Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

EPS8 supports pancreatic cancer growth by inhibiting BMI1 mediated proteasomal degradation of ALDH7A1.

Aldehyde dehydrogenase 7 family member A1 (ALDH7A1) is an enzyme catalyzing lipid peroxidation of fatty aldehydes. It plays a critical role in sustaining high oxygen consumption rate (OCR) and ATP production in pancreatic ductal adenocarcinoma (PADC). However, why PADC cells maintain a relatively high level of ALDH7A1 concentration is still not well understood. In the current study, we explored the interplay between epidermal growth factor receptor kinase substrate 8 (EPS8) and ALDH7A1 in PADC cells. PADC cell lines MIA PaCa-2 and AsPANC-1 were used for in vitro and in vivo studies. The co-IP assay showed mutual interactions between Flag-EPS8 and Myc-ALDH7A1 in both MIA PaCa-2 and AsPANC-1 cells. EPS8 knockdown resulted in decreased ALDH7A1 protein levels and increased poly-ubiquitination. An interaction was observed between ALDH7A1 and BMI1 but not between BMI1 and EPS8. BMI1 knockdown reduced ALDH7A1 poly-ubiquitination and degradation caused by EPS8 knockdown. Dual EPS8 and ALDH7A1 knockdown had a synergistic effect on suppressing PADC cell proliferation in vitro and in vivo. In conclusion, this study revealed that EPS8 supports PADC growth by interacting with ALDH7A1 and inhibiting BMI1 mediated proteasomal degradation of ALDH7A1.

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-1ß (IL-1ß), 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-1ß 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.

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.

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

BACKGROUND: Polarity 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. RESULTS: The 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. CONCLUSIONS: Our 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.

LncRNA DSCAM-AS1 acts as a sponge of miR-137 to enhance Tamoxifen resistance in breast cancer.

OBJECTIVE: To investigate the influence of long noncoding RNA (lncRNA) DSCAM-AS1 on the propagation and apoptosis of Tamoxifen-resistant (TR) breast cancer cells via regulation of mircoRNA (miR)-137 and epidermal growth factor receptor pathway substrate 8 (EPS8). METHODS: Data of GSE5840 downloaded from the Gene Expression Omnibus database were utilized to screen out aberrantly expressed lncRNA and messenger RNA in breast cancer tissue samples. The expressions of DSCAM-AS1, miR-137, and EPS8 were determined by quantitative real time polymerase chain reaction (qRT-PCR). Cell lines were screened by half maximal inhibitory concentration (IC 50 ). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and the flow cytometry assay were used to detect cell proliferation, apoptosis, and cell cycle. The relationship among DSCAM-AS1, miR-137, and EPS8 was studied by miRcode, TargetScan, and Pearson correlation coefficient. A xenograft mouse model experiment was performed to demonstrate the effect of DSCAM-AS1 and EPS8 on tumor growth in vivo. RESULTS: LncRNA DSCAM-AS1 and EPS8 were significantly upregulated, whereas miR-137 was downregulated in TR tissues. DSCAM-AS1 could promote the Tamoxifen resistance of breast cancer, and it was negatively correlated with miR-137, whereas positively correlated with the expression of EPS8 in TR breast cancer tissues. Furthermore, miR-137 could inhibit tumor development and arrest cell cycle at the G0/G1 phase by targeting the 3'-UTR of EPS8. DSCAM-AS1 targeted miR-137 and EPS8 to promote propagation of TR breast cancer cells and inhibit cell apoptosis. CONCLUSION: LncRNA DSCAM-AS1 acts as a competing endogenous RNA of miR-137 and regulates EPS8 to promote cell reproduction and suppresses cell apoptosis in TR breast cancer.

Inhibitory short peptides targeting EPS8/ABI1/SOS1 tri-complex suppress invasion and metastasis of ovarian cancer cells.

BACKGROUND: We aimed to develop inhibitory short peptides that can prevent protein interactions of SOS1/EPS8/ABI1 tri-complex, a key component essential for ovarian cancer metastasis. METHODS: Plasmids containing various regions of HA-tagged ABI1 were co-transfected into ovarian cancer cells with Flag-tagged SOS1 or Myc-tagged EPS8. Co-immunoprecipitation and GST-pulldown assay were used to identify the regions of ABI1 responsible for SOS1 and EPS8 binding. Inhibitory short peptides of these binding regions were synthesized and modified with HIV-TAT sequence. The blocking effects of the peptides on ABI1-SOS1 or ABI1-EPS8 interactions in vitro and in vivo were determined by GST-pulldown assay. The capability of these short peptides in inhibiting invasion and metastasis of ovarian cancer cell was tested by Matrigel invasion assay and peritoneal metastatic colonization assay. RESULTS: The formation of endogenous SOS1/EPS8/ABI1 tri-complex was detected in the event of LPA-induced ovarian cancer cell invasion. In the tri-complex, ABI1 acted as a scaffold protein holding together SOS1 and EPS8. The SH3 and poly-proline+PxxDY regions of ABI1 were responsible for SOS1 and EPS8 binding, respectively. Inhibitory short peptides p + p-8 (ppppppppvdyedee) and SH3-3 (ekvvaiydytkdkddelsfmegaii) could block ABI1-SOS1 and ABI1-EPS8 interaction in vitro. TAT-p + p-8 peptide could disrupt ABI1-EPS8 interaction and suppress the invasion and metastasis of ovarian cancer cells in vivo. CONCLUSIONS: TAT-p + p-8 peptide could efficiently disrupt the ABI1-EPS8 interaction, tri-complex formation, and block the invasion and metastasis of ovarian cancer cells.

A RhoA and Rnd3 cycle regulates actin reassembly during membrane blebbing.

The actin cytoskeleton usually lies beneath the plasma membrane. When the membrane-associated actin cytoskeleton is transiently disrupted or the intracellular pressure is increased, the plasma membrane detaches from the cortex and protrudes. Such protruded membrane regions are called blebs. However, the molecular mechanisms underlying membrane blebbing are poorly understood. This study revealed that epidermal growth factor receptor kinase substrate 8 (Eps8) and ezrin are important regulators of rapid actin reassembly for the initiation and retraction of protruded blebs. Live-cell imaging of membrane blebbing revealed that local reassembly of actin filaments occurred at Eps8- and activated ezrin-positive foci of membrane blebs. Furthermore, we found that a RhoA-ROCK-Rnd3 feedback loop determined the local reassembly sites of the actin cortex during membrane blebbing.

The Rho GTPase Rif signals through IRTKS, Eps8 and WAVE2 to generate dorsal membrane ruffles and filopodia.

Rif induces dorsal filopodia but the signaling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS (also known as BAIAP2L1) through its I-BAR domain. Rif also interacts with Pinkbar (also known as BAIAP2L2) in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant-negative Rif inhibits the formation of IRTKS-induced morphological structures, and Rif activity is blocked in IRTKS-knockout (KO) cells. To further define the Rif-IRTKS signaling pathway, we identify Eps8 and WAVE2 (also known as WASF2) as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS signaling, whereas WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic Escherichia coli infection, might compete for Rif for interaction with the I-BAR domain of IRTKS. Based on this evidence, we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53 (also known as BAIAP2), IRTKS and Pinkbar, as a central mechanism to modulate cell morphology.

Effect of trichostatin A on Burkitt's lymphoma cells: Inhibition of EPS8 activity through Phospho-Erk1/2 pathway.

Histone deacetylase inhibitors (HDACi) manifest great potential for treatment of Burkitt's lymphoma (BL), an aggressive B-cell lymphoma. Epidermal growth factor receptor pathway substrate 8 (EPS8) is confirmed overexpressed and associated with poor prognosis in solid tumors and leukemia. However, EPS8 expression and the relationship between EPS8 and HDACi on BL remains obscure. Here, we hypothesized that trichostatin A (TSA), a pan-HDACi, could inhibit BL cells by downregulating EPS8. We demonstrated that TSA reduced cell viability, induced apoptosis and cell arrest at G0/G1. Mechanismly, TSA attenuated EPS8 and downstream Phospho-Erk1/2 pathway. Knockdown of EPS8 resulted in a significant reduction in cellular proliferation and suppressed Phospho-Erk1/2 pathway activity, particularly when combined with TSA. Conversely, overexpression of EPS8 rescued this phenomenon. Then we showed that the combination of TSA and Epirubicin had a more significant effect when compared with TSA or Epirubicin alone. Finally, knockdown of EPS8 and TSA had a synergistic suppression effect on BALB/c nude mice. In conclusion, this study reveals that TSA affects BL cells by suppressing Phospho-Erk1/2 pathway through downregulating EPS8.

Pro-migratory and TGF-ß-activating functions of αvß6 integrin in pancreatic cancer are differentially regulated via an Eps8-dependent GTPase switch.

The integrin αvß6 is up-regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvß6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF-ß1; this latter function complicates therapeutic targeting of αvß6, since TGF-ß1 has both tumour-promoting and -suppressive effects. It is unclear how these different αvß6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF-ß1 by exerting mechanical tension on the ECM-bound latent complex. We examined the functional relationship between cell invasion and TGF-ß1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvß6-dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF-ß1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvß6, we found that Eps8 was up-regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvß6-dependent cell migration through activation of Rac1. Down-regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvß6-dependent TGF-ß1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvß6-dependent functions, resulting in a pro-migratory (Rac1-dependent) or a pro-TGF-ß1 activation (Rho-dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PTK6 Localized at the Plasma Membrane Promotes Cell Proliferation and MigratiOn Through Phosphorylation of Eps8.

Protein tyrosine kinase 6 (PTK6; also known as Brk) is closely related to the Src family kinases, but lacks a membrane-targeting myristoylation signal. Sublocalization of PTK6 at the plasma membrane enhances its oncogenic potential. To understand the mechanism(s) underlying the oncogenic property of plasma---membrane-associated PTK6, proteins phosphorylated by membrane-targeted myristoylated PTK6 (Myr-PTK6) were enriched and analyzed using a proteomics approach. Eps8 which was identified by this method is phosphorylated by Myr-PTK6 in HEK293 cells. Mouse Eps8 expressed in HEK293 cells is phosphorylated by Myr-PTK6 at residues Tyr497, Tyr524, and Tyr534. Compared to wild-type Eps8 (Eps8 WT), the phosphorylation-defective 3YF mutant (Eps8 3YF) reverts the increased proliferation, migration, and phosphorylation of ERK and FAK mediated by Eps8 WT in HEK293 cells overexpressing PTK6. PTK6 knockdown in T-47D breast cancer cells decreased EGF-induced phosphorylation of Eps8. Endogenous PTK6 phosphorylates ectopically expressed Eps8 WT, but not Eps8 3YF mutant, in EGF-stimulated T-47D cells. The EGF-induced Eps8 phosphorylation enhances activation of ERK and FAK, cell adhesion, and anchorage-independent colony formation in T-47D cells, but not in the PTK6-knokdown T-47D cells. These results indicate that plasma-membrane-associated PTK6 phosphorylates Eps8, which promotes cell proliferation, adhesion, and migration and, thus, tumorigenesis. J. Cell. Biochem. 118: 2887-2895, 2017. © 2017 Wiley Periodicals, Inc.

Intersectin-1s deficiency in pulmonary pathogenesis.

Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFßRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EHITSN and SH3A-EITSN fragments. The EHITSN fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EHITSN plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.

Identification and first characterization of SmEps8, a potential interaction partner of SmTK3 and SER transcribed in the gonads of Schistosoma mansoni.

In eukaryotes the roles of protein kinases (PKs) regulating important biological processes such as growth and differentiation are well known. Molecular, biochemical, and physiological analyses trying to unravel principles of schistosome development have substantiated the importance for PKs also in this parasite. Amongst others the role of SmTK3 was studied, one of the first cellular PKs characterized from Schistosoma mansoni. Its function was demonstrated in mitogenic and differentiation processes in the gonads. Furthermore, first insights were obtained for the downstream part of a signal transduction cascade SmTK3 is involved in, which includes the diaphanous homolog SmDia. Here we attempted to further unravel the SmTK3 signaling cascade by searching for upstream interaction partners. Using yeast three-hybrid (Y3H) analyses we detected the epidermal growth factor receptor (EGFR) pathway substrate 8 of S. mansoni (SmEps8) as the most interesting candidate. By detailed interaction analyses we showed a contribution of the Src homology (SH) domains SH2 and SH3 of SmTK3 to binding, with a clear bias towards SH2. Compared to full-length SmEps8, binding was enhanced when only its 5' part including the phosphotyrosine binding domain (PTB) was used for interaction analyses including the SH2 domain of SmTK3, although phosphorylation seemed not to play a decisive role for binding. RT-PCR analyses and in situ hybridization experiments demonstrated similar transcription patterns of SmTK3 and SmEPS8, which co-localize in the reproductive organs. Furthermore, first evidence was obtained for SmEps8 interaction and colocalization with SER, one of the epidermal growth factor receptor (EGFR) homologs detected in S. mansoni. The results of this study provide first evidence for a SER-SmEps8-SmTK3-SmDia signal transduction pathway controlling differentiation processes in the gonads of S. mansoni.

Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis.

BACKGROUND: The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. Intersectin-1s (ITSN-1s) is an important multidomain adaptor protein implicated in the pathophysiology of numerous pulmonary diseases. To date, the role of ITSN-1s in LC has not been studied. METHODS: Human LC cells, human LC tissue and A549 LC cells stable transfected with myc-ITSN-1s construct (A549 + ITSN-1s) were used in correlation with biochemical, molecular biology and morphological studies. In addition scratch assay with time lapse microscopy and in vivo xenograft tumor and mouse metastasis assays were performed. RESULTS: ITSN-1s, a prevalent protein of lung tissue, is significantly downregulated in human LC cells and LC tissue. Restoring ITSN-1s protein level decreases LC cell proliferation and clonogenic potential. In vivo studies indicate that immunodeficient mice injected with A549 + ITSN-1s cells develop less and smaller metastatic tumors compared to mice injected with A549 cells. Our studies also show that restoring ITSN-1s protein level increases the interaction between Cbl E3 ubiquitin ligase and Eps8 resulting in enhanced ubiquitination of the Eps8 oncoprotein. Subsequently, downstream unproductive assembly of the Eps8-mSos1 complex leads to impaired activation of the small GTPase Rac1. Impaired Rac1 activation mediated by ITSN-1s reorganizes the cytoskeleton (increased thick actin bundles and focal adhesion (FA) complexes as well as collapse of the vimentin filament network) in favor of decreased LC cell migration and metastasis. CONCLUSION: ITSN-1s induced Eps8 ubiquitination and impaired Eps8-mSos1 complex formation, leading to impaired activation of Rac1, is a novel signaling mechanism crucial for abolishing the progression and metastatic potential of LC cells.

Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells. It forms a complex with both focal adhesion kinase (FAK, also known as PTK2) and Src in SCC cells derived from skin carcinomas induced by administration of the chemical DMBA followed by TPA (the DMBA/TPA model). Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with Src and drives trafficking of Src to autophagic structures that SCC cells use to cope with high levels of active Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, thereby meaning active Src becomes tethered at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

The angiopoietin1-Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8.

In mammalian central nervous system (CNS), the integrity of the blood-spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood-brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1-Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8.

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.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

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, ß-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.