Target For Anticancer Therapy Research Articles

Targeting MYC for the treatment of breast cancer: use of the novel MYC-GSPT1 degrader, GT19630.

Since MYC is one of the most frequently altered driver genes involved in cancer formation, it is a potential target for new anti-cancer therapies. Historically, however, MYC has proved difficult to target due to the absence of a suitable crevice for binding potential low molecular weight drugs. The aim of this study was to evaluate a novel molecular glue, dubbed GT19630, which degrades both MYC and GSPT1, for the treatment of breast cancer. The antiproliferative potential of GT19630 was evaluated in 14 breast cancer cell lines representing the main molecular subtypes of breast cancer. In addition, we also investigated the effects of GT19630 on apoptosis, cell cycle progression, cell migration, and degradation of the negative immune checkpoint protein, B7-H3. GT19630 inhibited cell proliferation, blocked cell cycle progression, promoted apoptosis, and decreased cell migration at low nanomolar concentrations in breast cancer cell lines. By contrast, previously described MYC inhibitors such as specific MYC-MAX antagonists affected these processes at micromolar concentrations. Consistent with the ability of MYC to promote immune evasion, we also found that GT19630 degraded the negative immune checkpoint inhibitor, B7-H3. We conclude that the novel molecular glue, GT19630, is a potent mediator of endpoints associated with cancer formation/progression. Its ability to degrade B7-H3 suggests that GT19630 may also promote host immunity against cancer. To progress GT19630 as a therapy for breast cancer, our finding should now be confirmed in an animal model system.

Complement regulators as novel targets for anti-cancer therapy: A comprehensive review.

Cancer remains a formidable global health challenge requiring the continued exploration of innovative therapeutic approaches. While traditional treatment strategies including surgery, chemotherapy, and radiation therapy have had some success, primarily in early-stage disease, the quest for more targeted, personalized, safer, and effective therapies remains an ongoing pursuit. Over the past decade, significant advances in the field of tumor immunology have dramatically shifted a focus towards immunotherapy, although the ability to harness and coopt the immune system to treat cancer is still just beginning to be realized. One important area that has yet to be fully explored is the complement system, an integral part of innate immunity that has gathered attention recently as a source of potential targets for anti-cancer therapy. The complement system has a complex and context dependent role in cancer biology in that it not only contributes to immune surveillance but also may promote tumor progression. Complement regulators, including CD46, CD55, CD59, and complement factor H, exercise defined control over complement activation, and have also been acknowledged for their role in the tumor microenvironment. This review explores the intricate role of complement regulators in cancer development and progression, examining their potential as therapeutic targets, current strategies, challenges, and the evolving landscape of clinical research.

Claudins as diagnostic tools and therapeutic targets-Glimpse of the horizon.

Claudins (CLDNs) play a crucial and indispensable role as fundamental components within the structure of tight junctions. Due to the distinct and unique distribution pattern exhibited by CLDNs in both normal and malignant tissues, these proteins have garnered significant attention as pivotal targets for systemic anti-cancer therapy and as noteworthy diagnostic markers. This review provides a comprehensive and detailed elucidation of the fundamental understanding surrounding CLDNs, their intricate expression patterns, the potential role they play in cancer diagnosis and therapeutic potentials; all encapsulated within a succinct summary of the cutting-edge advancements and the information derived from various clinical trials.

PET Imaging of Solid Tumors with a G-Quadruplex-Targeting 18F-Labeled Peptide Probe.

Positron emission tomography (PET) is a common imaging technique and can provide accurate information about the size, shape, and location of tumors. Recent evidence has shown that G-quadruplex structures (G4s) are identified in human oncogenes, and these special structures are recognized as diagnostic cancer markers and drug targets for anticancer therapies. Although a number of techniques for in vivo imaging of G4s have been developed, achieving sufficient sensitivity and selectivity in vivo remains challenging. Herein, we have engineered and developed a radiolabeled peptide probe [18F]AlF-NOTA-RHAU18 targeting mitochondrial DNA G4s for in vivo PET imaging. The results of the study indicate that this probe is able to visualize and detect solid tumors in living homozygous mice. In addition, the distribution of the probe in cancer cells was investigated using FITC-RHAU18. This work may offer new insights into the development of cancer diagnostic tools by targeting in vivo G4s.

I-motif formation in the promoter region of the B-MYB proto-oncogene.

Understanding the mechanisms of carcinogenesis is essential to combat cancer. The search for alternative targets for anticancer therapy has gained interest, particularly when focused on upstream pathways. This strategy is particularly relevant when the encoded target proteins are known - or believed - to be "undruggable", as has been reported for the B-MYB oncogene. This gene, which regulates survival and cell cycle regulation, is overexpressed in cancer and correlates with an unfavorable prognosis. In this study, we focused on the identification of the i-motif (iM) structures in the promoter region of B-MYB as a possible anticancer target, with a complete biophysical characterization and in cell formation assessment using iM-CUT&Tag. Additionally, the interaction of the iM structures with a library of small molecules was investigated.

Differential Response to Local Stimulator of Interferon Genes Agonist Administration in Tumors with Various Stimulator of Interferon Genes Statuses.

Background/Objectives: The stimulator of interferon genes (STING) is currently accepted as a relevant target for anti-cancer therapies. Besides encouraging results showing STING agonist-induced tumor growth inhibition, in some types of tumors the effect is less prominent. We hypothesized that higher STING levels in cancer cells and the possibility of its activation determine a greater anti-cancer response. As the local administration of STING agonists induces a systemic reaction, we emphasized the importance of host tumor-induced hematological disruption in the efficiency of the therapeutic response. Methods: We investigated the response to STING stimulation in murine cancer cell lines-melanoma (B16-F10) and breast carcinoma (4T1)-and murine normal cell lines: fibroblast cells (NIH/3T3), endothelial cells (H5V), and macrophages (J774A.1). We assessed STING agonist-induced tumor growth inhibition and the therapy's impact on the hematological system parameters and systemic cytokine release. Results: Our results underlined the improved therapeutic effect of STING activation in melanoma (B16-F10) over breast carcinoma (4T1) tumors. The outcomes reflected a high dysregulation of the hematological system in mice with developed 4T1 tumors, which may support persistent inflammation and impede STING-induced therapeutic effects. Moreover, among typical cytokines produced following STING activation, CCL2 fold change was the one that increased the most in the serum of B16-F10-bearing mice and differentiated the observed response to the STING agonist between investigated tumor models. Conclusions: The current study provides new evidence of the different responses to STING activation among two poorly immunogenic tumor models. The high abundance of STING in B16-F10 cells and the possibility of its activation is linked with improved therapeutic response in vivo compared to 4T1. The effect also seems to be connected with a less dysregulated hematological system in mice with B16-F10 tumors over mice with 4T1 tumors. This highlighted the need for general insight into tumor-induced local and systemic responses to the efficiency of the proposed therapy.

Advances and current concepts on Eph receptors and ephrins in upper digestive tract cancers.

Erythropoietin-producing hepatocellular (Eph) receptors comprise the largest group of surface receptors and are responsible for cellular signals. Eph/ephrin signaling has been identified to play a role in key cancer development and progression processes, especially in the upper gastrointestinal tract. The Eph/ephrin system has been described as a tumor suppressor in duodenal cancer, while in esophageal, gastric, hepatic, and pancreatic cancer, the system has been related to tumor progression. For their significant role in developing a wide range of malignancies, Eph receptors and their ligands have proven to be an important target for new anticancer therapies. In this review, we present an overview of the literature and highlight evidence supporting the role of the Eph/ephrin system in upper digestive tract cancers. In addition, we discuss molecular findings that represent promising therapeutic targets for these cancers.

TRIM25: A Global Player of Cell Death Pathways and Promising Target of Tumor-Sensitizing Therapies.

Therapy resistance still constitutes a common hurdle in the treatment of many human cancers and is a major reason for treatment failure and patient relapse, concomitantly with a dismal prognosis. In addition to "intrinsic resistance", e.g., acquired by random mutations, cancer cells typically escape from certain treatments ("acquired resistance") by a large variety of means, including suppression of apoptosis and other cell death pathways via upregulation of anti-apoptotic factors or through inhibition of tumor-suppressive proteins. Therefore, ideally, the tumor-cell-restricted induction of apoptosis is still considered a promising avenue for the development of novel, tumor (re)sensitizing therapies. A growing body of evidence has highlighted the multifaceted role of tripartite motif 25 (TRIM25) in controlling different aspects of tumorigenesis, including chemotherapeutic drug resistance. Accordingly, overexpression of TRIM25 is observed in many tumors and frequently correlates with a poor patient survival. In addition to its originally described function in antiviral innate immune response, TRIM25 can play critical yet context-dependent roles in apoptotic- and non-apoptotic-regulated cell death pathways, including pyroposis, necroptosis, ferroptosis, and autophagy. The review summarizes current knowledge of molecular mechanisms by which TRIM25 can interfere with different cell death modalities and thereby affect the success of currently used chemotherapeutics. A better understanding of the complex repertoire of cell death modulatory effects by TRIM25 is an essential prerequisite for validating TRIM25 as a potential target for future anticancer therapy to surmount the high failure rate of currently used chemotherapies.

Prognostic value of CD163+ macrophages in solid tumor malignancies: A scoping review.

Tumor-associated macrophages (TAMs) play crucial roles in development and progression of malignant diseases. Notably, CD163+ TAMs likely perform specific pro-tumorigenic functions, suggesting that this subset may serve as both prognostic biomarkers and targets for future anti-cancer therapy. We conducted a scoping review to map the current knowledge on the prognostic role of CD163+ TAMs in the five most lethal cancers worldwide: Lung, colorectal, gastric, liver, and breast cancer. For all cancer types, most studies showed that high tumoral presence of CD163+ cells was associated with poor patient outcome, and this association was more frequently observed when CD163+ cells were measured at the tumor periphery compared to more central parts of the tumor. These results support that CD163+ TAMs represent a biomarker of poor patient outcome across a variety of solid tumors, and highlight the relevance of further investigations of CD163+ TAMs as targets of future immunotherapies.

Unraveling the mechanism of microRNA-134 in colon cancer progression: Targeting KRAS and PIK3CA for cell cycle control and histone deacetylase regulation.

Colon cancer is the leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are key regulators of gene expression, often dysregulated in colon cancer. This study aims to elucidate the therapeutic role of miR-134-5p as a tumor suppressor miRNA in colon cancer cells. We analyzed miRNA expression profiles in primary and metastatic colon cancer cells. The clinical significance of miR-134-5p was evaluated using the TCGA database. Bioinformatics tools (HADDOCK) predicted miRNA-mRNA interactions and the molecular docking of miRNA-mRNA-AGO2 complexes. Luciferase reporter assays, cell proliferation, immunofluorescence, colony forming unit assays, and qRT-PCR analysis assessed miR-134-5p effects on KRAS, PIK3CA, and downstream signaling pathways in primary and metastatic colon cancer cells. miR-134-5p was downregulated in colon cancer cells. Bioinformatics analysis suggested KRAS, PIK3CA, EGFR, and HDAC5 as potential targets. HADDOCK analysis revealed strong binding affinity and structural stability between KRAS, PIK3CA, miR-134-5p, and AGO2. Gene-reporter assays confirmed miR-134-5p-mediated degradation of KRAS and PIK3CA. miR-134-5p transfection reduced KRAS and PI3K protein levels, suppressed EGFR/RTK signaling and its downstream targets, and inhibited HDAC expression, ultimately reducing colon cancer cell proliferation. The results of this study confirm that miR-134-5p acts as a potential tumor suppressor miRNA in colon cancer cells by inhibiting KRAS and PI3K expression through AGO2-mediated gene silencing. It deregulates downstream EGFR signaling and HDACs, thereby reducing colon cancer cell proliferation. These findings highlight miR-134-5p as a promising therapeutic target for miRNA-mediated anticancer therapy.

Distinctive roles of aquaporins and novel therapeutic opportunities against cancer.

Aquaporins (AQPs) are integral membrane proteins responsible for facilitating the transmembrane transport of water and small solutes. Their involvement in diverse physiological functions extends to pathological conditions, including cancer, positioning them as promising targets for anticancer therapy. Tumor cells, particularly those with high metastatic potential, exhibit elevated AQP expression, reinforcing their critical role in tumor biology. Emerging evidence highlights AQPs' involvement in key oncogenic processes such as cell migration, proliferation, and tumor-associated edema, suggesting their potential as novel therapeutic targets. Despite this, the development of selective and potent AQP inhibitors has proven challenging. Efforts to produce small-molecule AQP inhibitors have largely been unsuccessful. However, recent advancements include monoclonal human IgG antibodies targeting extracellular domains of aquaporin-4, offering new therapeutic strategies, particularly in glioblastoma, where AQP-4 is overexpressed. However, recent advancements include monoclonal human IgG antibodies targeting extracellular domains of aquaporin-4, offering new therapeutic strategies, particularly in glioblastoma, where AQP-4 is over expressed. These antibodies hold promise for selectively targeting and eradicating AQP-4-expressing cells in malignant brain tumors. This review discusses the critical role AQPs play in cancer, including their contributions to tumor cell proliferation, migration, angiogenesis, and edema formation. Additionally, we explore innovative therapeutic approaches, such as antibody-based interventions, and outline potential future research directions in AQP-targeted cancer therapies.

PLK1 overexpression suppresses homologous recombination and confers cellular sensitivity to PARP inhibition

The overexpression of Polo-like kinase 1 (PLK1) is associated with poor clinical outcomes in various malignancies, making it an attractive target for anticancer therapies. Although recent studies suggest PLK1’s involvement in homologous recombination (HR), the impact of its overexpression on HR remains unclear. In this study, we investigated the effect of PLK1 overexpression on HR using bioinformatics and experimental approaches. Analyzing The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) datasets with the Homologous Recombination Deficiency (HRD) score, we found a positive correlation between PLK1 expression and HRD score, indicating that increased PLK1 expression suppresses HR. To validate these findings, we performed cell line-based experiments, demonstrating that PLK1 overexpression attenuates RAD51 focus formation and HR, as measured by ASHRA in T47D cells. Since HR-deficient cells are hypersensitive to PARP inhibitors, we further confirmed that PLK1 overexpression increases sensitivity to PARP inhibitors, both in CCLE dataset analysis and experiments using T47D cells. Additionally, we found that the effects of PLK1 overexpression on HR suppression and increased PARP inhibitor sensitivity were mitigated by either a PLK1 kinase inhibitor or the kinase-dead mutant [T210A]. This suggests that PLK1’s impact on HR and PARP inhibitor sensitivity is mediated through its kinase activity. Moreover, analysis of clinical ovarian cancer samples revealed that higher PLK1 expression correlates with increased sensitivity to PARP inhibitors. Our results suggest that PLK1 overexpression suppresses homologous recombination, leading to enhanced sensitivity to PARP inhibition, presenting a potential therapeutic strategy for targeting cancers with overexpression of PLK1.

TLR4 Targeting: A Promising Therapeutic Approach Across Multiple Human Diseases.

TLR4 stands at the forefront of innate immune responses, recognizing various pathogen- associated molecular patterns and endogenous ligands, thus serving as a pivotal mediator in the immune system's defense against infections and tissue damage. Beyond its canonical role in infection, emerging evidence highlights TLR4's involvement in numerous non-infectious human diseases, ranging from metabolic disorders to neurodegenerative conditions and cancer. Targeting TLR4 signaling pathways presents a promising therapeutic approach with broad applicability across these diverse pathological states. In metabolic disorders such as obesity and diabetes, dysregulated TLR4 activation contributes to chronic low-grade inflammation and insulin resistance, driving disease progression. In cardiovascular diseases, TLR4 signaling promotes vascular inflammation and atherogenesis, implicating its potential as a therapeutic target to mitigate cardiovascular risk. Neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, exhibit aberrant TLR4 activation linked to neuroinflammation and neuronal damage, suggesting TLR4 modulation as a strategy to attenuate neurodegeneration. Additionally, in cancer, TLR4 signaling within the tumor microenvironment promotes tumor progression, metastasis, and immune evasion, underscoring its relevance as a target for anticancer therapy. Advances in understanding TLR4 signaling cascades and their contributions to disease pathogenesis have spurred the development of various pharmacological agents targeting TLR4. These agents range from small molecule inhibitors to monoclonal antibodies, with some undergoing preclinical and clinical evaluations. Furthermore, strategies involving TLR4 modulation through dietary interventions and microbiota manipulation offer additional avenues for therapeutic exploration. Hence, targeting TLR4 holds significant promise as a therapeutic strategy across a spectrum of human diseases, offering the potential to modulate inflammation, restore immune homeostasis, and impede disease progression.

A Phosphatidyl Conjugated Telomerase-Dependent Telomere-Targeting Nucleoside Demonstrates Colorectal Cancer Direct Killing and Immune Signaling.

Telomerase and telomeres are crucial in cancer cell immortalization, making them key targets for anticancer therapies. Currently, 6-thio-dG (THIO) combined with the anti-PD-1 inhibitor Cemiplimab is under phase II clinical investigation (NCT05208944) in NSCLC patients resistant to prior immunotherapies. This study presents the design, synthesis, and evaluation of novel bimodular conjugate molecules combining telomere-targeting nucleoside analogs and phosphatidyl diglyceride groups. Among them, dihexanoyl-phosphatidyl-THIO (diC6-THIO) showed high anticancer activity with sub-µM EC50 values in vitro across various cancer cell lines. In mouse colorectal cancer models, diC6-THIO demonstrated strong anticancer effects alone and in combination with PD1/PD-L1 inhibitors. Administration of this compound resulted in the efficient formation of Telomere dysfunction Induced Foci (TIFs) in vitro, indicating an on-target, telomerase-mediated telomere-modifying mechanism of action for the molecule. Systemic treatment also activated CD4+ and CD8+ T cells while reducing regulatory T cells, indicating immune system enhancement. Notably, diC6-THIO exhibits an improved solubility profile while maintaining comparable anticancer properties, further supporting its potential as a promising therapeutic candidate. These findings highlight diC6-THIO as a promising telomere-targeting prodrug with dual effects on telomere modification and immune activation.

Targeting EZH2 in Cancer: Mechanisms, Pathways, and Therapeutic Potential.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase involved in cell cycle regulation, cell differentiation, and cell death and plays a role in modulating the immune response. Although it mainly functions by catalyzing the tri-methylation of H3 histone on K27 (H3K27), to inhibit the transcription of target genes, EZH2 can directly methylate several transcription factors or form complexes with them, regulating their functions. EZH2 expression/activity is often dysregulated in cancer, contributing to carcinogenesis and immune escape, thereby representing an important target in anti-cancer therapy. This review summarizes some of the mechanisms through which EZH2 regulates the expression and function of tumor suppressor genes and oncogenic molecules such as STAT3, mutant p53, and c-Myc and how it modulates the anti-cancer immune response. The influence of posttranslational modifications on EZH2 activity and stability and the possible strategies leading to its inhibition are also reviewed.

THUMPD3 regulates alternative splicing of ECM transcripts in human lung cancer cells and promotes proliferation and migration.

RNA-modifying enzymes have recently garnered considerable attention due to their relevance in cancer biology, identifying them as potential targets for novel therapeutic intervention. THUMPD3 was recently identified as an RNA methyltransferase catalysing N2-methylguanosine (m2G) within certain tRNAs. In this study, we unveil a novel role for THUMPD3 in lung cancer cells. Depletion of the enzyme from lung cancer cells significantly impairs their fitness, negatively impacting key cellular processes such as proliferation and migration. Notably, exogenous expression of THUMPD3 in normal lung fibroblasts stimulates their proliferation rate. Additionally, transcriptome-wide analyses reveal that depletion of THUMPD3 from lung cancer cells induces substantial changes in the expression of cell surface proteins, including those comprising the extracellular matrix (ECM). We further demonstrate that THUMPD3 maintains expression of an extra-domain B (EDB) containing pro-tumour isoform of Fibronectin-1 mRNA, encoding FN1, an important ECM protein. Crucially, depletion of THUMPD3 promotes an alternative splicing event that removes the EDB-encoding exon from Fibronectin-1. This is consistent with THUMPD3 depletion reducing cellular proliferation and migration. Moreover, depletion of THUMPD3 selectively and preferentially affects the alternative splicing of ECM and cell adhesion molecule encoding transcripts, as well as those encoding neurodevelopmental proteins. Overall, these findings highlight THUMPD3 as an important player in regulating cancer-relevant alternative splicing and they provide a rationale for further investigations into THUMPD3 as a candidate target in anti-cancer therapy.

Suppression of microtubule acetylation mediates the anti-leukemic effect of CDK9 inhibition

Cyclin-dependent kinase 9 (CDK9) is a crucial component of transcription and potential target for anti-cancer therapies, particularly for hematological malignancies. However, the precise mechanisms underlying the therapeutic effects of CDK9 inhibitors remain not fully understood. Here, we found that inhibiting CDK9 either pharmacologically or through gene downregulation, significantly reduced the levels of α-tubulin protein in a time- and dose-dependent manner. We further discovered that CDK9 inhibition led to increased susceptibility of α-tubulin to proteasomal degradation due to reduced acetylation at lysine 40 (K40), an important modification for microtubule stability. An acetylation-mimicking mutant of α-tubulin mitigated the anti-tumor effects of CDK9 inhibition. Mechanically, we identified that CDK9 inhibition downregulated the expression of ATAT1, the acetyltransferase responsible for α-tubulin acetylation, further compromising microtubule stability. We also conducted in vivo studies in a leukemic xenograft model, where AZD4573 treatment led to significant tumor regression, decreased ATAT1 expression, and α-tubulin degradation. Our study unravels a novel molecular mechanism by which CDK9 inhibition disrupts α-tubulin stability and provides valuable insights for exploring effective treatment regimens involving CDK9 inhibitors.

Supervised Screening of EGFR Inhibitors Validated through Computational Structural Biology Approaches.

One of the prominent challenges in breast cancer (BC) treatment is human epidermal growth factor receptor (EGFR) overexpression, which facilitates tumor proliferation and presents a viable target for anticancer therapies. This study integrates multiomics data to pinpoint promising therapeutic compounds and employs a machine learning (ML)-based similarity search to identify effective alternatives. We used BC cell line data from the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) databases and single-cell RNA sequencing (scRNA-seq) information that established afatinib as an efficacious candidate demonstrating superior IC50 values. Next, ML models, including support vector machine (SVM), artificial neural networks (ANN), and random forest (RF), were trained on ChEMBL data to classify compounds with similar activity to the reference drug as active or inactive. The promising candidates underwent computational structural biology assessments for their molecular interactions and conformational dynamics. Our findings indicate that compounds ChEMBL233324, ChEMBL233325, ChEMBL234580, and ChEMBL372692 exhibit potent repressive action against EGFR, underscoring their potential as active antibreast cancer agents.

Exosomal miR-502-5p suppresses the progression of gastric cancer by repressing angiogenesis through the Wnt/β-catenin pathway.

Gastric cancer (GC) is a significant global health concern, ranking as the fifth most common cancer and the third leading cause of cancer-related deaths. The role of miR-502-5p in various cancers has been studied, but its specific impact on gastric cancer through exosomes is not well understood. This study aimed to investigate the role and mechanism of exosome-derived miR-502-5p in gastric cancer. Differential expression of miR-502-5p in tissues or serum of GC patients was determined using qRT-PCR. The impact of miR-502-5p on cell proliferation, migration, and invasion was assessed through in vitro and in vivo experiments. The potential of exosome-miR-502-5p to inhibit metastatic ability was also explored by using vivo and vitro assay. Furthermore, the underlying mechanism of miR-502-5p in gastric cancer was investigated using western blotting. It was found that miR-502-5p suppressed the proliferation, migration, and invasion of gastric cancer cells. Exosome-miR-502-5p expression was negatively linked to metastatic ability and demonstrated inhibition of metastasis in vitro and in vivo. Additionally, miR-502-5p appeared to inhibit angiogenesis through the Wnt/β-catenin pathway in gastric cancer. Exosomal miR-502-5p acts as a suppressor in the development and progression of gastric cancer, suggesting its potential as a target for anti-cancer therapy or as a diagnostic biomarker.

Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography.

Atomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains ∼3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.