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Meningioma is the most common primary intracranial tumor in adults, with up to 10% manifesting as multiple tumors. Data on the genomic and molecular changes in sporadic multiple meningiomas are scarce, leading to ongoing debates regarding their evolutionary processes. A comprehensive genetic analysis of a large number of lesions, including precursor lesions, is necessary to explore these two possible origins: clonal and independent. In the present study, we performed whole-exome sequencing and analyzed somatic single-nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number alterations (CNAs) in a patient with sporadic multiple meningiomas. These meningiomas included two mass-forming lesions of different histological subtypes (transitional and chordoid) and two small meningothelial nests. Genetic analysis revealed CNAs on chromosomes 22q and Y as common abnormalities in the two largest tumors. Furthermore, we identified SNV/INDELs unique to each focus, with NF2 mutation prevalent in the transitional meningioma and CREBBP mutation in the chordoid meningioma. Loss of chromosome 22 was detected in two small meningothelial nests. Overall, we elucidated the clonal origin and subtype-specific evolution of multiple meningiomas in this case. CNAs may serve as the initial driving event in meningioma development., (© 2024. The Author(s).)

Metastasis to the liver is a leading cause of death in patients with colorectal cancer. To investigate the characteristics of cancer cells prone to metastasis, we utilized an isogenic model of BALB/c and colon tumor 26 (C26) cells carrying an active KRAS mutation. Liver metastatic (LM) 1 cells were isolated from mice following intrasplenic transplantation of C26 cells. Subsequent injections of LM1 cells generated LM2 cells, and after four cycles, LM4 cells were obtained. In vitro, using a perfusable capillary network system, we found comparable extravasation frequencies between C26 and LM4 cells. Both cell lines showed similar growth rates in vitro. However, C26 cells showed higher glucose consumption, whereas LM4 cells incorporated more fluorescent fatty acids (FAs). Biochemical analysis revealed that LM4 cells had higher cholesterol levels than C26 cells. A correlation was observed between fluorescent FAs and cholesterol levels detected using filipin III. LM4 cells utilized FAs as a source for cholesterol synthesis through acetyl-CoA metabolism. In cellular analysis, cholesterol accumulated in punctate regions, and upregulation of NLRP3 and STING proteins, but not mTOR, was observed in LM4 cells. Treatment with a cholesterol synthesis inhibitor (statin) induced LM4 cell death in vitro and suppressed LM4 cell growth in the livers of nude mice. These findings indicate that colorectal cancer cells prone to liver metastasis show cholesterol-dependent growth and that statin therapy could help treat liver metastasis in immunocompromised patients., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Nodal T-follicular helper cell lymphoma (TFHL) is a subset of T-cell lymphoma and frequently co-occurs with Epstein-Barr virus (EBV)-positive B-cell lymphoma but not with T/NK-cell lymphoma. Recently, a new entity with a worse prognosis, called EBV-positive nodal T/NK-cell lymphoma (NTNKL) has been established. Here, we report an autopsy case of synchronous multiple lymphomas, including TFHL and NTNKL. The patient was a 78-year-old female admitted with pneumonia. Although pneumonic symptoms were improved, fever, pancytopenia, and disseminated intravascular coagulation emerged, implicating lymphoma. She died on the 21st hospital day without a definitive diagnosis. The autopsy revealed the enlargement of multiple lymph nodes throughout her body. Histological analysis revealed three distinct regions in the left inguinal lymph node. The first region consists of small-sized lymphocytes with T-follicular helper phenotype and extended follicular dendritic cell meshwork, indicating TFHL. The second region included EBV-positive large B cells. The third region comprised EBV-positive large cells with cytotoxic T/NK cell phenotype, indicating NTNKL. Clonality analysis of the first and the third regions showed different patterns. Since various hematopoietic malignancies progress from common clonal hematopoiesis according to existing literature, this case may help to understand TFHL and NTNKL., (© 2024 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Cancer cells at the invasive front are believed to be responsible for invasion/metastasis. This has led to examining various morphological features and protein expressions at the invasive front. However, accurate assessment of the pathological section requires long-time training, and inter-observer disagreement is problematic. Immunohistochemistry and digital imaging analysis may mitigate these problems; however, the choice of which proteins to stain and the best analysis method remains controversial. We used the "go-or-grow" hypothesis to select markers with the greatest prognostic relevance. Importantly, nonproliferating cells can migrate. We used Ki67 as a proliferation marker, with p16 and p21 designating nonproliferating cells. We established a semi-automated quantification workflow to study protein expression in serial pathological sections. A total of 51 patients with completely resected colorectal cancer (stages I-IV) were analyzed, and 44 patients were followed up. Patients with cancer cells with p16-high/p21-low or p21-low/Ki67-low at the deepest invasive front demonstrated a significantly worse prognosis than those who did not display these characteristics. These results suggest that the nonproliferating cancer cells at the invasion front possess invasion/metastatic property with heterogeneity of senescence., (© 2022 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Tropomyosin (Tpm) 1 and 2 are important in the epithelial mesenchymal transition of lens epithelial cells; however, the effect of Tpm1 depletion during aging remains obscure. We analyzed the age-related changes in the crystalline lens of Tpm1- conditional knockout mice (Tpm1-CKO). Floxed alleles of Tpm1 were conditionally deleted in the lens, using Pax6-cre transgenic mice. Lenses of embryonic day (ED) 14, postnatal 1-, 11-, and 48-week-old Tpm1-CKO and wild type mice were dissected to prepare paraffin sections, which subsequently underwent histological and immunohistochemical analysis. Tpm1 and α smooth muscle actin (αSMA) mRNA expression were assessed using RT-PCR. The homozygous Tpm1-CKO (Tpm1 -/- ) lenses displayed a dramatic reduction in Tpm1 transcript, with no change to αSMA mRNA expression. Tpm1 -/- mice had small lenses with disorganized, vesiculated fiber cells, and loss of epithelial cells. The lenses of Tpm1 -/- mice had abnormal and disordered lens fiber cells with cortical and peri-nuclear liquefaction. Expression of filamentous-actin was reduced in the equator region of lenses derived from ED14, 1-, 11-, and 48-week-old Tpm1 -/- mice. Therefore, Tpm1 plays an integral role in mediating the integrity and fate of lens fiber differentiation and lens homeostasis during aging. Age-related Tpm1 dysregulation or deficiency may induce cataract formation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)β-2 showed a decrease in DCN expression. The expression of tropomyosin 1 ( Tpm1 ), a marker of lens EMT increased after the addition of TGFβ-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN ( hDCN-Tg ). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFβ-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow., Competing Interests: The authors have declared that no competing interests exist.

A sight threatening, pterygium is a common ocular surface disorders identified by fibrovascular growth of the cornea and induced by variety of stress factors, like ultraviolet (UV) exposure. However, the genes involved in the etiopathogenesis of this disease is not well studied. Herein, we identified the gene expression pattern of pterygium and examined the expression of pterygium-related genes in UV-B-induced human primary cultured corneal epithelial cells (HCEpCs), telomerase immortalized human corneal epithelial (hTCEpi), primary conjunctival fibroblast (HConFs) and primary pterygium fibroblast cells (HPFCs). A careful analysis revealed that the expression of 10 genes was significantly modulated (by > 10-fold). Keratin 24 (KRT24) and matrix metalloproteinase 9 (MMP-9) were dramatically upregulated by 49.446- and 24.214-fold, respectively. Intriguingly, UV-B exposure (50 J/m 2 ) induced the upregulation of the expressions of MMP-9 in corneal epithelial cells such as HCEpCs and hTCEpi. Furthermore, UV-B exposure (100 and/or 200 J/m 2 ) induced the upregulation of the expressions of MMP-9 in fibroblast such as HConFs and HPFCs. The exposure of HCEpCs to 100 and 200 J/m 2 UV-B induced significant expressions of KRT24 mRNA. Nevertheless, no expression of KRT24 mRNA was detected in HConFs and HPFCs. The findings provide evidence that the progression of pterygium may involve the modulation of extracellular matrix-related genes and vasculature development and the up-regulation of KRT24 and MMP-9 by UV stress. UV radiation may promote the modulation of these pterygium-related genes and induce the initiation and progression of human pterygium., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Among the members of the DOCK family, DOCK1-5 function as guanine-nucleotide exchange factors for small GTPase Rac1, which regulates the actin cytoskeleton. It has been reported that in model organisms the Dock-Rac axis is required for myoblast fusion. We examined the role of DOCK1-5 in trophoblast fusion herein., Methods: We used a quantitative polymerase chain reaction (qPCR) to examine the mRNA expressions of DOCK1-5 and differentiation-related genes, i.e., fusogenic genes, in human trophoblastic cell lines, BeWo and JEG-3. We treated BeWo cells with TBOPP and C21 to inhibit DOCK1 and DOCK5. Cell dynamics and cell fusion were assessed by live imaging and immunostaining. The signaling pathways induced by DOCK1/5 inhibition were examined by western blotting., Results: DOCK1 and DOCK5 were expressed in BeWo cells. The inhibition of DOCK1 or DOCK5 did not prevent the cell fusion induced by forskolin (a common reagent for cell fusion); it induced cell fusion. DOCK1 inhibition induced cell death, as did forskolin. DOCK1 and DOCK5 inhibition for 24 and 48 h increased the expression of the genes ASCT2 and SYNCYTIN2, which code responsive proteins of trophoblast cell fusion, respectively., Conclusion: DOCK1 and DOCK5 inhibition participates in BeWo cell fusion, probably via pathways independent from forskolin-mediated pathways., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. All of the authors contributed significantly and are in agreement with the contents of the manuscript. This study did not involve human participants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brain metastasis is an ineffective process, and many cancer cells enter into an indolent state following extravasation in the brain. Single cell RNA sequencing of melanoma brain metastases reveals that non-proliferating brain metastatic melanoma cells exhibit a pattern of gene expression associated with inhibition of DNA methyltransferase 1 (DNMT1). The brain microenvironment, specifically the combination of reactive astrocytes and mechanically soft surroundings, suppressed DNMT1 expression in various cancer types and caused cell cycle delay. Somewhat unexpectedly, we find that DNMT1 suppression not only induces cell cycle delay but also activates pro-survival signals in brain metastatic cancer cells, including L1CAM and CRYAB . Our results demonstrate that transcriptional changes triggered by DNMT1 suppression is a key step for cancer cells to survive in the brain microenvironment and that they also restrict cancer cell proliferation. The dual consequences of DNMT1 suppression can explain the persistence of indolent cancer cells in the brain microenvironment., Competing Interests: S.Y. obtained commercial research grants from AstraZeneca, Chugai Pharm, and Boehringer-Ingelheim and has received speaking honoraria from AstraZeneca, Chugai Pharma, and Boehringer-Ingelheim. Other authors declare no competing interests., (© 2020 The Author(s).)

Multicellular structures, such as tumor buddings and poorly differentiated clusters (PDC), exist at the invasive front of colorectal cancers (CRC). Although it has been reported that CRC with PDC showed frequent lymph node metastases with a worse prognosis, the molecular markers of PDC that are responsible for prognosis have not been identified. We here noticed for the first time that Ezrin, a regulator of the actin cytoskeleton, is expressed in the corner cells of PDC. We then aimed to verify whether heterogeneous Ezrin expression in PDC predicts the prognosis of CRC patients. We immunohistochemically analyzed Ezrin expression in PDC of 184 patients with completely resected stages I-III CRC. We established the Ezrin corner score (ECS), which quantifies the tendency of Ezrin-positive cells to accumulate at the corners of PDC. On the basis of ECS values, 2 indices, the mean ECS and the number of PDC with high ECS, were obtained. Both indices were significantly higher in CRC with lymphatic invasion, higher PDC grade, and presence of micropapillary (MP) PDC. The mean ECS-high group showed shorter recurrence-free survival than the mean ECS-low group but without significance. The other index, the number of ECS-high PDC, was significantly associated with recurrence-free survival. These results suggest that Ezrin is involved in PDC progression and lymphatic invasion, and that ECS may be a marker for aggressive PDC., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ.

Cancer stem-like cells (CSCs) are expanded in the CSC niche by increased frequency of symmetric cell divisions at the expense of asymmetric cell divisions. The symmetric division of CSCs is important for the malignant properties of cancer; however, underlying molecular mechanisms remain largely elusive. Here, we show a cytokine, semaphorin 3 (Sema3), produced from the CSC niche, induces symmetric divisions of CSCs to expand the CSC population. Our findings indicate that stimulation with Sema3 induced sphere formation in breast cancer cells through neuropilin 1 (NP1) receptor that was specifically expressed in breast CSCs (BCSCs). Knockdown of MICAL3 , a cytoplasmic Sema3 signal transducer, greatly decreased tumor sphere formation and tumor-initiating activity. Mechanistically, Sema3 induced interaction among MICAL3, collapsin response mediator protein 2 (CRMP2), and Numb. It appears that activity of MICAL3 monooxygenase (MO) stimulated by Sema3 is required for tumor sphere formation, interaction between CRMP2 and Numb, and accumulation of Numb protein. We found that knockdown of CRMP2 or Numb significantly decreased tumor sphere formation. Moreover, MICAL3 knockdown significantly decreased Sema3-induced symmetric divisions in NP1/Numb-positive BCSCs and increased asymmetric division that produces NP1/Numb negative cells without stem-like properties. In addition, breast cancer patients with NP1-positive cancer tissues show poor prognosis. Therefore, the niche factor Sema3-stimulated NP1/MICAL3/CRMP2/Numb axis appears to expand CSCs at least partly through increased frequency of MICAL3-mediated symmetric division of CSCs., Competing Interests: Conflict of interest statement: N.G., K.T., and A.T. applied for patent pending (2015-132122) in Japan., (Copyright © 2019 the Author(s). Published by PNAS.)

At the invasive front of adenocarcinomas, single cells and multicellular structures exist; the latter include glands and cell clusters, such as tumor buddings and poorly differentiated clusters. Recent reports suggest the importance of collective cell migration in metastasis; however, it is technically difficult to observe the movement of multicellular structures in vivo. We utilized MDCK cells as a model for epithelial cells and established a method to quantify their motility in 3D structures in vitro. A single MDCK cell grows as a cell cluster in the gel and later proliferates and forms a cyst. Active K-RAS expression induced rotation of both the cell clusters and the cysts. The rotation speed of cell clusters was 4 times higher than that of cysts. The screening of inhibitors for their effects on cell clusters and cysts revealed that cyclin B1 and β-catenin were the key molecules for their rotation, respectively. Regulators for cyst rotation, such as vorinostat and β-catenin, were not effective for inducing cell cluster rotation. These results indicate that the signaling pathways of cell dynamics are different between cell clusters and cysts. As cell clusters are related to lymph node involvement and the prognosis of various carcinomas, our in vitro quantitative system may be useful for the screening of drugs to prevent lymphatic invasion., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The process of epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 43.2 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The enteric nervous system (ENS) is a network of neurons and glia that are derived from enteric neural crest cells (ENCCs) and essential for regulating peristaltic activity of the colon. ENCCs migrate along the gastrointestinal tract to form the ENS, and disruption of ENCC motility leads to ENS disorders, such as Hirschsprung's disease. Previous ENCC-transplant experiments show that ENCCs can invade into isolated mouse intestines by age E13.5, but not after E15.5. We hypothesized that altered age-specific micro-environments in the intestine are responsible for ENCC invasion/migration. Here, we compared gene expression in the intestine between at E11.5 and E15.5 and identified 1355 differentially expressed transcripts. Among these, we found that genes encoding extracellular matrix (ECM) proteins were enriched. Notably, collagen VI (ColVI) family members were upregulated in the E15.5 mouse intestine at the mRNA and protein levels, whereas fibronectin (FN) was downregulated; however, both proteins showed colocalization at E15.5. To understand the mechanisms of ColVI and FN in ENCC migration, we examined neurosphere or individual ENCC-adherence capabilities toward the ECM. ColVI suppressed FN-induced ENCC spreading/migration, whereas ColVI induced morphologically narrow ENCC spreading and weak stress-fiber formation as compared with those with FN. Additionally, in ENCCs cultured on plates containing ColVI, the expression and phosphorylation of p130 Cas , a members of focal adhesion complexes, was reduced. These data indicated an inhibitory role of ColVI in ENCC migration and suggested that ColVI suppression in the intestine might represent a novel therapeutic strategy for aganglionic colonic diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Rupture of lens cataract (RLC) is a hereditary mouse model that shows spontaneous rupture of the lens at the posterior pole at 45-100 days of age. The responsible gene for this phenotype was identified as Dock5 , a guanine nucleotide exchange factor for small GTPase Rac1. This study was performed to elucidate the pathway initiating this phenotype., Methods: We examined the RNA expression by microarray in lens epithelial cells (LECs) from wild-type and RLC mice at the pre-rupture age of 21 days. We applied the list of altered genes to an Ingenuity Pathway Analysis (IPA) to predict the pathways that are altered upon dedicator of cytokinesis-5 (Dock5) protein loss. The activation status of the predicted pathways was examined by western blotting in the cultured epithelial cells treated with a Dock5 inhibitor., Results: The highest-scored network was "Antimicrobial Response, Inflammatory Response, Dermatological Diseases and Conditions." In that network, it is predicted that extracellular signal-regulated kinase (Erk) is activated in LECs from RLC mice. Our test confirmed that Erk was more phosphorylated in the LECs at the equator in both Dock5-knockout mice and RLC mice. In an in vitro experiment of the cultured epithelial cells, the inhibition of Dock5 activity significantly induced Erk activation. It was also confirmed that Akt (cellular homolog of murine thymoma virus akt8 oncogene, also called protein kinase B) and nuclear factor-kappa B (NFκB), predicted to be the key molecules in two other high-scoring networks by IPA, were activated upon Dock5 inhibition in the cultured epithelial cells., Conclusions: Dock5 participates in epithelial cell maintenance by regulating gene expression.

Intestinal epithelial cells function as a barrier to protect our body from various agents; therefore, any damage to these cells must be immediately repaired. Several in vivo and vitro studies have shown the involvement of Erk (extracellular signal-regulated kinase) in the regeneration process; however, the spatial regulation of Erk related to tissue morphology has not been well documented. Using two-photon microscopy and mice carrying a Förster resonance energy transfer-based biosensor, we here monitored the Erk activity in the ileal epithelial cells of living mice. Forty-eight h after ischemia-induced injury, epithelial cells were observed as a monolayer covering the injured area. The Erk activity in these cells was higher than that in the epithelial cells at the surrounding crypts, and treatment with an epidermal growth factor receptor inhibitor cancelled the higher Erk activity. The resealing epithelial cells were not in the G 2 /M phase of the cell cycle, and Yap (Yes-associated protein) was localized to the nucleus. Immunostaining of intestinal ulcers from patients revealed ERK phosphorylation and nucleus localization of YAP without Ki-67 staining in the resealing epithelial cells. These findings led us to propose that the YAP-EGFR-ERK axis is involved in migration, but not in proliferation, of the resealing epithelial cells.

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial-mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up-regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F-actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up-regulation of α-smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock-down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ-treated LECs perturbed EMT by reactivating the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2-mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs-driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to conventional cancer therapy. However, it is still unclear how they are maintained in tumor tissues. Here, we show that the growth differentiation factor 15 (GDF15), a member of the TGFβ family, may maintain cancer stem-like cells in breast cancer tissues by inducing its own expression in an autocrine/paracrine manner. We found that GDF15, but not TGFβ, increased tumor sphere formation in several breast cancer cell lines and patient-derived primary breast cancer cells. As expected, TGFβ strongly stimulated the phosphorylation of Smad2. GDF15 also stimulated the phosphorylation of Smad2, but the GDF15-induced tumor sphere forming efficiency was not significantly affected by treatment with SB431542, an inhibitor of the TGFβ signaling. Although TGFβ transiently activated ERK1/2, GDF15 induced prolonged activation of ERK1/2. Treatment with U0126, an inhibitor of the MEK-ERK1/2 signaling, greatly inhibited the GDF15-induced tumor sphere formation. Moreover, cytokine array experiments revealed that GDF15, but not TGFβ, is able to induce its own expression; furthermore, it appears to form an autocrine/paracrine circuit to continuously produce GDF15. In addition, we found heterogeneous expression levels of GDF15 among cancer cells and in human breast cancer tissues using immunohistochemistry. This may reflect a heterogeneous cancer cell population, including cancer stem-like cells and other cancer cells. Our findings suggest that GDF15 induces tumor sphere formation through GDF15-ERK1/2-GDF15 circuits, leading to maintenance of GDF15high cancer stem-like cells. Targeting GDF15 to break these circuits should contribute to the eradication of tumors.

Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functioning. The roles of lipids are not only to generate the membrane, but also to provide the specific domains for signal transduction, or to transmit signals as second messengers. By using a liquid chromatography-electrospray ionization mass spectrometry (LC-MS)/MS method, we here analyzed sphingolipids in MDCK cysts under various conditions. Our result showed that, compared to the three-dimensional cyst, the two-dimensional MDCK sheet is relatively enriched in sphingolipids. During cystogenesis, the contents of sphingomyelin (SM) and lactocylceramide (LacCer)-but, none those of ceramide, hexocylceramide, or GM3-are altered depending on their acyl chains. While the total SM is decreased more efficiently by SMS-1 knockdown than by SMS-2 knockdown, depletion of SMS-2, but not SMS-1, inhibits cyst growth. Finally upon the switching on of activated K-Ras expression which induces luminal cell filling, ceramide and LacCer are increased. Our parallel examinations of the microarray data for mRNA of sphingolipid metabolic enzymes failed to fully explain the remodelling of the sphingolipids of MDCK cysts. However, these results should be useful to investigate the cell-type- and structure-specific lipid metabolism., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Purpose. This study investigated the effects of oral propolis on the progression of galactose-induced sugar cataracts in rats and the in vitro effects of propolis on high-glucose-induced reactive oxygen species (ROS) and cell death in cultured rat lens cells (RLECs). Methods. Galactose-fed rats and RLECs cultured in high glucose (55 mM) medium were treated with propolis or vehicle control. Relative lens opacity was assessed by densitometry and changes in lens morphology by histochemical analysis. Intracellular ROS levels and cell viability were measured. Results. Oral administration of propolis significantly inhibited the onset and progression of cataract in 15% and 25% of galactose-fed rats, respectively. RLECs cultured with high glucose showed a significant increase in ROS expression with reduced cell viability. Treatment of these RLECs with 5 and 50 μg/mL propolis cultured significantly reduced ROS levels and increased cell viability, indicating that the antioxidant activity of propolis protected cells against ROS-induced damage. Conclusion. Propolis significantly inhibited the onset and progression of sugar cataract in rats and mitigated high-glucose-induced ROS production and cell death. These effects may be associated with the ability of propolis to inhibit hyperglycemia-evoked oxidative or osmotic stress-induced cellular insults.

Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology must be maintained for their proper function. To examine the genes that are specifically expressed in the late stages of cystogenesis and are involved in maintaining the morphology of the mature cysts, we performed a microarray analysis comparing the mRNA expression between the early and late stages of Madin-Darby Canine Kidney (MDCK) cystogenesis. We found that one of the gene candidates, Ripply1, was expressed higher in the late stages, and its expression was also transiently much higher in the early stages. Although the protein expression showed similar kinetics, depletion of Ripply1 had only a slight effect on organoid growth. Unexpectedly, we found that the Ripply1 protein is degraded by the proteasome system. Mutant analysis suggests that Ripply1 is not ubiquitinated directly, but rather is degraded only after binding to Transducin-like Enhancer of Split (TLE)1, a transcriptional repressor. Ripply1 is degraded in the nucleus, and this degradation is inhibited during the mitosis. These data indicate for the first time that Ripply1 expression is regulated at the protein level., (Copyright © 2015 Elsevier Inc. All rights reserved.)

RAS genes are frequently mutated in various human tumours. These mutations cause GTPase RAS to remain locked in constitutively active signals through the downstream cascades leading to proliferation. A series of earlier studies reported on the morphological appearance of cells upon RAS activation. Classically, morphologic changes of fibroblasts have been used to confirm the oncogenic activity of RAS. Recent works found that the active RAS induces benign and malignant morphological changes in organoids, which are an in vitro model system for epithelial tissue. The studies of animal models support the basic oncogenic features of RAS revealed in vitro, while also providing evidence that the effects of RAS activation in vivo are different from those in vitro. The pathological observation of the various human materials indicates that the oncogenic RAS participates in metaplasia, which occurs before proliferation, and that RAS promotes mucin production in various organs. These morphological analyses may shed light on important signalling pathways that merit investigation in vitro., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each chemical mediator have been demonstrated with neutrophils in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue. ERK activity in neutrophils rapidly increased during spreading on the endothelial cells and showed positive correlation with the migration velocity on endothelial cells or in interstitial tissue. Meanwhile, in the neutrophils migrating in the interstitial tissue, high PKA activity correlated negatively with migration velocity. In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils. The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs). Therefore, NSAID-induced enteritis may be caused at least partially by the inhibition of EP4 receptor signaling of neutrophils. Our results demonstrate that ERK positively regulates the neutrophil recruitment cascade by promoting adhesion and migration steps., (© 2014 Mizuno et al.)

Rac1b is frequently expressed in a number of human cancer cells. It is still unclear, however, whether Rac1b causes morphological abnormalities in epithelial tissues. To investigate whether Rac1b induces morphological changes in 3-dimensional epithelial structures, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate Rac1b function in MDCK (Madin-Darby Canine Kidney) cysts, a model for polarized epithelial structure. Cells carrying the wild-type Rac1, Rac1b and constitutively active Rac1V12 gene were morphologically indistinguishable from normal, when their coding proteins were not expressed. However, upon protein induction, Rac1V12, but not the wild-type Rac1 or Rac1b, significantly induced the luminal cell accumulation. Live cell imaging with cell cycle indicators showed that expression of Rac1V12, but not the wild-type Rac1 or Rac1b, promoted cell cycle progression. From these results, we concluded that the expression of Rac1b per se cannot induce cell proliferation. Rather, it is considered that Rac1b expression may participate in progression of malignancy.

In a number of human cancer cells, K-RAS is frequently mutated and activated constitutively, culminating in the induction of continuous cell growth, a hallmark of cancer cells. It is still unclear, however, how the mutated K-RAS induces morphological abnormalities in cancerous tissues. To investigate the mechanism underlying the K-RAS-induced morphological changes, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate constitutively active K-Ras in MDCK (Madin-Darby canine kidney) cysts, a model for polarized epithelial structure. Cells carrying the constitutively active KRasV12 gene were morphologically indistinguishable from normal cells in two-dimensional culture. However, in a gel of extracellular matrix, KRasV12-expressing cells failed to form a spherical cyst. When KRasV12 induction was delayed until after cyst formation, some cells in the cyst wall lost polarity and were extruded into and accumulated in the luminal space. With effector-specific mutants of KRasV12 and inhibitors for MEK and PI3-kinase, we found that both the Raf-MEK-ERK and PI3-kinase axes are necessary and sufficient for this phenotype. Live cell imaging with cell cycle indicators showed that KRasV12 expression promoted cell cycle progression, which was prevented by either MEK or PI3-kinase inhibitors. From these results, we provide a model wherein active-Ras induces cell cycle progression leading to apical cell extrusion through Raf and PI3-kinase in a cooperative manner. The system developed here can be applied to drug screening for various cancers originating from epithelial cells.

Using MDCK cells that constitutively express a Förster resonance energy transfer biosensor, we found that Rac1 activity is homogenous at the entire plasma membrane in early stages of cystogenesis, whereas in later stages Rac1 activity is higher at the lateral membrane than at the apical plasma membrane. If Rac1 is activated at the apical membrane in later stages, however, the monolayer cells move into the luminal space. In these cells, tight junctions are disrupted, accompanied by mislocalization of polarization markers and disorientation of cell division. These observations indicate that Rac1 suppression at the apical membrane is essential for the maintenance of cyst structure.

Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functions. Previously, we showed that Rac1 activation is suppressed at the apical membrane in the mature organoid, and that such spatially biased Rac1 activity is required for the polarity maintenance. Here we identify Chimaerin, a GTPase activating protein for Rac1, as a suppressor of Rac1 activity at the apical membrane. Depletion of Chimaerin causes over-activation of Rac1 at the apical membrane in the presence of hepatocyte growth factor (HGF), followed by luminal cell accumulation. Importantly, Chimaerin depletion did not inhibit extension formation at the basal membrane. These observations suggest that Chimaerin functions as the apical-specific Rac1 GAP to maintain epithelial morphology.

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P(2) was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P(2) accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP(3) distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Low molecular weight ("small") GTPases are key regulators of a number of signaling cascades. Each GTPase is regulated by numerous guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and each GTPase binds to numerous effector proteins in a GTP-dependent manner. In many instances, individual regulators activate more than one GTPase, and each effector binds to one or more GTPases belonging to the same family. To untangle these complex networks, probes based on the principle of Förster resonance energy transfer (FRET) are widely used. Here, we provide an overview of the probes based on FRET and examples of discoveries achieved with them. In the process, we attempt to delineate the merits, current limitations, and future applications of this technique to pharmacological studies.

Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Förster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factor-induced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.

Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.

DOCK180 is an atypical guanine nucleotide exchange factor of Rac1 identified originally as one of the two major proteins bound to the SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. Recently, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and found that ANKRD28, a protein with twenty-six ankyrin domain-repeats, interacts with the SH3 domain of DOCK180. Knockdown of ANKRD28 reduced the migration velocity and altered the distribution of focal adhesion proteins such as Crk, paxillin and p130(Cas). On the other hand, the expression of ANKRD28, p130(Cas), Crk and DOCK180 induced hyper-phosphorylation of p130(Cas), which paralleled the induction of multiple long cellular processes. Depletion of ELMO, another protein bound to the SH3 domain of DOCK180, also retarded cell migration, but its expression together with p130(Cas), Crk and DOCK180 induced extensive lamellipodial protrusion around the entire circumference without 130(Cas) hyperphosphorylation. These data suggest the dual modes of DOCK180-Rac regulation for cell migration.

DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130(Cas). On the other hand, expression of ANKRD28, p130(Cas), Crk, and DOCK180 induced hyper-phosphorylation of p130(Cas), and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway.