Intercellular adhesion molecule-1 suppresses TMZ chemosensitivity in acquired TMZ-resistant gliomas by increasing assembly of ABCB1 on the membrane.

AIMS: Despite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood. METHODS: The TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates. RESULTS: Acquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane. CONCLUSIONS: Our findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma.

Directing with restraint: Mechanisms of protrusion restriction in collective cell migrations.

Cell migration is necessary for morphogenesis, tissue homeostasis, wound healing and immune response. It is also involved in diseases. In particular, cell migration is inherent in metastasis. Cells can migrate individually or in groups. To migrate efficiently, cells need to be able to organize into a leading front that protrudes by forming membrane extensions and a trailing edge that contracts. This organization is scaled up at the group level during collective cell movements. If a cell or a group of cells is unable to limit its leading edge and hence to restrict the formation of protrusions to the front, directional movements are impaired or abrogated. Here we summarize our current understanding of the mechanisms restricting protrusion formation in collective cell migration. We focus on three in vivo examples: the neural crest cell migration, the rotatory migration of follicle cells around the Drosophila egg chamber and the border cell migration during oogenesis.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Moesin deficiency leads to lupus-like nephritis with accumulation of CXCL13-producing patrolling monocytes.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins to the cortical cytoskeleton and thus regulate diverse cellular processes. Mutations in the human moesin gene cause a primary immunodeficiency called X-linked moesin-associated immunodeficiency (X-MAID), which may be complicated by an autoimmune phenotype with kidney involvement. We previously reported that moesin-deficient mice exhibit lymphopenia similar to that of X-MAID and develop a lupus-like autoimmune phenotype with age. However, the mechanism through which moesin defects cause kidney pathology remains obscure. Here, we characterized immune cell infiltration and chemokine expression in the kidney of moesin-deficient mice. We found accumulation of CD4+ T and CD11b+ myeloid cells and high expression of CXCL13, whose upregulation was detected before the onset of overt nephritis. CD4+ T cell population contained IFN-γ-producing effectors and expressed the CXCL13 receptor CXCR5. Among myeloid cells, Ly6Clo patrolling monocytes and MHCIIlo macrophages markedly accumulated in moesin-deficient kidneys and expressed high CXCL13 levels, implicating the CXCL13-CXCR5 axis in nephritis development. Functionally, Ly6Clo monocytes from moesin-deficient mice showed reduced migration toward sphingosine 1-phosphate. These findings suggest that moesin plays a role in regulating patrolling monocyte homeostasis, and that its defects lead to nephritis associated with accumulation of CXCL13-producing monocytes and macrophages.

The role of moesin in diagnosing and assessing severity of lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare disease which is easily misdiagnosed. Vascular endothelial growth factor D (VEGF-D), as the most common biomarker, however, is not so perfect for the diagnosis and severity assessment of LAM. MATERIALS AND METHODS: The isobaric tags for relative and absolute quantitation (iTRAQ)-based method was used to identify a cytoskeleton protein, moesin. 84 patients with LAM, 44 patients with other cystic lung diseases (OCLDs), and 37 healthy control subjects were recruited for collecting blood samples and clinical data. The levels of moesin in serum were evaluated by ELISA. The relationships of moesin with lymphatic involvement, lung function, and treatment decision were explored in patients with LAM. RESULTS: The candidate protein moesin was identified by the proteomics-based bioinformatic analysis. The serum levels of moesin were higher in patients with LAM [219.0 (118.7-260.5) pg/mL] than in patients with OCLDs (125.8 ± 59.9 pg/mL, P < 0.0001) and healthy women [49.6 (35.5-78.9) ng/mL, P < 0.0001]. Moesin had an area under the receiver operator characteristic curve (AUC) of 0.929 for predicting LAM diagnosis compared to healthy women (sensitivity 81.0%, specificity 94.6%). The combination of moesin and VEGF-D made a better prediction in differentiating LAM from OCLDs than moesin or VEGF-D alone. Moreover, elevated levels of moesin were related to lymphatic involvement in patients with LAM. Moesin was found negatively correlated with FEV1%pred, FEV1/FVC, and DLCO%pred (P = 0.0181, r = - 0.3398; P = 0.0067, r = - 0.3863; P = 0.0010, r = - 0.4744). A composite score combining moesin and VEGF-D improved prediction for sirolimus treatment, compared with each biomarker alone. CONCLUSION: Higher levels of moesin in serum may indicate impaired lung function and lymphatic involvement in patients with LAM, suggest a more serious condition, and provide clinical guidance for sirolimus treatment.

Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real time.

Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

Intracellular and extracellular moesins differentially regulate Src activity and ß-catenin translocation to the nucleus in breast cancer cells.

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and ß-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as ß-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, ß-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and ß-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and ß-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and ß-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during Caenorhabditiselegans development.

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.

Identification of Moesin (MSN) as a Potential Therapeutic Target for Colorectal Cancer via the ß-Catenin-RUNX2 Axis.

CRC is the second leading cause of cancer-related death. The complex mechanisms of metastatic CRC limit available therapeutic choice. Thus, identifying new CRC therapeutic targets is essential. Moesin (MSN), a member of the ezrin-radixin-moesin family, connects the cell membrane to the actin-based cytoskeleton and regulates cell morphology. We investigated the role of MSN in the progression of CRC. GENT2 and oncomine were used to study MSN expression and CRC patient outcomes. MSN-specific shRNAs or MSN-overexpressed plasmid were used to establish MSN-KD and MSN overexpressed cell lines, respectively. SRB, migration, wound healing, and flow cytometry were used to test cell survival and migration. Propidium iodide and annexin V stain were used to analyze the cell cycle and apoptosis. MSN expression was found to be higher in CRC tissues than in normal tissues. Higher MSN expression is associated with poor overall survival, disease-free survival, and relapse-free survival rates in CRC patients. MSN silencing inhibits cell proliferation, adhesion, migration, and invasion in vitro, whereas MSN overexpression accelerates cell proliferation, adhesion, migration, and invasion. RNA sequencing was used to investigate differentially expressed genes, and RUNX2 was discovered as a possible downstream target for MSN. In CRC patients, RUNX2 expression was significantly correlated with MSN expression. We also found that MSN silencing decreased cytoplasmic and nuclear ß-catenin levels. Additionally, pharmacological inhibition of ß-catenin in MSN-overexpressed cells led to a reduction of RUNX2, and activating ß-catenin signaling by inhibiting GSK3ß rescued the RUNX2 downregulation in MSN-KD cells. This confirms that MSN regulates RUNX2 expression via activation of ß-catenin signaling. Finally, our result further determined that RUNX2 silencing reduced the ability of MSN overexpression cells to proliferate and migrate. MSN accelerated CRC progression via the ß-catenin-RUNX2 axis. As a result, MSN holds the potential to become a new target for CRC treatment.

Potential Role of Moesin in Regulating Mast Cell Secretion.

Mast cells have existed for millions of years in species that never suffer from allergic reactions. Hence, in addition to allergies, mast cells can play a critical role in homeostasis and inflammation via secretion of numerous vasoactive, pro-inflammatory and neuro-sensitizing mediators. Secretion may utilize different modes that involve the cytoskeleton, but our understanding of the molecular mechanisms regulating secretion is still not well understood. The Ezrin/Radixin/Moesin (ERM) family of proteins is involved in linking cell surface-initiated signaling to the actin cytoskeleton. However, how ERMs may regulate secretion from mast cells is still poorly understood. ERMs contain two functional domains connected through a long α-helix region, the N-terminal FERM (band 4.1 protein-ERM) domain and the C-terminal ERM association domain (C-ERMAD). The FERM domain and the C-ERMAD can bind to each other in a head-to-tail manner, leading to a closed/inactive conformation. Typically, phosphorylation on the C-terminus Thr has been associated with the activation of ERMs, including secretion from macrophages and platelets. It has previously been shown that the ability of the so-called mast cell "stabilizer" disodium cromoglycate (cromolyn) to inhibit secretion from rat mast cells closely paralleled the phosphorylation of a 78 kDa protein, which was subsequently shown to be moesin, a member of ERMs. Interestingly, the phosphorylation of moesin during the inhibition of mast cell secretion was on the N-terminal Ser56/74 and Thr66 residues. This phosphorylation pattern could lock moesin in its inactive state and render it inaccessible to binding to the Soluble NSF attachment protein receptors (SNAREs) and synaptosomal-associated proteins (SNAPs) critical for exocytosis. Using confocal microscopic imaging, we showed moesin was found to colocalize with actin and cluster around secretory granules during inhibition of secretion. In conclusion, the phosphorylation pattern and localization of moesin may be important in the regulation of mast cell secretion and could be targeted for the development of effective inhibitors of secretion of allergic and inflammatory mediators from mast cells.

Comparative Study of Immunohistochemical Expression of Moesin and FLOT 1 in OSCC and Their Correlation with Histopathological Prognostic Factors.

AIM: To study immunohistochemical (IHC) expression patterns of Moesin and FLOT 1 in oral squamous cell carcinoma (OSCC) and to correlate it with histopathological prognostic factors. MATERIALS AND METHODS: A cross-sectional study design was conducted on histopathologically diagnosed cases of OSCC. The inclusion criteria were carcinoma of buccal mucosa, tongue, alveolar mucosa, palate, gingiva, the floor of the mouth, retromolar area, and soft palate. The exclusion criteria included cases of squamous cell carcinoma from sites other than the oral cavity, potentially malignant disorders (PMDs), and any pseudomalignancies of the head and neck. Tissue sections were subjected to IHC staining for Moesin and FLOT 1 and the results were subjected to statistical analysis. RESULTS: Moesin showed strong positivity and was significantly associated with the histopathological variables such as lymph nodes and the worst pattern of invasion, whereas FLOT 1 was not associated with any clinical, histopathological, or demographical variable in this study. CONCLUSION: Cytoplasmic detection of Moesin (35.19%) was higher than FLOT 1 (15.74%). There was no statistically significant relationship between the grade of the lesion and Moesin and FLOT 1. CLINICAL SIGNIFICANCE: New emerging prognostic biomarkers can aid to assess the rate of malignant transformation (epigenetic and molecular changes), thereby resulting in early prophylactic conciliation of the disease progression in OSCC. There is an urgent need for introducing these as an interventional therapy for effectively addressing OSCC at an early stage, thus preventing it from further proceeding to the advanced severe stage.

The ERM protein moesin regulates natural killer cell homeostasis in vivo.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins with actin filaments in the cell cortex. Hemizygous mutations in the X-linked moesin gene are associated with primary immunodeficiency with T and B cell lymphopenia, which also affects natural killer (NK) cells in most cases. We previously showed that moesin deficiency in mice substantially affects lymphocyte homeostasis, but its impact on NK cells remains unexplored. Here, we found that in moesin-deficient mice, NK cells were decreased in the peripheral blood and bone marrow but increased in the spleen. Analysis of female heterozygous mice showed a selective advantage for moesin-expressing NK cells in the blood. Moesin-deficient NK cells exhibited increased cell death and impaired signaling in response to IL-15, suggesting that moesin regulates NK cell survival through IL-15-mediated signaling. Our findings thus identify moesin as an NK cell homeostasis regulator in vivo.

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme's activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.

Astragalus polysaccharide (APS) attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis in HCC.

CONTEXT: Astragalus polysaccharide (APS) is a new tumour therapeutic drug, that has an inhibitory effect on a variety of solid tumours. Tumour cell immunosuppression is related to the up-regulation of programmed death ligand 1 (PD-L1). However, whether APS exerts its antitumor effect by regulating PD-L1 remains unclear. OBJECTIVE: To explore whether APS exerts its antineoplastic effect via regulating PD-L1-mediated immunosuppression in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: SMMC-7721 cells were subcutaneous injected into BALB/C mice for HCC model establishment. Mice were intraperitoneally injected with 100, 200 and 400 mg/kg APS for 12 days. Immunohistochemistry (IHC) was performed to assess CD8+ T cells' rate and PD-L1 level in HCC tissues. HCC cells were pre-treated with 0.1, 0.5 and 1 mg/mL APS for 4 h, then were treated with 10 ng/mL IFN-γ 24 h. PD-L1 level and cell apoptosis was detected by flow cytometry. PD-L1 and Moesin (MSN) proteins were measured by western blot. MiR-133a-3p and MSN mRNA levels were assessed by qRT-PCR. The targets of miR-133a-3p were predicted by starBase, and which was verified by dual-luciferase reporter assay. RESULTS: Our findings illustrated that APS dose-dependently inhibited HCC growth tested with IC50 values of 4.2 mg/mL, and IFN-γ-induced PD-L1 expression and attenuated PD-L1-mediated immunosuppression in HCC cells. APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p in HCC cells. Besides, miR-133a-3p targeted to MSN, and MSN inhibited the antitumor effect of APS by maintaining the stability of PD-L1. Moreover, APS attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis. CONCLUSIONS: APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p/MSN axis to develop an antitumor effect. APS may be an effective drug for HCC treatment.

Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction.

During morphogenesis, epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key for the localisation of the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. Here, we applied quantitative fluorescence techniques to show that, during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage-dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain-binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin (also known as Shotgun) turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity.This article has an associated First Person interview with the first author of the paper.

The cell adhesion molecule TMIGD1 binds to moesin and regulates tubulin acetylation and cell migration.

BACKGROUND: The cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell-cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated. RESULTS: TMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of α-tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of α-tubulin and filamentous (F)-actin organization. CONCLUSIONS: TMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of α-tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.

Rapid Increase of Gastrointestinal P-Glycoprotein Functional Activity in Response to Etoposide Stimulation.

We previously reported that exposure of human colon adenocarcinoma (Caco-2) cells to the bitter substance phenylthiocarbamide (PTC) rapidly enhanced the transport function of P-glycoprotein (P-gp). In this study, we investigated the short-term effect of etoposide, another bitter-tasting P-gp substrate, on P-gp transport function in the same cell line. We found that etoposide exposure significantly increased both the P-gp protein level in the plasma membrane fraction and the efflux rate of rhodamine123 (Rho123) in Caco-2 cells within 10 min. The efflux ratio (ratio of the apparent permeability coefficient in the basal-to-apical direction to that in the apical-to-basal direction) of Rho123 in etoposide-treated cells was also significantly increased compared with the control. These results indicated that etoposide rapidly enhances P-gp function in Caco-2 cells. In contrast, P-gp expression in whole cells at both the mRNA and protein level was unchanged by etoposide exposure, compared with the levels in non-treated cells. Furthermore, etoposide increased the level of phosphorylated ezrin, radixin and moesin (P-ERM) proteins in the plasma membrane fraction of Caco-2 cells within 10 min. P-gp functional changes were blocked by YM022, an inhibitor of cholecystokinin (CCK) receptor. These results suggest that etoposide induces release of CCK, causing activation of the CCK receptor followed by phosphorylation of ERM proteins, which recruit intracellular P-gp for trafficking to the gastrointestinal membrane, thereby increasing the functional activity of P-gp.

Moesin as a prognostic indicator of lung adenocarcinoma improves prognosis by enhancing immune lymphocyte infiltration.

BACKGROUND: Ezrin-radixin-moesin (ERM) have been explored in many cancer processes. Moesin, as its component, has also been found to play an important role in the prognosis of cancer patients, tumor metastasis, drug resistance, and others. Especially in regulating the immunity, but most results came from direct studies on immune cells, there is no clear conclusion on whether moesin has similar effects in tumor cells. And moesin has certain research results in many cancers in other aspects, but there are few about moesin in lung adenocarcinoma (LUAD). METHODS: We detect the expression of moesin in 82 LUAD and matched normal tissue samples by immunohistochemistry. Besides, for the pathological feature, we did a detailed statistical analysis. And with the help of various databases, we have done in-depth exploration of moesin's ability to enhance the extent of immune lymphocyte infiltration. RESULTS: Moesin is a poor expression in lung cancer tissues than the corresponding normal samples. And this phenomenon had a strongly associated with the prognosis and TNM stage of these LUAD patients. Moesin can enhance the infiltration of multiple immune lymphocytes in lung cancer. And this may be related to the interaction between moesin and various inflammatory molecules. CONCLUSIONS: Moesin is a newly index for the prognosis of LUAD and improves the prognosis of LUAD patients by regulating a variety of inflammation-related molecules to enhance immune lymphocytes infiltration.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

Ezrin Modulates the Cell Surface Expression of Programmed Cell Death Ligand-1 in Human Cervical Adenocarcinoma Cells.

Cancer cells employ programmed cell death ligand-1 (PD-L1), an immune checkpoint protein that binds to programmed cell death-1 (PD-1) and is highly expressed in various cancers, including cervical carcinoma, to abolish T-cell-mediated immunosurveillance. Despite a key role of PD-L1 in various cancer cell types, the regulatory mechanism for PD-L1 expression is largely unknown. Understanding this mechanism could provide a novel strategy for cervical cancer therapy. Here, we investigated the influence of ezrin/radixin/moesin (ERM) family scaffold proteins, crosslinking the actin cytoskeleton and certain plasma membrane proteins, on the expression of PD-L1 in HeLa cells. Our results showed that all proteins were expressed at mRNA and protein levels and that all ERM proteins were highly colocalized with PD-L1 in the plasma membrane. Interestingly, immunoprecipitation assay results demonstrated that PD-L1 interacted with ERM as well as actin cytoskeleton proteins. Furthermore, gene silencing of ezrin, but not radixin and moesin, remarkably decreased the protein expression of PD-L1 without affecting its mRNA expression. In conclusion, ezrin may function as a scaffold protein for PD-L1; regulate PD-L1 protein expression, possibly via post-translational modification in HeLa cells; and serve as a potential therapeutic target for cervical cancer, improving the current immune checkpoint blockade therapy.