Epithelial Cell Motility Research Articles

Aquaporin-3 mediates ovarian steroid hormone-induced motility of endometrial epithelial cells.

STUDY QUESTIONHow does aquaporin-3 (AQP3) affect endometrial receptivity?SUMMARY ANSWERAQP3, which is regulated by the combination and estrogen (E2) and progesterone (P4), induces epithelial–mesenchymal transition (EMT) of endometrial epithelial cells.WHAT IS KNOWN ALREADYEmbryo implantation is an extremely complex process, and endometrial receptivity is essential for successful embryo implantation. Estrogen and progesterone regulate endometrial receptivity. AQP3, which is regulated by estrogen (E2), increases cell migration and invasion ability by regulating the expression of EMT-related factors and influencing the reorganization of the actin cytoskeleton.STUDY DESIGN, SIZE, DURATIONThis study investigated the pathophysiological significance of AQP3 in human endometrial function during different phases of the menstrual cycle.PARTICIPANTS/MATERIALS, SETTING, METHODSAQP3 expression levels during different phases of the menstrual cycle were measured using immunohistochemical assays. In cells of different receptivity (high-receptive RL95-2 cells and low-receptive HEC-1A cells), the expression of AQP3 was measured using western blotting, qRT-PCR and immunofluorescence assays. Activities of AQP3, and its regulation by E2 and P4, were studied through in-vitro experiments using RL95-2 cells.MAIN RESULTS AND THE ROLE OF CHANCEAQP3 expression in the mid- and late-secretory phases of the human endometrium is significantly higher than in other phases. Since AQP3 expression levels were higher in RL95-2 cells than in HEC-1A cells, mechanisms of AQP3 regulation by E2 and P4 were studied using RL95-2 cells. We provided the first report that P4 up-regulates AQP3 by directly targeting the promoter of the AQP3 gene. The up-regulation of AQP3 expression by a combination of E2 and P4 is significantly higher than that caused by either E2 or P4 alone. Together E2 and P4 promote RL95-2 cell migration and invasion by inducing EMT through AQP3. We also found that AQP3 co-localizes with ezrin and affects the formation of filopodia and lamellipodia during the E2 and P4-induced EMT process but has no effect on the expression of ezrin and F-actin.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONIt is still unclear whether AQP3 is a main regulator of endometrial receptivity or one of several factors influencing the process.WIDER IMPLICATIONS OF THE FINDINGSFurther investigation on AQP3 may contribute to a greater understanding of endometrial receptivity.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the National Natural Scientific Grants of China (No. 31570798), the Program for Liaoning Excellent Talents in University (LR2017042), the Doctoral Scientific Research Foundation of Liaoning province (201601236), and the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences. There are no conflicts of interest.

DPY30 is required for the enhanced proliferation, motility and epithelial-mesenchymal transition of epithelial ovarian cancer cells.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecological malignancies and is known to be associated with the accumulation of various genetic and epigenetic alterations. As a member of the human histone-lysine N-methyltransferase SETD1A (SET1)/histone-lysine N-methyltransferase 2A (MLL) complexes that are required for full SET1/MLL methyltransferase activity, protein dpy-30 homolog (DPY30) catalyzes histone H3K4 methylation, and its dysfunction has been associated with the occurrence of cancer. Therefore, the present study investigated the role of DPY30 in EOC and the potential association between DPY30 expression and the clinicopathological characteristics of EOC. The expression of DPY30 was examined in EOC tissues and cell lines to identify any correlations between the clinico-pathological characteristics of EOC and DPY30 expression, and to determine the effects of DPY30 on EOC cell proliferation, migration and invasion. DPY30 was highly expressed in EOC tissues and cell lines, and high DPY30 expression was significantly associated with notable clinicopathological variables in EOC patients, including International Federation of Gynecology and Obstetrics stage, pathological grade and lymph node metastasis. Functional studies on EOC cell lines demonstrated that DPY30 significantly promoted cell proliferation, migration, and invasion, accelerated cell cycle progression, and promoted epithelial-mesenchymal transition. Chromatin immunoprecipitation assay results revealed that DPY30 regulates histone H3K4 modification via interaction with the vimentin gene promoter, suggesting that DPY30 promotes the transcription of vimentin. Finally, high expression of DPY30 was significantly associated with reduced survival in patients with EOC. The results indicated that DPY30 may act as an oncogene in EOC and thus represents a potential therapeutic target and prognostic marker in EOC.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Circulating monocytes from prostate cancer patients promote invasion and motility of epithelial cells.

BackgroundRecruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor‐derived signals.MethodsBlood monocytes from advanced and stable PCa patients were cultured, and the conditioned media (CM) were collected and analyzed using standard invasion and wound closure assays to measure effects on invasion and motility of PCa tumor cells. We then identified the proteome profile of these monocytes using proteome array and ELISA.ResultsConditioned media from circulating monocytes in patients with metastatic prostate cancer (PCa‐M) increased invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte‐derived CM from metastatic castration‐resistant (mCRPC) patients presented high levels of chitinase‐3‐like 1 (CHI3L1, YKL‐40) when compared to patients with stable disease (PCa‐N) and healthy control individuals (HC). The only described receptor for CHI3L1, interleukin‐13 receptor α2 (IL‐13Rα2), was significantly up‐regulated in the human metastatic PCa cell line, ARCaPM. Accordingly, we observed that the activation of IL‐13Rα2 from PCa‐M CM increased the invasiveness of ARCaPM cells while siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa‐M patients.ConclusionsThus, we show that circulating monocytes from metastatic PCa patients exert a tumor‐promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.

Abstract 5208: Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (PCa) patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced PCa patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behaviors of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte® Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5208.

Prospects for Drugs Based on the Mitochondria-Targeting Antioxidant SkQ1 in Treatment of Wounds with Impaired Healing

Chronic wounds with impared wound healing that require prolong time for healing remain unsolved problem of modern medicine. Excessive oxidative stress plays an important role in the pathogenesis of chronic wounds caused by aging, diabetes and other pathologies. This review is aimed at the role of mitochondria in oxidative stress and to the future prospects for using the innovative mitochondria targeted antioxidants for treatment of impaired wounds. Recent studies in old mice and mice with type 2 diabetes showed that the mitochondrial antioxidant SkQ1 [10- (6'- plastoquinonyl) decyltriphenylphosphonium] stimulates healing of full-thickness dermal wounds. SkQ1 accelerates inflammatory stage of wound healing, maturation of granulation tissue, angiogenesis and epithelization of wounds. The anti-inflammatory effect of SkQ1 is possibly connected to decreased inflammatory activation of the vascular endothelium, which is typical for aging, diabetes and other pathologies. Local administration of SkQ1 also accelerates wound healing and provides strong anti-inflammatory effect in the model of acute aseptic inflammation. In addition, SkQ1 to stimulate apoptosis of neutrophils and suppresses their activation, as well as suppresses inflammatory activation of mast cells. In the wound model in vitro, SkQ1 accelerates movement of epithelial cells and fibroblasts into the «wound» and stimulates differentiation of human subcutaneous fibroblasts to myofibroblasts. Reviewed data suggest that SkQ1-based topical drugs have a great potential to treat wounds that exhibit impaired healing also in patients suffering from chronic critical illness.

Alpha-Tocopheryl Succinate Selectively Affects the Morphology and Motility of Normal and Tumor Epithelial Cells

Alpha-tocopheryl succinate (α-TS) is a vitamin-E derivative with potential antitumor properties. In this study, we have investigated the effect of α-TS on the morphology and motility of the A431 human epidermoid carcinoma cell line and HaCaT immortalized nontumorigenic human keratinocytes. Phase contrast and fluorescent microscopy, in vitro wound healing assay (scratch test), and scanning electron microscopy have been used. In both cell lines, alteration of cell shape, actin-cytoskeleton reorganization, cell-surface smoothing, disappearance of microvillae, and cell-motility inhibition have been shown. These effects are similar in both cell lines, but in the HaCaT cell line they occur after treatment with higher α-TS doses than for A431cells. Thus, we have demonstrated the selectivity of the effect of α-TS on tumor cells derived from human-skin stratified epithelia. The results of this work may be useful for further investigations focused on development of antitumor agents with selective action toward tumor cells.

Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing.

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.

Collective Movement and 3D Morphogenesis of Epithelial Cells Induced by Softness of the Extracellular Matrix

Collective Movement and 3D Morphogenesis of Epithelial Cells Induced by Softness of the Extracellular Matrix

Inhibition of CX3CR1 reduces cell motility and viability in pancreatic adenocarcinoma epithelial cells

Increased expression of the chemokine CX3CL1 and its sole receptor, CX3CR1 have been correlated with poor pancreatic cancer patient survival and time to recurrence, as well as with pancreatic perineural invasion. We have previously shown that metastasis of prostate and breast cancer is in part driven by CX3CL1, and have developed small molecule inhibitors against the CX3CR1 receptor that diminish metastatic burden. Here we ask if inhibition of this chemokine receptor affects the phenotype of PDAC tumor cells. Our findings demonstrate that motility, invasion, and contact-independent growth of PDAC cells all increase following CX3CL1 exposure, and that antagonism of CX3CR1 by the inhibitor JMS-17-2 reduces each of these phenotypes and correlates with a downregulation of AKT phosphorylation. These data suggest that PDAC tumor cell migration and growth, elements critical in metastatic progression, may susceptible to pharmacologic intervention.

Periostin promotes malignant potential by induction of epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma.

Periostin, a secreted matricellular protein, has been reported to induce epithelial‐mesenchymal transition (EMT), which increases motility and invasiveness in various epithelial cancer cells. Periostin is also overexpressed in intrahepatic cholangiocarcinoma (ICC) and suggested to be a biomarker for tumor progression and poor prognosis; however, its functional role in ICC is not fully understood. Here, we investigated whether periostin influences malignant potential through the induction of EMT in ICC. Analyses of surgical resected ICC specimens revealed that the gene expression of periostin was significantly higher in ICC tumors than in adjacent nontumor liver tissues and was closely correlated with the expression of mesenchymal markers, including N‐cadherin, vimentin, and fibronectin. However, the expression level of periostin varied in each case. Consistently, the expression of periostin in HuH28 (an undifferentiated ICC cell) was markedly higher than in HuCCT‐1 (a moderately differentiated ICC cell). In addition, high‐level secretion of periostin into culture media was observed in HuH28 but not in HuCCT‐1. To identify the biological significance of periostin in EMT, gene silencing of periostin by small interfering RNA was performed in HuH28 cells. Periostin knockdown in HuH28 cells significantly down‐regulated mesenchymal markers and up‐regulated epithelial markers, suggesting the reversal of EMT, namely mesenchymal‐epithelial transition. Along with these changes, cell proliferation was significantly suppressed by 52%. In addition, cell migration and invasion were significantly suppressed by 62% and 61%, respectively, with reduced gene expression of matrix metalloproteinase 2. Interestingly, chemosensitivity to gemcitabine was also significantly improved by periostin depletion. Conclusion: Periostin plays an important role in the regulation of malignant potential through EMT and is suggested to be a novel target for the treatment of ICC. (Hepatology Communications 2017;1:1099–1109)

Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus mediated by an IL-13-driven epithelial cell transcriptional program. Herein, we show that the cytoskeletal protein synaptopodin (SYNPO), previously associated with podocytes, is constitutively expressed in esophageal epithelium and induced during allergic inflammation. In addition, we show that the SYNPO gene is transcriptionally and epigenetically regulated by IL-13 in esophageal epithelial cells. SYNPO was expressed in the basal layer of homeostatic esophageal epithelium, colocalized with actin filaments, and expanded into the suprabasal epithelium in EoE patients, where expression was elevated 25-fold compared with control individuals. The expression level of SYNPO in esophageal biopsies correlated with esophageal eosinophil density and was improved following anti-IL-13 treatment in EoE patients. In esophageal epithelial cells, SYNPO gene silencing reduced epithelial motility in a wound healing model, whereas SYNPO overexpression impaired epithelial barrier integrity and reduced esophageal differentiation. Taken together, we demonstrate that SYNPO is induced by IL-13 in vitro and in vivo, is a nonredundant regulator of epithelial cell barrier function and motility, and is likely involved in EoE pathogenesis.

Biological roles of hepatocyte growth factor-Met signaling from genetically modified animals.

Hepatocyte growth factor (HGF) is produced by stromal and mesenchymal cells, and it stimulates epithelial cell proliferation, motility, morphogenesis and angiogenesis in various organs via tyrosine phosphorylation of its cognate receptor, Met. The HGF-Met signaling pathway contributes in a paracrine manner to the development of epithelial organs, exerts regenerative effects on the epithelium, and promotes the regression of fibrosis in numerous organs. Additionally, the HGF-Met signaling pathway is correlated with the biology of cancer types, neurons and immunity. In vivo analyses using genetic modification have markedly increased the profound understanding of the HGF-Met system in basic biology and its clinical applications. HGF and Met knockout (KO) mice are embryonically lethal. Therefore, amino acids in multifunctional docking sites of Met have been exchanged with specific binding motifs for downstream adaptor molecules in order to investigate the signaling potential of the HGF-Met signaling pathway. Conditional Met KO mice were generated using Cre-loxP methodology and characterization of these mice indicated that the HGF-Met signaling pathway is essential in regeneration, protection, and homeostasis in various tissue types and cells. Furthermore, the results of studies using HGF-overexpressing mice have indicated the therapeutic potential of HGF for various types of disease and injury. In the present review, the phenotypes of Met gene-modified mice are summarized.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

Knockdown of sodium–calcium exchanger 1 induces epithelial-to-mesenchymal transition in kidney epithelial cells

Mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT) are important processes in kidney development. Failure to undergo MET during development leads to the initiation of Wilms tumor, whereas EMT contributes to the development of renal cell carcinomas (RCC). The role of calcium regulators in governing these processes is becoming evident. We demonstrated earlier that Na+/Ca2+ exchanger 1 (NCX1), a major calcium exporter in renal epithelial cells, regulates epithelial cell motility. Here, we show for the first time that NCX1 mRNA and protein expression was down-regulated in Wilms tumor and RCC. Knockdown of NCX1 in Madin-Darby canine kidney cells induced fibroblastic morphology, increased intercellular junctional distance, and induced paracellular permeability, loss of apico-basal polarity in 3D cultures, and anchorage-independent growth, accompanied by expression of mesenchymal markers. We also provide evidence that NCX1 interacts with and anchors E-cadherin to the cell surface independent of NCX1 ion transport activity. Consistent with destabilization of E-cadherin, NCX1 knockdown cells showed an increase in β-catenin nuclear localization, enhanced transcriptional activity, and up-regulation of downstream targets of the β-catenin signaling pathway. Taken together, knockdown of NCX1 in Madin-Darby canine kidney cells alters epithelial morphology and characteristics by destabilization of E-cadherin and induction of β-catenin signaling.

Abstract 3931: Sodium-calcium exchanger-1 regulates the epithelial phenotype and is lost in renal cancers

Abstract Epithelial cells that line the renal tubules possess apico-basal polarity and a defined cellular structure maintained by junctional proteins. The underlying mesenchymal cells are fibroblastic with a non-uniform cell structure and secrete extracellular matrix proteins. Mesenchymal to epithelial transition (MET) and epithelial to mesenchymal transition (EMT) are required for renal tubule formation during kidney development. Failure of renal mesenchymal cells to undergo epithelial transformation leads to the initiation of Wilms tumor, the most frequently occurring renal cancer in children. Conversely, aberrant mesenchymal transformation of tubular epithelial cells in the nephron contributes to the development of renal cell carcinoma, which constitutes 85-90% of the adult tubular malignancies. The role of calcium regulators in governing EMT and MET is becoming evident. Sodium calcium exchanger 1 (NCX1), located on the basolateral surface of tubular epithelial cells, is the principal calcium regulator that mediates calcium reabsorption in these cells. NCX1 mediates the extrusion of one calcium ion and the influx of three sodium ions in one exchange movement. We demonstrated earlier that NCX1 regulates epithelial cell motility. However, the role of NCX1 in EMT was undetermined. We observed that knockdown of NCX1 in renal epithelial cells (MDCK) induced fibroblastic morphology, and increased permeability to rhodamine indicative of leaky cell-cell junctions. Electron micrographs of these cells displayed increased inter-cellular junctional distance also suggesting that NCX1 knockdown altered junctions between adjacent cells. Cells with NCX1 knockdown showed loss of apico-basal polarity in three-dimensional cultures accompanied by expression of mesenchymal markers. Furthermore, NCX1 knockdown cells were capable of anchorage independent growth suggesting that these cells had acquired tumorigenic potential. Interestingly, we show for the first time that NCX1 mRNA and protein expression is greatly reduced in both Wilms tumor and renal cell carcinoma demonstrating a direct correlation between NCX1 expression and the epithelial phenotype. Mechanistically, we provide evidence that NCX1 interacts with and anchors E-cadherin, a classical adhesion molecule, to the cell surface independent of NCX1 ion transport activity. MDCK cells with NCX1 knockdown exhibited β-catenin nuclear localization and enhanced transcriptional activity. Taken together, knockdown of NCX1 in MDCK cells induces mesenchymal transition by destabilization of E-cadherin and induction of β-catenin transcriptional activity. Citation Format: Sona Lakshme Balasubramaniam, Anilkumar Gopalakrishnapillai, Nicholas J. Petrelli, Sonali P. Barwe. Sodium-calcium exchanger-1 regulates the epithelial phenotype and is lost in renal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2017-3931

Sonic Hedgehog Signaling and Development of the Dentition.

Sonic hedgehog (Shh) is an essential signaling peptide required for normal embryonic development. It represents a highly-conserved marker of odontogenesis amongst the toothed vertebrates. Signal transduction is involved in early specification of the tooth-forming epithelium in the oral cavity, and, ultimately, in defining tooth number within the established dentition. Shh also promotes the morphogenetic movement of epithelial cells in the early tooth bud, and influences cell cycle regulation, morphogenesis, and differentiation in the tooth germ. More recently, Shh has been identified as a stem cell regulator in the continuously erupting incisors of mice. Here, we review contemporary data relating to the role of Shh in odontogenesis, focusing on tooth development in mammals and cartilaginous fishes. We also describe the multiple actions of this signaling protein at the cellular level.

The role of small GTPases of the Rho/Rac family in TGF‐β‐induced EMT and cell motility in cancer

This article focuses on the role of Rho family GTPases, particularly Rac1 and Rac1b in TGF-β-induced epithelial-mesenchymal transition (EMT) and EMT-associated responses such as cell migration, invasion, and metastasis in cancer. EMT is considered a prerequisite for cells to adopt a motile and invasive phenotype and eventually become metastatic. A major regulator of EMT and metastasis in cancer is TGF-β, and its specific functions on tumor cells are mediated beside Smad proteins and mitogen-activated protein kinases (MAPKs) by small GTPases of the Rho/Rac1 family. Available data point to extensive signaling crosstalk between TGF-β and various Rho GTPases, and in particular a synergistic role of Rho and Rac1 during EMT and cell motility in normal and neoplastic epithelial cells. In contrast, the Rac1-related isoform, Rac1b, emerges as an endogenous inhibitor of Rac1 in TGF-β signaling, at least in pancreatic carcinoma cells. Given the tumor-promoting role of TGF-β in late-stage carcinomas and the intimate crosstalk of Rho/Rac1/Rac1b and TGF-β signaling in various tumor cell responses, targeting specific Rho GTPases may allow for selective interference with prooncogenic TGF-β responses to aid in anticancer treatments. Developmental Dynamics 247:451-461, 2018. © 2017 Wiley Periodicals, Inc.

Epithelial cell behaviours during neurosensory organ formation.

Perception of the environment in vertebrates relies on a variety of neurosensory mini-organs. These organs develop via a multi-step process that includes placode induction, cell differentiation, patterning and innervation. Ultimately, cells derived from one or more different tissues assemble to form a specific mini-organ that exhibits a particular structure and function. The initial building blocks of these organs are epithelial cells that undergo rearrangements and interact with neighbouring tissues, such as neural crest-derived mesenchymal cells and sensory neurons, to construct a functional sensory organ. In recent years, advances in in vivo imaging methods have allowed direct observation of these epithelial cells, showing that they can be displaced within the epithelium itself via several modes. This Review focuses on the diversity of epithelial cell behaviours that are involved in the formation of small neurosensory organs, using the examples of dental placodes, hair follicles, taste buds, lung neuroendocrine cells and zebrafish lateral line neuromasts to highlight both well-established and newly described modes of epithelial cell motility.