Growth Factor Receptor Pathway Substrate Research Articles

HNF1A induces glioblastoma by upregulating EPS8 and activating PI3K/AKT signaling pathway

Despite the exact biological role of HNF1 homolog A (HNF1A) in the regulatory mechanism of glioblastoma (GBM), the molecular mechanism, especially the downstream regulation as a transcription factor, remains to be further elucidated. Immunohistochemistry was used to detect the expression and clinical relevance of HNF1A in GBM patients. CCK8, TUNEL, and subcutaneous tumor formation in nude mice were used to evaluate the effect of HNF1A on GBM in vitro and in vivo. The correction between HNF1A and epidermal growth factor receptor pathway substrate 8 (EPS8) was illustrated by bioinformatics analysis and luciferase assay. Further mechanism was explored that the transcription factor HNF1A regulated the expression of EPS8 and downstream signaling pathways by directly binding to the promoter region of EPS8. Our comprehensive analysis of clinical samples in this study showed that upregulated expression of HNF1A was associated with poor survival in GBM patients. Further, we found that knockdown of HNF1A markedly suppressed the malignant phenotype of GBM cells in vivo and in vitro as well as promoted apoptosis of tumor cells, which was reversed by upregulation of HNF1A. Mechanistically, HNF1A could significantly activate PI3K/AKT signaling pathway by specifically binding to the promoter regions of EPS8. Moreover, overexpression of EPS8 was able to reverse the apoptosis of tumor cells caused by HNF1A knockdown, thereby exacerbating the GBM progression. Correctively, our study has clarified the explicit mechanism by which HNF1A promotes GBM malignancy and provides a new therapeutic target for further clinical application.

Abstract B006: Exosomal protein Eps8 as a new biomarker for pancreatic cancer

Abstract Exosomes are extracellular vesicles containing nucleic acids, proteins, and lipids of approximately 100 nm, which are released from cancer cells to create a microenvironment favorable for their survival. Epidermal growth factor receptor pathway substrate 8 (Eps8), an exosomal protein, is reportedly associated with metastasis. We investigated the relationship between Eps8 in the serum exosomes of patients with pancreatic cancer and clinicopathological factors and examined the usefulness of exosomal Eps8 as a biomarker for pancreatic cancer. Methods: Serum exosomes of 93 patients with pancreatic disease (64 cases of pancreatic cancer, 12 cases of chronic pancreatitis, and 17 cases of IPMN) were extracted via ultracentrifugation, and Eps8 in exosomes were evaluated by western blotting. We analyzed 1) the clinicopathological factors of patients with pancreatic cancer, 2) the Eps8 positive rate in each disease, 3) the sensitivity, specificity, and positive predictive value of Eps8 for the diagnosis of pancreatic cancer, 4) the relationship between Eps8 and clinicopathological factors, and 5) compared and discussed between exosomal Eps8 and serum CA19-9. Results: The median patient age was 72 years (47–85), with 35 male and 29 female patients included in the study. The median body mass index was 20.8 kg/m2 (12–26.7 kg/m2). 1) Site Ph33/Pbt31, cStageIV/III/II/I: 28/11/14/11; 2) pancreatic cancer 59.4%, chronic pancreatitis 33%, IPMN 12%; 3) sensitivity 59.4. 4) In patients with pancreatic cancer, Eps8-positive patients had significantly higher ALT (p = 0.03), significantly less cancer (p = 0.04), lower IPMN complication rates, and more chronic pancreatitis and autoimmune diseases than the exosomal Eps8-negative patients. Stage I 64% (7/11), Stage II 36% (5/14), Stage III 64% (7/11) Stage IV 68% (19/28) 5) Exosomal Eps8 positivity (64%) was higher than serum CA19-9 positivity (45%) in Stage I. We also found some patients were negative for serum CA19-9 but positive for exosomal Eps8. Conclusion: Exosomal Eps8 has a higher positive rate in Stage I than serum CA19-9. Diagnosis with exosomal Eps8 may improve diagnostic performance for pancreatic cancer. Citation Format: Kazunao Hayashi, Atsunori Tsuchiya, Shuji Terai. Exosomal protein Eps8 as a new biomarker for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B006.

Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4.

Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4's liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.

Exploration of the common pathogenic link between COVID‐19 and diabetic foot ulcers: An in silico approach

AbstractBackground and AimsThe Coronavirus Disease‐19 (COVID‐19) is posing an ongoing threat to human health. Patients of diabetic foot ulcer (DFU) are susceptible to COVID‐19‐induced adverse outcomes. Nevertheless, investigations into their mutual molecular mechanisms have been limited to date. In the present work, we tried to uncover the shared pathogenesis and regulatory gene targets of COVID‐19 and DFU.MethodsIn this study, we chose GSE161281 as the COVID‐19 data set, which contained severe acute respiratory syndrome coronavirus 2 infected human induced embryonic stem cell‐derived peripheral neurons (n = 2) with uninfected controls (n = 2). The GSE134431 designated as the DFU data set, comprising full‐thickness DFU (n = 13) and diabetic foot skin (n = 8) samples from diabetic patients. The differential expressed genes (DEGs) were identified from GSE161281 and GSE134431, and the common DEGs between COVID‐19 and DFU were extracted. Multifactor regulatory network and co‐expression network of the common DEGs were analyzed, along with candidate drug prediction.ResultsAltogether, six common DEGs (dickkopf‐related protein 1 [DKK1], serine proteinase inhibitor A3 [SERPINA3], ras homolog family member D [RHOD], myelin protein zero like 3 [MPZL3], Claudin‐11 [CLDN11], and epidermal growth factor receptor pathway substrate 8‐like 1 [EPS8L1]) were found between COVID‐19 and DFU. Functional analyses indicated that pathways of apoptotic and Wnt signaling may contribute to progression of COVID‐19. Gene co‐expression network implied the shared pathways of immune regulation and cytokine response participated collectively in the development of DFU and COVID‐19. A multifactor regulatory network was constructed integrating the corresponding microRNAs (miRNAs) and transcription factors. Additionally, we proposed potential drug objects for the combined therapy.ConclusionOur study revealed the shared molecular mechanisms underlying COVID‐19 and DFU. The identified pivotal targets and common pathways can provide new perspectives for further research and assist the development of management strategies in patients of DFU complicated with COVID‐19.

Comparative immunohistochemical analysis suggests a conserved role of EPS8L1 in epidermal and hair follicle barriers of mammals.

The mammalian skin and its appendages depend on tightly coordinated differentiation of epithelial cells. Epidermal growth factor receptor (EGFR) pathway substrate 8 (EPS8) like 1 (EPS8L1) is enriched in the epidermis among human tissues and has also been detected in the epidermis of lizards. Here, we show by the analysis of single-cell RNA-sequencing data that EPS8L1 mRNA is co-expressed with filaggrin and loricrin in terminally differentiated human epidermal keratinocytes. Comparative genomics indicated that EPS8L1 is conserved in all main clades of mammals, whereas the orthologous gene has been lost in birds. Using a polyclonal antibody against EPS8L1, we performed an immunohistochemical screening of skin from diverse mammalian species and immuno-electron microscopy of human skin. EPS8L1 was detected predominantly in the granular layer of the epidermis in monotremes, marsupial, and placental mammals. The labeling was partly associated with cell membranes, and it was evident along the perimeter of keratinocytes at the transition with the cornified layer of the epidermis, similar to involucrin distribution. Basal, spinous, and the fully mature cornified layers lacked immunolabeling of EPS8L1. In addition to the epidermis, the hair follicle inner root sheath (IRS) was immunolabeled. Both epidermal granular layer and IRS contribute to the barrier function of the skin, suggesting that EPS8L1 is involved in the regulation of these barriers.

MTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

NTRK Gene Fusions in Non-Small-Cell Lung Cancer: Real-World Screening Data of 1068 Unselected Patients.

(1) Background: The main objectives of our study are (i) to determine the prevalence of NTRK (neurotrophic tyrosine kinase) fusions in a routine diagnostic setting in NSCLC (non-small cell lung cancer) and (ii) to investigate the feasibility of screening approaches including immunohistochemistry (IHC) as a first-line test accompanied by fluorescence in situ hybridization (FISH) and RNA-(ribonucleic acid-)based next-generation sequencing (RNA-NGS). (2) Methods: A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n = 973) or direct FISH testing (n = 95). (3) Results: One hundred and thirty-three patients (14.8%) were IHC positive; consecutive RNA-NGS testing revealed two patients (0.2%) with NTRK fusions (NTRK1-EPS15 (epidermal growth factor receptor pathway substrate 15) and NTRK1-SQSTM1 (sequestosome 1)). Positive RNA-NGS was confirmed by FISH, and NTRK-positive patients benefited from targeted treatment. All patients with direct FISH testing were negative. RNA-NGS- or FISH-positive results were mutually exclusive with alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (proto-oncogene B-Raf), RET (rearranged during transfection) or KRAS (kirsten rat sarcoma viral oncogene). Excluding patients with one of these alterations raised the prevalence of NTRK-fusion positivity among panTrk-(tropomyosin receptor kinase-) IHC positive samples to 30.5%. (4) Conclusions: NTRK fusion-positive lung cancers are exceedingly rare and account for less than 1% of patients in unselected all-comer populations. Both RNA-NGS and FISH are suitable to determine clinically relevant NTRK fusions in a real-world setting. We suggest including panTrk-IHC in a diagnostic workflow followed by RNA-NGS. Excluding patients with concurrent molecular alterations to EGFR/ALK/ROS1/BRAF/RET or KRAS might narrow the target population.

Loss of EPS8 sensitizes non-small-cell lung carcinoma to chemotherapy-induced DNA damage.

Epidermal growth factor receptor pathway substrate number 8 (EPS8) has been reported to be critical in mediating tumor progression. However, the molecular and biological consequences of EPS8 overexpression remain unclear. Here we evaluated whether EPS8 increased DNA damage repair in non-small-cell lung carcinoma (NSCLC) cells and the mechanism of EPS8-mediated DNA damage repair which influenced chemosensitivity. Serial studies of functional experiments revealed that EPS8 knockdown inhibited cell growth, induced cell-cycle arrest and increased cisplatin therapeutic effects on NSCLC. EPS8 was found to induce DNA damage repair via upregulation of phosphorylated-ATM and downregulation of the tumor suppressor p53 and G1 cell kinase inhibitor p21. Moreover, in conjunction with cisplatin, decreasing EPS8 protein levels further increased p53 protein level and inhibited ATM signaling. Transplanted tumor studies were also performed to demonstrate that EPS8 knockdown inhibited tumor growth and sensitized tumors to cisplatin treatment. In conclusion, we have described a novel molecular mechanism through which EPS8 is likely to be involved in cancer progression and chemoresistance via DNA damage repair, indicating that EPS8 expression may influence the response to chemotherapy. Therefore, targeting EPS8 may be a potential therapeutic approach for patients with NSCLC.

Es-Arp3 and es-Eps8 regulate spermatogenesis via microfilaments in the seminiferous tubule of Eriocheir sinensis

Spermatogenesis is a complicated process that includes spermatogonia differentiation, spermatocytes meiosis, spermatids spermiogenesis and final release of spermatozoa. Actin-related protein 3 (Arp3) and epidermal growth factor receptor pathway substrate 8 (Eps8) are two actin binding proteins that regulate cell adhesion in seminiferous tubules during mammalian spermatogenesis. However, the functions of these two proteins during spermatogenesis in nonmammalian species, especially Crustacea, are still unknown. Here, we cloned es-Arp3 and es-Eps8 from the testis of Chinese mitten crab Eriocheir sinensis. es-Arp3 and es-Eps8 were located in spermatocytes, spermatids and spermatozoa. Knockdown of es-Arp3 and es-Eps8 in vivo caused morphological changes to seminiferous tubules including delayed spermatozoa release, shedding of germ cells and vacuoles. Filamentous-actin (F-actin) filaments network was disorganized due to deficiency of es-Arp3 and es-Eps8. Accompanying this, four junctional proteins (α-catenin, β-catenin, pinin and ZO1) displayed abnormal expression levels as well as penetrating biotin signals in seminiferous tubules. We also used the Arp2/3 complex inhibitor CK666 to block es-Arp3 activity and supported es-Arp3 knockdown results. In summary, our study demonstrated for the first time that es-Arp3 and es-Eps8 are important for spermatogenesis via regulating microfilament-mediated cell adhesion in Eriocheir sinensis.

Circulating cancer-associated extracellular vesicles as early detection and recurrence biomarkers for pancreatic cancer.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is essential for improving patient survival rates, and noninvasive biomarkers are urgently required to identify patients who are eligible for curative surgery. Here, we examined extracellular vesicles (EVs) from the serum of PDAC patients to determine their ability to detect early‐stage disease. EV‐associated proteins purified by ultracentrifugation and affinity columns underwent proteomic analysis to identify novel PDAC markers G protein‐coupled receptor class C group 5 member C (GPRC5C) and epidermal growth factor receptor pathway substrate 8 (EPS8). To verify the potency of GPRC5C‐ or EPS8‐positive EVs as PDAC biomarkers, we analyzed EVs from PDAC patient blood samples using ultracentrifugation in two different cohorts (a total of 54 PDAC patients, 32 healthy donors, and 22 pancreatitis patients) by immunoblotting. The combination of EV‐associated GPRC5C and EPS8 had high accuracy, with area under the curve values of 0.922 and 0.946 for distinguishing early‐stage PDAC patients from healthy controls in the two cohorts, respectively, and could detect PDAC patients who were negative for CA19‐9. Moreover, we analyzed 30 samples taken at three time points from 10 PDAC patients who underwent surgery: before surgery, after surgery, and recurrence as an early‐stage model. These proteins were detected in EVs derived from preoperative and recurrence samples. These results indicated that GPRC5C‐ or EPS8‐positive EVs were biomarkers that have the potential to detect stage I early pancreatic cancer and small recurrent tumors detected by computed tomography.

Gene expression profile associated with Asmt knockout-induced depression-like behaviors and exercise effects in mouse hypothalamus.

Sleep disorder caused by abnormal circadian rhythm is one of the main symptoms and risk factors of depression. As a known hormone regulating circadian rhythms, melatonin (MT) is also namely N-acetyl-5-methoxytryptamine. N-acetylserotonin methyltransferase (Asmt) is the key rate-limiting enzyme of MT synthesis and has been reportedly associated with depression. Although 50–90% of patients with depression have sleep disorders, there are no effective treatment ways in the clinic. Exercise can regulate circadian rhythm and play an important role in depression treatment. In the present study, we showed that Asmt knockout induced depression-like behaviors, which were ameliorated by swimming exercise. Moreover, swimming exercise increased serum levels of MT and 5-hydroxytryptamine (5-HT) in Asmt knockout mice. In addition, the microarray data identified 10 differentially expressed genes (DEGs) in KO mice compared with WT mice and 29 DEGs in KO mice after swimming exercise. Among the DEGs, the direction and magnitude of change in epidermal growth factor receptor pathway substrate 8-like 1 (Eps8l1) and phospholipase C-β 2 (Plcb2) were confirmed by qRT-PCR partly. Subsequent bioinformatic analysis showed that these DEGs were enriched significantly in the p53 signaling pathway, long-term depression and estrogen signaling pathway. In the protein–protein interaction (PPI) networks, membrane palmitoylated protein 1 (Mpp1) and p53-induced death domain protein 1 (Pidd1) were hub genes to participate in the pathological mechanisms of depression and exercise intervention. These findings may provide new targets for the treatment of depression.

The roles and prognostic significance of ABI1-TSV-11 expression in patients with left-sided colorectal cancer

Abnormally expressed and/or phosphorylated Abelson interactor 1 (ABI1) participates in the metastasis and progression of colorectal cancer (CRC). ABI1 presents as at least 12 transcript variants (TSVs) by mRNA alternative splicing, but it is unknown which of them is involved in CRC metastasis and prognosis. Here, we firstly identified ABI1-TSV-11 as a key TSV affecting the metastasis and prognosis of left-sided colorectal cancer (LsCC) and its elevated expression is related to lymph node metastasis and shorter overall survival (OS) in LsCC by analyzing data from The Cancer Genome Atlas and TSVdb. Secondly, ABI1-TSV-11 overexpression promoted LoVo and SW480 cells adhesion and migration in vitro, and accelerated LoVo and SW480 cells lung metastasis in vivo. Finally, mechanism investigations revealed that ABI1-isoform-11 interacted with epidermal growth factor receptor pathway substrate 8 (ESP8) and regulated actin dynamics to affect LoVo and SW480 cells biological behaviors. Taken together, our data demonstrated that ABI1-TSV-11 plays an oncogenic role in LsCC, it is an independent risk factor of prognosis and may be a potential molecular marker and therapeutic target in LsCC.

Non-SUMOylated CRMP2 decreases NaV1.7 currents via the endocytic proteins Numb, Nedd4-2 and Eps15

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

Effects and mechanisms of Eps8 on the biological behaviour of malignant tumours (Review).

Epidermal growth factor receptor pathway substrate 8 (Eps8) was initially identified as the substrate for the kinase activity of EGFR, improving the responsiveness of EGF, which is involved in cell mitosis, differentiation and other physiological functions. Numerous studies over the last decade have demonstrated that Eps8 is overexpressed in most ubiquitous malignant tumours and subsequently binds with its receptor to activate multiple signalling pathways. Eps8 not only participates in the regulation of malignant phenotypes, such as tumour proliferation, invasion, metastasis and drug resistance, but is also related to the clinicopathological characteristics and prognosis of patients. Therefore, Eps8 is a potential tumour diagnosis and prognostic biomarker and even a therapeutic target. This review aimed to describe the structural characteristics, role and related molecular mechanism of Eps8 in malignant tumours. In addition, the prospect of Eps8 as a target for cancer therapy is examined.

EPS8 regulates an NLRP3 inflammasome-independent caspase-1 activation pathway in monosodium urate crystal-treated RAW264.7 macrophages

Gout is an inflammatory arthritis caused by the phagocytosis of monosodium urate (MSU) crystal deposition in joints. NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome-dependent caspase-1 activation is implicated in the processing of interleukin-1β (IL-1β), which is the major effector cytokine in the acute inflammatory response of gout. Mechanisms underlying caspase-1 activation remain unclear. Epidermal growth factor receptor pathway substrate no. 8 (Eps8) is a signal transducer and actin filament organizer that plays a key role in lipopolysaccharide-stimulated phagocytosis in macrophages. Here, RAW264.7 macrophages that have no intact NLRP3 inflammasomes were used to investigate the role of Eps8 in MSU crystal-mediated caspase-1 activation. A kinetic study revealed that the induction of Eps8 expression by MSU crystals occurred before NLRP3, p46/p33 caspase-1, and mature IL-1β in RAW 264.7 cells. In addition, actin cytoskeleton dynamics was required for Eps8 induction and caspase-1 activation in MSU crystal stimulation. Silencing Eps8 had no effect on the basal expression of p46/p33 caspase-1 and NLRP3, but nearly abolished MSU crystal-induced NLRP3 expression and caspase-1 activation. Furthermore, MSU crystals induced Eps8–pro-caspase-1 complex formation and Eps8 formed a stable complex with p33 caspase-1, but not with NLRP3. In summary, our results demonstrated for the first time the importance of Eps8 in MSU crystal-mediated caspase-1 activation without the involvement of NLRP3 inflammasomes.

Dexmedetomidine suppresses the progression of esophageal cancer via miR-143-3p/epidermal growth factor receptor pathway substrate 8 axis

Esophageal cancer is one of the fatal cancers around the world. Dexmedetomidine (DEX) is widely used during anesthesia of esophageal cancer surgery. Nevertheless, the role of DEX in the progression of esophageal cancer remains barely known. The proliferation, apoptosis and metastasis of esophageal cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, transwell migration and invasion assays and Western blot assay. The expression of miR-143-3p was measured by quantitative real-time PCR in esophageal cancer tissues and cells. The binding sites between miR-143-3p and epidermal growth factor receptor pathway substrate 8 (EPS8) were predicted by Starbase online software, and the combination was verified by dual-luciferase reporter assay. The murine xenograft model was established using KYSE150 cells to verify the function of DEX in vivo. DEX inhibited the proliferation and metastasis while accelerated the apoptosis of esophageal cancer cells. The abundance of miR-143-3p was lower in esophageal cancer tissues and cells than that in paring normal tissues and normal esophageal mucosal cells Het-1A. MiR-143-3p could be induced by DEX treatment in esophageal cancer cells, and miR-143-3p also suppressed the development of esophageal cancer. EPS8 was a functional target of miR-143-3p, and it played an oncogenic role in esophageal cancer. DEX inhibited the growth of tumor via miR-143-3p/EPS8 in vivo. DEX suppressed the growth and metastasis while facilitated the apoptosis of esophageal cancer cells through upregulating the abundance of miR-143-3p and reducing the level of EPS8 in vivo and in vitro, providing promising target for the treatment of esophageal cancer.

MiR-345 inhibits migration and stem-like cell phenotype in gastric cancer via inactivation of Rac1 by targeting EPS8.

Tumor metastasis is the main cause of treatment failure and death in patients with late stage of gastric cancer (GC). Studies showed that microRNAs (miRNAs) are important regulators in the process of tumor metastasis. In this study, we used miRNA array analysis to search for metastasis-associated miRNAs in primary and matched metastasis tissues of patients with GC and found that miR-345-5p (miR-345) was significantly higher in primary sites. Decreased expression of miR-345 was observed in GC tissues and cell lines, which was correlated with aggressive stage and grade. Patients with a higher level of miR-345 had a better prognosis. miR-345 could inhibit the migration and spheroid formation abilities in GC cell lines in transwell assay and spheroid formation assay. RNA sequencing and bioinformatics analysis revealed that miR-345 downregulated the epidermal growth factor receptor pathway substrate 8 (EPS8) and its downstream Rac1 signaling. Mechanistically, we confirmed that miR-345 could target EPS8 by directly binding to its 3' untranslated region by luciferase reporter assay. Further rescue assay showed that the ability of miR-345 in inhibiting the migration, stem-like cell phenotype, and epithelial-mesenchymal transition (EMT) in GC was partly dependent on targeting EPS8. In conclusion, miR-345 plays an inhibitory role in GC metastasis through inhibiting cell migration, EMT, and cancer stem cell phenotype via inactivation of Rac1 signaling by targeting EPS8, which provides the potential therapeutic and predictive value of miR-345 in GC.

Modulation of EPS8 functions by phosphorylation

Objectives Epidermal growth factor receptor pathway substrate 8 (EPS8) is a scaffolding protein involved in regulating cell proliferation, actin dynamics, and receptor trafficking in human cells. EPS8 expression is increased in a range of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previous studies have indicated that overexpression of EPS8 enhances mitogenesis and migration of tumor cells and is sufficient to convert nontumorigenic cells to a tumorigenic phenotype. The nonreceptor tyrosine kinase Src is reported to phosphorylate EPS8 at 4 tyrosine residues, although the impact of this on EPS8 function is unknown. The purpose of this study was to investigate the role of tyrosine phosphorylation of EPS8 at Src target sites in modulating biochemical functions, cell growth, and motility in HNSCC. Study Design Expression plasmids encoding EPS8 with amino acid substitutions to phenylalanine (F) at the 4 putative Src phosphorylation sites (Y485 F, Y525 F, Y602 F, Y774 F), and all 4 combined (4 F), were prepared by site-directed mutagenesis. To evaluate the effect of unphosphorylated EPS8 on downstream signals and biologic behavior, plasmids were transduced into a model cell line expressing a normal endogenous level of EPS8. Additionally, cells were treated with dasatinib, a Src inhibitor, to block phosphorylation of Src substrates. Expression of downstream targets was evaluated by Western blotting. Wound closure and proliferation assays were used to investigate the impact of these mutations on cell motility and growth. Results FOXM1, AURKA, and AURKB levels were decreased in cells expressing the nonphosphorylatable 4 F and Y602 F EPS8 mutants, whereas cells harboring the Y485 F, Y525 F, and Y774 F EPS8 mutants showed no differences in the expression of these proteins compared with controls. Moreover, dasatinib treatment resulted in a significant decrease in the expression of EPS8 downstream targets. In addition, both Y602 F and 4 F mutants elicited a significant reduction in tumor cell proliferation and migration. Conclusions The nonphosphorylatable Y602 F-EPS8 and 4 F-EPS8 mutants decreased the expression of EPS8 downstream targets, as well as reducing tumor cell growth and motility, implying a crucial role for phosphorylation of EPS8, principally at Y602, in mediating protumorigenic signal transduction.

SATB1 establishes ameloblast cell polarity and regulates directional amelogenin secretion for enamel formation

BackgroundPolarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes’ processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs.ResultsThe higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1−/− mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1−/− ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes’ processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1−/− SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1−/− SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1−/− mice as well, displaying defective intestinal microvilli.ConclusionsOur data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1−/− SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

EPS8 Facilitates Uncoating of Influenza A Virus

SUMMARYAll viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We use gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identifies the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirm its importance in enhancing FLUAV infection and titers. Loss of EPS8 does not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associates with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes is significantly delayed in the absence of EPS8. Our study identifies EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.