates the delineation of many pathways that could determine the strength and duration of the signaling, as well as the location and destination of the receptor.