EH domain-containing protein 2 (EHD2): Overview, biological function, and therapeutic potential.

EH domain-containing protein 2 (EHD2) is a member of the EHD protein family and is mainly located in the plasma membrane, but can also be found in the cytoplasm and endosomes. EHD2 is also a nuclear-cytoplasmic shuttle protein. After entering the cell nuclear, EHD2 acts as a corepressor of transcription to inhibit gene transcription. EHD2 regulates a series of biological processes. As a key regulator of endocytic transport, EHD2 is involved in the formation and maintenance of endosomal tubules and vesicles, which are critical for the intracellular transport of proteins and other substances. The N-terminal of EHD2 is attached to the cell membrane, while its C-terminal binds to the actin-binding protein. After binding, EHD2 connects with the actin cytoskeleton, forming the curvature of the membrane and promoting cell endocytosis. EHD2 is also associated with membrane protein trafficking and receptor signaling, as well as in glucose metabolism and lipid metabolism. In this review, we highlight the recent advances in the function of EHD2 in various cellular processes and its potential implications in human diseases such as cancer and metabolic disease. We also discussed the prospects for the future of EHD2. EHD2 has a broad prospect as a therapeutic target for a variety of diseases. Further research is needed to explore its mechanism, which could pave the way for the development of targeted treatments.

EHD2, a novel HIF target gene, is a promising biomarker in clear cell renal cell carcinoma.

OBJECTIVES: The aim of the present study was to determine the clinical value of a novel hypoxia-inducible factor (HIF) target EH domain-containing protein 2 (EHD2) for predicting the outcome of patients with clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: GEPIA public database was searched to determine a possible association between HIF2Α and EHD protein family members, and kidney renal clear cell carcinoma data were used to find the expression profile of EHD proteins in ccRCC samples. A tissue microarray from 70 ccRCC samples was used for immunohistochemical analysis to determine the specific expression pattern of EHD2 in ccRCC samples. In addition, univariate and multivariate analyses were performed to assess the utility of EHD2 as an independent prognostic factor for ccRCC. RESULTS: EHD protein family members were all found to be significantly correlated with HIF2Α expression in ccRCC. However, EHD2 was the only protein that was observed to be overexpressed in ccRCC cancer tissues compared with normal tissues. EHD2 and HIF2Α mRNA expression levels were found to be higher in cancer tissues compared with those in adjacent normal tissue according to reverse transcription-quantitative PCR analysis. Among the 70 patients with ccRCC, EHD2 was overexpressed in 52.8% (37/70). Subsequently, EHD2 was found to be significantly associated with both overall survival (P=0.016) and disease-free survival (P=0.029). Furthermore, by multivariate analysis, EHD2 was an independent prognostic factor for patients with ccRCC. CONCLUSION: EHD2 is a novel HIF target, based on a relatively large sample of EHD2 research in patients with ccRCC. Furthermore, our study provided evidence that EHD2 can serve as a promising biomarker for predicting ccRCC outcome.

EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry.

With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2- and CAV1/2-overexpressing BC.

Novel MYCBP::EHD2 and RUNX1::ZNF780A Fusion Genes in T-cell Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: T-cell acute lymphoblastic leukemia (T-ALL) is a rare malignancy characterized by proliferation of early T-cell precursors that replace normal hematopoietic cells. T-ALL cells carry non-random chromosome aberrations, fusion genes, and gene mutations, often of prognostic significance. We herein report the genetic findings in cells from a T-ALL patient. MATERIALS AND METHODS: Bone marrow cells from a patient with T-ALL were examined using G-banding, array comparative genomic hybridization (aCGH), RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing, and fluorescence in situ hybridization. RESULTS: G-banding revealed del(1)(p34), add(5)(q14), trisomy 8, and monosomy 21 in the leukemic cells. aCGH detected the gross unbalances inferred from the karyotyping results, except that heterozygous loss of chromosome 21 did not include its distal part; 21q22.12-q22.3 was undeleted. In addition, aCGH detected a submicroscopic interstitial 7.56 Mbp deletion in the q arm of chromosome 19 from 19q13.2 to 19q13.33. RNA sequencing detected and RT-PCR/Sanger sequencing confirmed the presence of two novel chimeras, MYCBP::EHD2 and RUNX1::ZNF780A. They were generated from rearrangements involving subbands 1p34.3 (MYCBP), 19q13.2 (ZNF780A), 19q13.33 (EHD2), and 21q22.12 (RUNX1), i.e., at the breakpoints of chromosomal deletions. CONCLUSION: The leukemic cells showed the heterozygous loss of many genes as well as the generation of MYCBP::EHD2 and RUNX1::ZNF780A chimeras. Because the partner genes in the chimeras were found at the breakpoints of the chromosomal deletions, we believe that both the heterozygous losses and the generation of the two chimeras occurred simultaneously, and that they were pathogenetically important.

EHD2 inhibits the invasive ability of lung adenocarcinoma and improves the prognosis of patients.

Background: EH domain contains protein 2 (EHD2) may be involved in tumorigenesis and development. However, the role of EHD2 in lung adenocarcinoma (LUAD) is unknown. Methods: The link between EHD2 and LUAD and the associated underlying mechanism was determined using bioinformatics analysis. Then, immunohistochemistry (IHC) was employed to detect EHD2 expression level in LUAD patients. The stable transfection cell line was used to establish with lentivirus vector, and then the transfection efficiency was detected by western blot. Phagokinetic motility assays, transwell assays, and western blotting were also employed to investigate EHD2 impacts on cell viability. Results: The results indicated that EHD2 protein expression in human LUAD samples was significantly lower than that in the adjacent normal tissues. Low EHD2 expression was significantly linked to lymph node metastasis as well as advanced tumor-node-metastasis (TNM) staging (P<0.05). The Kaplan-Meier survival curve showed that low EHD2 expression was significantly associated with low survival (P=0.01). The multivariate Cox regression analysis confirmed that EHD2 expression and TNM stage were independent prognostic factors for LUAD patients (all P<0.05). The in vitro experiments demonstrated that EHD2 knockdown markedly contributed to an increase in migration and invasion in A549 cells. Overexpression of EHD2 substantially suppressed H1299 cell migration and invasion. Furthermore, decreased E-cadherin expression was observed in A549 cells with EHD2 knockdown, as well as increased N-cadherin and vimentin expressions. By contrast, E-cadherin expression was increased in H1299 cells, whereas N-cadherin and vimentin expressions were decreased as a result of EHD2 overexpression. Conclusions: Our study demonstrated that EHD2 reduces LUAD migration and invasion by preventing the epithelial-mesenchymal transition (EMT) process. Furthermore, the results suggest that EHD2 may be a novel biomarker for prognosis prediction.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells.

GlycoTAIL and FlexiTAIL as Half-Life Extension Modules for Recombinant Antibody Fragments.

Many therapeutic proteins are small in size and are rapidly cleared from circulation. Consequently, half-life extension strategies have emerged to improve pharmacokinetic properties, including fusion or binding to long-lasting serum proteins, chemical modifications with hydrophilic polymers such as PEGylation, or, more recently, fusion to PEG mimetic polypeptides. In the present study, two different PEG mimetic approaches, the GlycoTAIL and the FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, including scFv, diabody, and scFv-EHD2 fusion proteins. The GlycoTAIL and FlexiTAIL sequences of varying lengths are composed of aliphatic and hydrophilic residues, with the GlycoTAIL furthermore comprising N-glycosylation sites. All modified proteins could be produced in a mammalian expression system without reducing stability and antigen binding, and all modified proteins exhibited a prolonged half-life and increased drug disposition in mice. The strongest effects were observed for proteins comprising a FlexiTAIL of 248 residues. Thus, the GlycoTAIL and FlexiTAIL sequences represent a flexible and modular system to improve the pharmacokinetic properties of proteins.

EHD2 modulates Dll4 endocytosis during blood vessel development.

OBJECTIVE: Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta-like ligand 4 (Dll4) endocytosis during Notch activation. APPROACH AND RESULTS: Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane-bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE-cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. CONCLUSIONS: Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.

Ese-3 contributes to colon cancer progression by downregulating EHD2 and transactivating INPP4B.

Epithelium-specific Ets protein 3 (Ese-3), a member of the Ets family of transcription factors, plays an important role in the development of cancers. However, little is known concerning its role in colon cancer (CC). In this study, we demonstrate that the expression of Ese-3 is upregulated in CC tissues and elevated Ese-3 expression is relationship with advanced T stage (P=0.037) and poor disease-free survival (DFS, P=0.044). Univariate and multivariate cox regression analyses show that Ese-3 expression may be an independent prognostic value for CC patients. Moreover, Ese-3 knockdown suppresses CC cell proliferation in vitro and in vivo, while Ese-3 overexpression has the opposite result. Further, we first demonstrate that EHD2 and INPP4B are the downstream genes of Ese-3. Subsequent investigation find that EHD2 is downregulated in CC tissues and knockdown of EHD2 significantly increase CC cell proliferation in vitro and vivo. Our findings reveal that Ese-3 promotes CC cell proliferation by downregulating EHD2 and transactivating INPP4B, and targeting the pathway may be a promising therapeutic target for CC patients.

EHD2 Overexpression Suppresses the Proliferation, Migration, and Invasion in Human Colon Cancer.

Background: To investigate how EHD2 influences the development of colon cancer.Methods: Immunohistochemistry of 90 colon cancer tissue specimens were determined the expression of EHD2. The lentivirus-EHD2-transfected colon cancer cells were conducted to evaluate the biological behaviors.Results: EHD2 was closely associated with clinic pathological parameters (p < 0.001). EHD2 upregulation was relative with a longer overall survival. The results of the univariate and multivariate analyses indicated that EHD2 could be an independent prognosis marker. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest.Conclusions: EHD2 might represent a therapeutic target of colon cancer.IMPACT STATEMENTWhat is already known on this subject? Membrane trafficking is crucial for cell proliferation, differentiation and apoptosis, especially tumorigenesis and development. EHD2 proteins play an important role in the regulation of membrane trafficking in endocytosis. EHD2 has been suggested to participate in the occurrence of some malignancies.What are the new findings? EHD2 could be an independent prognosis marker in colon cancer. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest in vitro. EHD2 overexpression markedly increased the expression of EMT marker E-cadherin in colon cancer.How might it impact on clinical practice in the foreseeable future? EHD2 overexpression may inhibit tumorigenesis in colon cancer through the modulation of E-cadherin, the critical marker of EMT which is closely related to invasion and distant metastasis of tumor cells.

Analysis of Protein and Lipid Interactions Using Liposome Co-sedimentation Assays.

The dynamic assembly of proteins at the membrane interphase is key to many cell biological processes such as the generation and stabilization of caveolae at the cell surface via coat proteins. The liposome co-sedimentation assay has been widely used for studies of protein and lipid interactions and has provided important information about binding mechanisms, lipid-binding specificity, and curvature preference of proteins. Here, we describe this technique in detail and how it can be used as a tool to address the membrane-binding ability and lipid specificity of caveolae-associated proteins.

Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology.

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.

Membrane tension buffering by caveolae: a role in cancer?

Caveolae are bulb-like invaginations made up of two essential structural proteins, caveolin-1 and cavins, which are abundantly present at the plasma membrane of vertebrate cells. Since their discovery more than 60 years ago, the function of caveolae has been mired in controversy. The last decade has seen the characterization of new caveolae components and regulators together with the discovery of additional cellular functions that have shed new light on these enigmatic structures. Early on, caveolae and/or caveolin-1 have been involved in the regulation of several parameters associated with cancer progression such as cell migration, metastasis, angiogenesis, or cell growth. These studies have revealed that caveolin-1 and more recently cavin-1 have a dual role with either a negative or a positive effect on most of these parameters. The recent discovery that caveolae can act as mechanosensors has sparked an array of new studies that have addressed the mechanobiology of caveolae in various cellular functions. This review summarizes the current knowledge on caveolae and their role in cancer development through their activity in membrane tension buffering. We propose that the role of caveolae in cancer has to be revisited through their response to the mechanical forces encountered by cancer cells during tumor mass development.

Lipid accumulation controls the balance between surface connection and scission of caveolae.

Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Caveolae: Formation, dynamics, and function.

Caveolae are abundant surface pits formed by the assembly of cytoplasmic proteins on a platform generated by caveolin integral membrane proteins and membrane lipids. This membranous assembly can bud off into the cell or can be disassembled releasing the cavin proteins into the cytosol. Disassembly can be triggered by increased membrane tension, or by stress stimuli, such as UV. Here, we discuss recent mechanistic studies showing how caveolae are formed and how their unique properties allow them to function as multifunctional protective and signaling structures.

Keeping in touch with the membrane; protein- and lipid-mediated confinement of caveolae to the cell surface.

Caveolae are small Ω-shaped invaginations of the plasma membrane that play important roles in mechanosensing, lipid homeostasis and signaling. Their typical morphology is characterized by a membrane funnel connecting a spherical bulb to the membrane. Membrane funnels (commonly known as necks and pores) are frequently observed as transient states during fusion and fission of membrane vesicles in cells. However, caveolae display atypical dynamics where the membrane funnel can be stabilized over an extended period of time, resulting in cell surface constrained caveolae. In addition, caveolae are also known to undergo flattening as well as short-range cycles of fission and fusion with the membrane, requiring that the membrane funnel closes or opens up, respectively. This mini-review considers the transition between these different states and highlights the role of the protein and lipid components that have been identified to control the balance between surface association and release of caveolae.

Energy and Dynamics of Caveolae Trafficking.

Caveolae are 70-100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study.

Caveolae: The FAQs.

Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round-table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.

Cardiomyocyte damage control in heart failure and the role of the sarcolemma.

The cardiomyocyte plasma membrane, termed the sarcolemma, is fundamental for regulating a myriad of cellular processes. For example, the structural integrity of the cardiomyocyte sarcolemma is essential for mediating cardiac contraction by forming microdomains such as the t-tubular network, caveolae and the intercalated disc. Significantly, remodelling of these sarcolemma microdomains is a key feature in the development and progression of heart failure (HF). However, despite extensive characterisation of the associated molecular and ultrastructural events there is a lack of clarity surrounding the mechanisms driving adverse morphological rearrangements. The sarcolemma also provides protection, and is the cell's first line of defence, against external stresses such as oxygen and nutrient deprivation, inflammation and oxidative stress with a loss of sarcolemma viability shown to be a key step in cell death via necrosis. Significantly, cumulative cell death is also a feature of HF, and is linked to disease progression and loss of cardiac function. Herein, we will review the link between structural and molecular remodelling of the sarcolemma associated with the progression of HF, specifically considering the evidence for: (i) Whether intrinsic, evolutionary conserved, plasma membrane injury-repair mechanisms are in operation in the heart, and (ii) if deficits in key 'wound-healing' proteins (annexins, dysferlin, EHD2 and MG53) may play a yet to be fully appreciated role in triggering sarcolemma microdomain remodelling and/or necrosis. Cardiomyocytes are terminally differentiated with very limited regenerative capability and therefore preserving cell viability and cardiac function is crucially important. This review presents a novel perspective on sarcolemma remodelling by considering whether targeting proteins that regulate sarcolemma injury-repair may hold promise for developing new strategies to attenuate HF progression.