Altered Cell Adhesion Research Articles

Arvcf Dependent Adherens Junction Stability is Required to Prevent Age-Related Cortical Cataracts.

The etiology of age-related cortical cataracts is not well understood but is speculated to be related to alterations in cell adhesion and/or the changing mechanical stresses occurring in the lens with time. The role of cell adhesion in maintaining lens transparency with age is difficult to assess because of the developmental and physiological roles that well-characterized adhesion proteins have in the lens. This report demonstrates that Arvcf, a member of the p120-catenin subfamily of catenins that bind to the juxtamembrane domain of cadherins, is an essential fiber cell protein that preserves lens transparency with age in mice. No major developmental defects are observed in the absence of Arvcf, however, cortical cataracts emerge in all animals examined older than 6-months of age. While opacities are not obvious in young animals, histological anomalies are observed in lenses at 4-weeks that include fiber cell separations, regions of hexagonal lattice disorganization, and absence of immunolabeled membranes. Compression analysis of whole lenses also revealed that Arvcf is required for their normal biomechanical properties. Immunofluorescent labeling of control and Arvcf-deficient lens fiber cells revealed a reduction in membrane localization of N-cadherin, β-catenin, and αN-catenin. Furthermore, super-resolution imaging demonstrated that the reduction in protein membrane localization is correlated with smaller cadherin nanoclusters. Additional characterization of lens fiber cell morphology with electron microscopy and high resolution fluorescent imaging also showed that the cellular protrusions of fiber cells are abnormally elongated with a reduction and disorganization of cadherin complex protein localization. Together, these data demonstrate that Arvcf is required to maintain transparency with age by mediating the stability of the N-cadherin protein complex in adherens junctions.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

NEDD9 Mediates the FAK/Src Signaling Pathway to Promote the Adhesion of Human Trabecular Meshwork Cells after Dexamethasone Treatment

Purpose The differential gene expression of neural precursor cell expressed developmentally downregulated 9 (NEDD9) in human trabecular meshwork (HTM) cells after dexamethasone (Dex) treatment was confirmed through gene expression profiling. However, the regulatory mechanism of NEDD9 expression in HTM cells remains unknown. In this study, we investigated NEDD9 expression in HTM cells and gained a better understanding of glucocorticoid-induced glaucoma (GIG) pathophysiology. Methods The Gene Expression Omnibus database and GEO2R tool were used to identify differentially expressed genes in the GSE37474 and GSE124114 datasets, and NEDD9 gene expression was found to be upregulated. Human corneoscleral segments and HTM cells were treated with 100 nM Dex or an equal volume of ethanol (0.01%) for 7 days. NEDD9 expression in TM tissues was evaluated by immunohistochemistry, and NEED9 expression in HTM cells was confirmed by RT-qPCR and western blotting. HTM cell adhesive behaviors were assessed with a cell adhesion detection kit. NEDD9 expression was knocked down with short hairpin RNA in HTM cells, and FAK/Src signaling pathway activation was found to be regulated by NEDD9. Results After 7 days of HTM cell Dex treatment, NEDD9 expression was upregulated to be approximately twice that of control. FAK, Src, phospho-FAK, and phospho-Src expression in Dex-treated HTM cells was markedly increased. Downregulation of NEDD9 expression reduced HTM cell adhesion to the surface of culture wells and simultaneously led to a reduction in FAK, Src, phospho-FAK and phospho-Src expression. Conclusions NEDD9 expression is upregulated in HTM cells after Dex treatment and promotes HTM cell adhesion. These findings underscore the contribution of NEDD9 overexpression to altered HTM cell adhesion during glucocorticoid therapy and may play a key role in GIG pathological progression. Considering the similarity between GIG and primary open-angle glaucoma (POAG), our findings suggest that targeting NEDD9 may be a new therapeutic strategy for POAG patients.

Implications of fibrotic extracellular matrix in diabetic retinopathy.

Fibrosis is an accumulation of extracellular matrix (ECM) proteins and fibers in a disordered fashion, which compromises cell and tissue functions. High glucose-induced fibrosis, a major pathophysiological change of diabetic retinopathy (DR), severely affects vision by compromising the retinal vasculature and ultimately disrupting retinal tissue organization. The retina is a highly vascularized, stratified tissue with multiple cell types organized into distinct layers. Chronically high blood glucose stimulates certain retinal cells to increase production and assembly of ECM proteins resulting in excess ECM deposition primarily in the capillary walls on the basal side of the endothelium. This subendothelial fibrosis of the capillaries is the earliest histological change in the diabetic retina and has been linked to the vascular dysfunction that underlies DR. Proteins that are not normally abundant in the capillary basement membrane (BM) matrix, such as the ECM protein fibronectin, are assembled in significant quantities, disrupting the architecture of the BM and altering its properties. Cell culture models have identified multiple mechanisms through which elevated glucose can stimulate fibronectin matrix assembly, including intracellular signaling pathways, alternative splicing, and non-enzymatic glycation of the ECM. The fibrotic subendothelial matrix alters cell adhesion and supports further accumulation of other ECM proteins leading to disruption of endothelial cell-cell junctions. We review evidence supporting the notion that these molecular changes in the ECM contribute to the pathogenesis of DR, including vascular leakage, loss of endothelial cells and pericytes, changes in blood flow, and neovascularization. We propose that the accumulation of ECM, especially fibronectin matrix, first around the vasculature and later in extravascular locations, plays a critical role in DR and vision loss. Strategies for DR prevention and treatment should consider the ECM a potential therapeutic target.

Lysophosphatidylcholine disrupts cell adhesion and induces anoikis in hepatocytes.

Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated lipoapoptosis. However, LPC-induced cytotoxicity in hepatocytes is not well understood. Here, we found for the first time that LPC-induced cell rounding occurred prior to apoptosis. LPC-induced rounding of cells reduced both cell-extracellular matrix (ECM) adhesion and cell-cell junctions, which promoted detachment-induced apoptosis (defined as anoikis) in hepatocytes. Further study revealed that LPC altered cellular morphology and cell adhesion by inhibiting integrin and cadherin signalling-mediated microfilament polymerization. We also found that ECM supplementation and microfilament cytoskeletal stabilization inhibited LPC-induced hepatocyte death by attenuating anoikis. Our data indicate a novel cytotoxic process and signalling pathway induced by LPC.

Matteucinol combined with temozolomide inhibits glioblastoma proliferation, invasion, and progression: an in vitro, in silico, and in vivo study.

Glioblastoma is the most prevalent and malignant brain tumor identified in adults. Surgical resection followed by radiotherapy and chemotherapy, mainly with temozolomide (TMZ), is the chosen treatment for this type of tumor. However, the average survival of patients is around 15 months. Novel approaches to glioblastoma treatment are greatly needed. Here, we aimed to investigate the anti-glioblastoma effect of the combination of matteucinol (Mat) (dihydroxyflavanone derived from Miconia chamissois Naudin) with the chemotherapeutic TMZ in vitro using tumor (U-251MG) and normal astrocyte (NHA) cell lines and in vivo using the chick embryo chorioallantoic membrane (CAM) assay. The combination was cytotoxic and selective for tumor cells (28 μg/mL Mat and 9.71 μg/mL TMZ). Additionally, the combination did not alter cell adhesion but caused morphological changes characteristic of apoptosis in vitro. Notably, the combination was also able to reduce tumor growth in the chick embryo model (CAM assay). The docking results showed that Mat was the best ligand to the cell death membrane receptor TNFR1 and to TNFR1/TMZ complex, suggesting that these two molecules may be working together increasing their potential. In conclusion, Mat-TMZ can be a good candidate for pharmacokinetic studies in view of clinical use for the treatment of glioblastoma.

Integration of data from the in vitro long-term exposure study on human endothelial cells and the in silico analysis: A case of dibutyl phthalate-induced vascular dysfunction

General population is exposed to dibutyl phthalate (DBP) through continuous use of various consumer products. DBP exhibits its effects mainly on the endocrine and reproductive system but it can also affect the function of the vasculature; however, the underlying mechanisms behind DBP-induced vascular dysfunction are not fully understood. To infer pathways, molecular functions, biological processes, and human diseases associated with DBP exposure, we integrated the toxicogenomic data obtained from the 4-week-long exposure of human vascular endothelial cells (ECs) to three environmentally relevant concentrations of DBP with the in silico analysis. Nine genes were affected by DBP exposure: six of the integrin family, VCAM1, ICAM1, and MMP2. As shown by the in silico analysis, changes in DBP-affected genes could affect extracellular matrix and binding of molecules and cells to ECs, thereby altering cell adhesion and migration. Several pathways, molecular functions, and biological processes were further identified to provide insight into the DBP-vascular disease relationships and the potential mechanism of action. The top three human disease categories associated with DBP exposure and vascular dysfunction include cardiovascular, cerebrovascular, and immune system diseases. Integration of experimental and in silico approaches may offer better understanding of the potential human health risks associated with DBP exposure.

In vitro Studies on Calcium Oxalate Induced Apoptosis Attenuated by Didymocarpus pedicellata

The present study focuses on exploring the antilithiatic potential of Didymocarpus pedicellata, which is valuable in managing renal disorders. Urolithiasis is an idiopathic disorder with a high recurrence and an incidence rate and is of major concern worldwide due to partial and unsatisfactory relief. Calcium oxalate crystals in contact with renal epithelial cells (HK2), causing reactive oxygen species overproduction, oxidative stress, apoptosis resulting in crystal adhesion and internalization. Crystals were modulated by cotreatment with ethanolic extract of D. pedicellata. Cell toxicity assay was assessed using flow cytometry. Cell-crystal interaction, adhesion, and internalization were visualized through Scanning electron microscopy (SEM) analysis and hematoxylin-eosin staining. The lithogenic induction caused impairment of renal function due to oxidative stress, measured by ROS levels. Cell death assays were detected by dual staining methods. Fluorimeter evaluation pointed to active caspase 3 mediated cell death (apoptotic) in oxalate injured cells was attenuated by Didymocarpus pedicellata extract. Alterations in cell adhesion were observed by immunocytochemistry. The current study revealed that the Didymocarpus pedicellata was endowed with antiurolithiatic activity as it displayed increased viability, reduced oxidative stress due to lowered production of intracellular reactive oxygen species (ROS), and decreased apoptosis when oxalate injured HK2 cells were cotreated with the extract.

Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells.

Glycogen-specific kinase (GSK3β) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3β is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3β has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3β kinase have been identified, the wider protein networks within which GSK3β participates are not well known, and the consequences of these interactions not well understood. In this study, LC-MS/MS expression analysis was performed using knockout GSK3β colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of β-catenin. Consistent with the role of GSK3β, we found that β-catenin levels and canonical Wnt activity are unaffected by knockout of GSK3β and therefore used this knockout cell model to identify other processes in which GSK3β is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterized the phenotype and altered proteomic profiles associated with this. We also characterized the perturbation of metabolic pathways resulting from GSK3β knockout and identified defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated β-catenin we identified several of the diverse pathways and processes associated with GSK3β function.

The Persistence of Hepatitis C Virus Infection in Hepatocytes Promotes Hepatocellular Carcinoma Progression by Pro-Inflammatory Interluekin-8 Expression.

Background: A large amount of epidemiological evidence indicates that persistent HCV infection is the main risk factor for HCC. We aimed to study the effects of persistent HCV infection on the interaction of the virus and host cell to identify cancer gene profiles. Methods: Next-generation sequencing (NGS) was used to identify differentially expressed genes between uninfected Huh7.5.1 control cells, short-term HCV (S-HCV), early long-term HCV (eL-HCV), and long-term HCV (L-HCV) infections, which were analyzed using different dynamic bioinformatics and analytic tools. mRNA expression was validated and quantified using q-PCR. One hundred ninety-six serum samples of HCV patients with IFN/RBV treatment were used to study chemokine levels. Results: S-HCV activates an inflammatory response and drives cell death and apoptosis through cell cycle arrest via MAPK signaling. L-HCV promotes cell growth and alters cell adhesion and chemokine signaling via CXCL8-mediated-SRC regulation. A total of 196 serum samples from the HCV and HCV-HCC cohorts demonstrated significantly upregulated pro-inflammatory CXCL8 in non-SVR (persistent HCV infection) patients in the HCV-HCC group. Conclusions: Persistent infection with HCV induced pro-inflammatory CXCL8 and the oncogene SRC, thereby triggering and promoting hepatocarcinogenesis. CXCL8 may be a potential biomarker for monitoring HCV-related HCC progression.

High shear stress enhances endothelial permeability in the presence of the risk haplotype at 9p21.3.

Single nucleotide polymorphisms (SNPs) are exceedingly common in non-coding loci, and while they are significantly associated with a myriad of diseases, their specific impact on cellular dysfunction remains unclear. Here, we show that when exposed to external stressors, the presence of risk SNPs in the 9p21.3 coronary artery disease (CAD) risk locus increases endothelial monolayer and microvessel dysfunction. Endothelial cells (ECs) derived from induced pluripotent stem cells of patients carrying the risk haplotype (R/R WT) differentiated similarly to their non-risk and isogenic knockout (R/R KO) counterparts. Monolayers exhibited greater permeability and reactive oxygen species signaling when the risk haplotype was present. Addition of the inflammatory cytokine TNFα further enhanced EC monolayer permeability but independent of risk haplotype; TNFα also did not substantially alter haplotype transcriptomes. Conversely, when wall shear stress was applied to ECs in a microfluidic vessel, R/R WT vessels were more permeable at lower shear stresses than R/R KO vessels. Transcriptomes of sheared cells clustered more by risk haplotype than by patient or clone, resulting in significant differential regulation of EC adhesion and extracellular matrix genes vs static conditions. A subset of previously identified CAD risk genes invert expression patterns in the presence of high shear concomitant with altered cell adhesion genes, vessel permeability, and endothelial erosion in the presence of the risk haplotype, suggesting that shear stress could be a regulator of non-coding loci with a key impact on CAD.

Identification of an Altered Matrix Signature in Kidney Aging and Disease.

Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.

Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death.

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

High ROBO3 expression predicts poor survival in non-M3 acute myeloid leukemia

Roundabout guidance receptor proteins are crucial components of the SLIT/ROBO signaling pathway. This pathway is important for the nervous system and in embryonic development. Recently, increasing evidence has shown that roundabout guidance receptor proteins and the SLIT/ROBO signaling pathway also participate in tumorigenesis. Here, by analyzing transcriptome data from the TCGA and GEO databases, we found that ROBO3 is highly expressed in non-M3 acute myeloid leukemia. High ROBO3 expression was associated with increased age at diagnosis and poorer risk classification (both P < 0.01). Patients with high ROBO3 expression had higher rates of TP53 and RUNX1 mutations (both P < 0.05). Significantly worse overall survival and event-free survival were observed in high ROBO3 expression patients compared with low ROBO3 expression patients (OS: P = 0.004; EFS: P= 0.012). High ROBO3 expression was also associated with poorer overall survival and event-free survival in a subgroup of patients who received intensive chemotherapy (OS: P = 0.024; EFS: P = 0.040). Moreover, multivariate analysis indicated that high ROBO3 expression was an independent risk factor for poor overall survival in non-M3 acute myeloid leukemia patients who are younger than 60 and received intensive chemotherapy during remission induction. Bioinformatics analysis by Kyoto Encyclopedia of Genes and Genomes and Gene Ontology revealed that high ROBO3 expression significantly altered cell adhesion and extracellular matrix-related pathways (adjusted P < 0.05). Taken together, the data demonstrate that ROBO3 is upregulated in non-M3 acute myeloid leukemia and may be a potent biomarker of inferior prognosis.

Response of breast cancer carcinoma spheroids to combination therapy with radiation and DNA-PK inhibitor: growth arrest without a change in α/β ratio

Purpose Agents that increase tumor radiosensitivity are of interest in improving outcomes in radiotherapy (XRT). DNA-PK inhibitors radiosensitize and alter cell adhesion proteins. We investigated combination radiation and a DNA-PK inhibitor in monolayers vs spheroids. Materials and methods Using HER2 positive mammary carcinoma cells, we investigated the impact of NU7441, a DNA-PK inhibitor, on irradiated monolayer and spheroid cultures. Colony formation assays were performed with monolayer culture cells and spheroids after irradiation with/without NU7441 (5 μM). Results In monolayer culture cells, α/β increased from 3.0 ± 0.2 Gy (XRT alone) to 6.9 ± 0.2 Gy (XRT+NU7441). Corresponding α/β values for cells obtained by disaggregating treated spheroids were 3.6 ± 0.7 Gy (XRT alone) and 3.5 ± 0.2 Gy (XRT+NU7441). However, spheroid survival was highly sensitive to NU7441 incubation. After 4 Gy XRT alone 75% of the irradiated spheroids remained intact; when NU7441 treatment was involved, 13% remained intact. No spheroids survived to 3 weeks at 6 Gy or more. The discrepancy between the minimal change in α/β from cells derived from spheroids and the spheroid growth response was not related to poor penetration of NU7441. Conclusions DNA-PK inhibitor NU7441 radiosensitized monolayer cells but not cells obtained from spheroids. NU7441 and radiation increased spheroid fragmentation.

Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma.

Phaeochromocytomas and paragangliomas (PPGLs) are neuroendocrine catecholamine-producing tumours that may progress into inoperable metastatic disease. Treatment options for metastatic disease are limited, indicating a need for functional studies to identify pharmacologically targetable pathophysiological mechanisms, which require biologically relevant experimental models. Recently, a human progenitor phaeochromocytoma cell line named “hPheo1” was established, but its genotype has not been characterised. Performing exome sequencing analysis, we identified a KIF1B T827I mutation, and the oncogenic NRAS Q61K mutation. While KIF1B mutations are recurring somatic events in PPGLs, NRAS mutations have hitherto not been detected in PPGLs. Therefore, we aimed to assess its implications for the hPheo1 cell line, and possible relevance for the pathophysiology of PPGLs. We found that transient downregulation of NRAS in hPheo1 led to elevated expression of genes associated with cell adhesion, and enhanced adhesion to hPheo1 cells’ extracellular matrix. Analyses of previously published mRNA data from two independent PPGL patient cohorts (212 tissue samples) revealed a subcluster of PPGLs featuring hyperactivated RAS pathway-signalling and under-expression of cell adhesion-related gene expression programs. Thus, we conclude that NRAS activity in hPheo1 decreases adhesion to their own extracellular matrix and mirrors a transcriptomic RAS-signalling-related phenomenon in PPGLs.

Fibrocystin Is Essential to Cellular Control of Adhesion and Epithelial Morphogenesis.

Mutations of the Pkhd1 gene cause autosomal recessive polycystic kidney disease (ARPKD). Pkhd1 encodes fibrocystin/polyductin (FPC), a ciliary type I membrane protein of largely unknown function, suggested to affect adhesion signaling of cells. Contributions of epithelial cell adhesion and contractility to the disease process are elusive. Here, we link loss of FPC to defective epithelial morphogenesis in 3D cell culture and altered cell contact formation. We study Pkhd1-silenced Madin-Darby Canine Kidney II (MDCKII) cells using an epithelial morphogenesis assay based on micropatterned glass coverslips. The assay allows analysis of cell adhesion, polarity and lumen formation of epithelial spheroids. Pkhd1 silencing critically affects the initial phase of the morphogenesis assay, leading to a reduction of correctly polarized spheroids by two thirds. Defects are characterized by altered cell adhesion and centrosome positioning of FPC-deficient cells in their 1-/2-cell stages. When myosin II inhibitor is applied to reduce cellular tension during the critical early phase of the assay, Pkhd1 silencing no longer inhibits formation of correctly polarized epithelia. We propose that altered sensing and cell interaction of FPC-deficient epithelial cells promote progressive epithelial defects in ARPKD.

Selection of established tumour cells through narrow diameter micropores enriches for elevated Ras/Raf/MEK/ERK MAPK signalling and enhanced tumour growth

ABSTRACT As normal cells become cancer cells, and progress towards malignancy, they become progressively softer. Advantages of this change are that tumour cells become more deformable, and better able to move through narrow constraints. We designed a positive selection strategy that enriched for cells which could move through narrow diameter micropores to identify cell phenotypes that enabled constrained migration. Using human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, we found that micropore selection favoured cells with relatively higher Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signalling, which affected actin cytoskeleton organization, focal adhesion density and cell elasticity. In this follow-up study, we provide further evidence that selection through micropores enriched for cells with altered cell morphology and adhesion. Additional analysis of RNA sequencing data revealed a set of transcripts associated with small cell size that was independent of constrained migration. Gene set enrichment analysis identified the ‘matrisome’ as the most significantly altered gene set linked with small size. When grown as orthotopic xenograft tumours in immunocompromised mice, micropore selected cells grew significantly faster than Parent or Flow-Sorted cells. Using mathematical modelling, we determined that there is an interaction between 1) the cell to gap size ratio; 2) the bending rigidity of the cell, which enable movement through narrow gaps. These results extend our previous conclusion that Ras/Raf/MEK/ERK MAPK signalling has a significant role in regulating cell biomechanics by showing that the selective pressure of movement through narrow gaps also enriches for increased tumour growth in vivo.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring.Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161–180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 μg IRBP161−180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization.Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis.Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

SAT-LB26 A Dual Role for IGF-1R in Mammary Tumorigenesis

The insulin-like growth factor type 1 receptor (IGF-1R) is now thought to have a dual function in breast cancer. Several studies have shown overexpression of the IGF-1R pathway results in increased tumor cell proliferation and survival. Recent loss-of-function models have shown decreased mammary tumorigenesis latency and increased metastasis. These recent studies correlate with analyses of human patient datasets identifying worse overall survival with low IGF-1R expression. Similarly, inhibition of IGF-1R in the clinic has had no affect or has led to worse outcomes supporting the hypothesis that the IGF-1R may have tumor suppressive properties. Our prior published studies revealed loss of IGF-1R function results in heightened tumor epithelial stress, which alters the tumor microenvironment to be permissive for metastasis. Therefore, we asked does the loss of IGF-1R inherently change the tumor epithelium or is it simply the alterations of the microenvironment that lead to a metastatic primary tumor? We first analyzed cell invasion of primary tumor epithelial cells from a mouse tumor model driven by the Wnt1 oncogene (MMTV-Wnt1) and with reduced IGF-1R signaling by expression of a dominant-negative transgene (MMTV-dnIGF-1R). Epithelial cells from the MMTV-Wnt1/dnIGF-1R (bigenic) tumors invaded at the same rate as MMTV-Wnt1 tumor epithelial cells by tail vein injection suggesting invasive capacity is unchanged with reduced IGF-1R signaling. Interestingly, size of lung micrometastases from tail vein injected bigenic tumor epithelial cells was significantly reduced and the number also decreased over time in part due to a proliferative defect determined by immunostaining for pH3. Similarly, bigenic primary tumor epithelial cells injected into the mammary gland fat pad failed to form tumors suggesting alterations in cell adhesion. Consistent with this observation, E-cadherin gene and protein expression were decreased in the bigenic tumor epithelium compared to MMTV-Wnt1 tumors. In vitro analysis of cell adhesion in MMTV-Wnt1 primary epithelial cells resulted in both K8+ (luminal) and K14+ (basal) tumor epithelial cells adherence, while only K14+ cells from bigenic tumors adhered to collagen. Similarly, the lung micrometastases from tail vein injections exhibited predominantly K14+ cells. Analysis of MMTV-Wnt1 and bigenic primary tumors using single cell RNAseq revealed alterations in both stromal and epithelial populations that may contribute to bigenic primary tumor growth and metastasis. These data support the conclusion that inhibiting IGF-1R signaling results in both alterations to the tumor epithelium (partial EMT) and microenvironment that result in metastasis, but also, that the IGF-1R deficient metastatic cells need a niche or paracine signaling to proliferate.