FAT Atypical Cadherin Research Articles

Interaction of atypical cadherin Fat1 with SoHo adaptor proteins CAP/ponsin and ArgBP2.

Mammalian Fat1 is a giant atypical cadherin/tumor suppressor involved in the regulation of cellular orientation, migration, and growth. Fat1 is implicated in the development of the brain, eye, and kidney. Altered expression or mutations of FAT1 are also associated with cancer and facioscapulohumeral muscular dystrophy (FSHD). Yet, the mechanistic functions of this pathway remain incompletely understood. Here, we report the identification of Sorbin-homology (SoHo) proteins as novel interaction partners of Fat1 by virtue of a yeast-two-hybrid screen. SoHo proteins play diverse roles as adaptor proteins in cell signaling, cell adhesion and sarcomere architecture, including altered expression in cancer and FSHD. Specifically, we found SoHo proteins CAP/ponsin-1 and -2 (Sorbs1) and ArgBP2 (Sorbs2) to interact with the cytoplasmic domain of Fat1. We mapped the interaction to a prolin-rich classic type II PXXP motif within Fat1 and to the three Src-homology (SH3) domains within SoHo proteins using mutant expression in yeast, pulldown assays, and cell culture. Functionally, endogenous ponsin-2 expression of NRK-52E cells at cellular leading edges was lost upon knockdown of Fat1. In summary, our data point to an interaction of Fat1 with SoHo proteins that is able to recruit SoHo proteins to sites of Fat1 expression.

Association between FAT1 mutation and overall survival in patients with human papillomavirus-negative head and neck squamous cell carcinoma.

BackgroundThe purpose of this study was to characterize the mutation profile of FAT atypical cadherin 1 (FAT1) and determine the prognostic significance of FAT1 mutation for overall survival in patients with human papillomavirus (HPV)‐negative head and neck squamous cell carcinoma (HNSCC).MethodsData were downloaded from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) data portals and used as discovery and validation sets. FAT1 mutational status was determined in 234 and 37 patients with HPV‐negative HNSCC, respectively, and overall survival analysis was performed. For comparison, HPV‐positive patients were also analyzed for overall survival.ResultsMost of the identified nonsynonymous somatic FAT1 mutations were loss‐of‐function mutations. FAT1 mutation was significantly associated with better overall survival in HPV‐negative patients from both the TCGA cohort (p = .026) and the ICGC cohort (p = .047), but not in HPV‐positive patients.Conclusion FAT1 mutational status is a strong independent prognostic factor in patients with HPV‐negative HNSCC. © 2016 The Authors Head & Neck Published by Wiley Periodicals, Inc. Head Neck 38: E2021–E2029, 2016

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

The Fat family of atypical cadherins, originally identified in Drosophila, play diverse roles during embryogenesis and adult tissue maintenance. Among four mammalian members, Fat1 is essential for kidney and muscle organization, and is also essential for eye development; Fat1 knockout causes partial penetrant microphthalmia or anophthalmia. To account for the partial penetrance of the Fat1 phenotype, involvement of Fat4 in eye development was assessed. Lens phenotypes in Fat1 and 4 knockouts were also examined. Fat1 and Fat4 mRNA expression was examined by in situ hybridization. Knockout phenotypes of Fat1 and Fat4 were analyzed by hematoxylin and eosin (H&E) and immunofluorescent staining. We found Fat4 knockout did not affect eye induction or enhance severity of Fat1 eye defects. Although Fat1 and Fat4 mRNAs are similarly expressed in the lens epithelial cells, only Fat1 knockout caused a fully penetrant lens epithelial cell defect, which was apparent at embryonic day 14.5 (E14.5). The columnar structure of the lens epithelial cells was disrupted and in some regions cell aggregates were formed. In these multilayered regions, apical cell junctions were fragmented and the apical-basal polarity was lost. EdU incorporation assay also showed enhanced proliferation in the lens epithelial cells. Interestingly, these defects were found mainly in the central zone of the epithelial layer. The lens epithelial cells of the germinative zone maintained their normal morphology and fiber differentiation occurred normally at the equator. These observations indicate that Fat1 is essential for lens epithelial cell polarity and proliferation but not for terminal differentiation.

Gene Expression Profile of Persistent Postoperative Hypertension Patients with Aldosterone-producing Adenomas.

Background:Hypertension often persists after adrenalectomy for primary aldosteronism (PA). Many studies have analyzed the outcomes of adrenalectomy for aldosterone-producing adenomas (APA) to identify predictive factors for persistent hypertension. However, differentially expressed genes in persistent postoperative hypertension remain unknown. Our aim was to describe gene expression profile of persistent postoperative hypertension patients with APA.Methods:In this study, we described and compared gene expression profiles in persistent postoperative hypertension and postoperative normotension in Chinese patients with APA using microarray analysis. Confirmation was performed with quantitative real time-polymerase chain reaction analysis. Bioinformatic analysis (gene ontology analysis, pathway analysis and network analysis) was used for further research.Results:Microarray analysis identified a total of 99 differentially expressed genes, including 18 up-regulated and 81 down-regulated genes. Among the dysregulated genes were fat atypical cadherin 1 as well as fatty acid binding protein 4 and other genes that have not been previously studied in persistent postoperative hypertension with APA. Bioinformatics analysis indicated that differentially expressed genes were associated with lipid metabolic process, metal ion binding, and cell differentiation. Pathway analysis determined that five pathways corresponded to the dysregulated transcripts. The mRNAs-ncRNAs co-expression network was composed of 49 network nodes and 72 connections between 18 coding genes and 31 noncoding genes.Conclusions:This study revealed differentially expressed genes in persistent postoperative hypertension with APA and provided a resource of candidate genes for exploration of possible drug targets and prognostic markers.

Abstract 432: The Atypical Cadherin Fat1 Suppresses Mitochondrial Function to Control Vascular Smooth Muscle Cell Growth After Vascular Injury

In response to vascular injury, vascular smooth muscle cells (SMCs) undergo phenotypic switching, with enhanced cell cycle entry and migration, and loss of contractile protein expression. Previously, we found that the atypical cadherin Fat1 is upregulated in SMCs after vascular injury. In cultured SMCs, Fat1 is induced by growth factor stimulation, but limits SMC proliferation. Recently, we found that Fat1 species accumulate in SMC mitochondria and interact with critical proteins, including Complex I subunits. The factors governing and significance of mitochondrial activity during SMC response to vascular injury are unclear. We hypothesized that Fat1 controls mitochondrial activity to regulate SMC growth. In cultured SMCs, loss of Fat1 increased mitochondrial oxygen consumption rate (24.8±2.2 vs. 48.1±5.4 pMoles/min, WT vs. Fat1 knockout (KO), p<0.001, n=10), maximal respiratory capacity (35.8±3.4 vs. 55.4±5.4 pMoles/min, WT vs. KO, p<0.01, n=10), oxygen consumed for ATP production (13.1±1 vs. 27.3±3.3 pMoles/min, WT vs. KO, p=0.0005, n=10), and Complex I activity (1.0±0.04 vs.1.3±0.09 RU, WT vs. KO, p<0.034, n=10). Reactive oxygen species (ROS) also increased in Fat1 KO SMCs (1426±313 vs.1848±329 dihydroethidium fluorescence intensity/cell, WT vs. KO, p<0.002, n=3). SMCs lacking Fat1 exhibited enhanced growth (p<0.0001, n=12) and dedifferentiation; adding Rotenone to inhibit Complex I function opposed this growth advantage (p<0.0001, n=6). In a mouse model of vascular injury, SMC Fat1 deletion caused early and exuberant medial hyperplasia, (0.23±0.23 vs.7.16±1.62%, WT n=5 vs. KO n=8, p<0.003, day 3 post-injury) and neointimal expansion (0.18±0.05 vs.0.55±0.1 I/M ratio, WT n=5 vs. KO n=8, p<0.02, day 14 post-injury). We also found higher neointimal cell proliferation (7.7±2 vs. 26±7.1% of pHH3 positive nuclei, WT vs. KO, p=0.041, n=6) and ROS production in Fat1-deleted vessels after injury. In conclusion, we show that the atypical cadherin Fat1 communicates with mitochondrial Complex I to repress energy and ROS production, acting as a molecular “brake” on mitochondrial activity to suppress SMC growth after vascular injury. This novel mechanism could serve as an effective target in the treatment of hyperproliferative vascular diseases.

Loss of FAT Atypical Cadherin 4 Expression Is Associated with High Pathologic T Stage in Radically Resected Gastric Cancer.

PurposeRecent studies have revealed recurrent alterations in the cell adhesion gene FAT4, a candidate tumor suppressor gene, in cancer. FAT atypical cadherin 4 (FAT4) is a transmembrane receptor involved in the Hippo signaling pathway, which is involved in the control of organ size. Here, we investigated the loss of FAT4 expression and its association with clinicopathological risk factors in gastric cancer.Materials and MethodsWe assessed the expression of FAT4 by using immunohistochemistry on three tissue microarrays containing samples from 136 gastric cancer cases, radically resected in the Soonchunhyang University Cheonan Hospital between July 2006 and June 2008. Cytoplasmic immunoexpression of FAT4 was semi-quantitatively scored using the H-score system. An H-score of ≥10 was considered positive for FAT4 expression.ResultsVariable cytoplasmic expressions of FAT4 were observed in gastric cancers, with 33 cases (24.3%) showing loss of expression (H-score <10). Loss of FAT4 expression was associated with an increased rate of perineural invasion (H-score <10 vs. ≥10, 36.4% vs. 16.5%, P=0.015), high pathologic T stage (P=0.015), high tumor-node-metastasis stage (P=0.017), and reduced disease-free survival time (H-score <10 vs. ≥10, mean survival 62.7±7.3 months vs. 79.1±3.1 months, P=0.025). However, no association was found between the loss of FAT4 expression and tumor size, gross type, histologic subtype, Lauren classification, lymphovascular invasion, or overall survival.ConclusionsLoss of FAT4 expression appears to be associated with invasiveness in gastric cancer.

Regulation and function of the atypical cadherin FAT1 in hepatocellular carcinoma

In human cancers, giant cadherin FAT1 may function both, as an oncogene and a tumor suppressor. Here, we investigated the expression and function of FAT1 in hepatocellular carcinoma (HCC). FAT1 expression was increased in human HCC cell lines and tissues compared with primary human hepatocytes and non-tumorous liver tissue as assessed by quantitative PCR and western blot analysis. Combined immunohistochemical and tissue microarray analysis showed a significant correlation of FAT1 expression with tumor stage and proliferation. Suppression of FAT1 expression by short hairpin RNA impaired proliferation and migration as well as apoptosis resistance of HCC cells in vitro. In nude mice, tumors formed by FAT1-suppressed HCC cells showed a delayed onset and more apoptosis compared with tumors of control cells. Both hepatocyte growth factor and hypoxia-mediated hypoxia-inducible factor 1 alpha activation were identified as strong inducers of FAT1 in HCC. Moreover, demethylating agents induced FAT1 expression in HCC cells. Hypoxia lead to reduced levels of the methyl group donor S-adenosyl-L-methionine (SAM) and hypoxia-induced FAT1 expression was inhibited by SAM supplementation in HCC cells. Together, these findings indicate that FAT1 expression in HCC is regulated via promotor methylation. FAT1 appears as relevant mediator of hypoxia and growth receptor signaling to critical tumorigenic pathways in HCC. This knowledge may facilitate the rational design of novel therapeutics against this highly aggressive malignancy.

Deficiency of the MicroRNA-31–MicroRNA-720 Pathway in the Plasma and Endothelial Progenitor Cells From Patients With Coronary Artery Disease

Defects in angiogenesis/vasculogenesis or vessel repair are major complications of coronary artery disease (CAD). Endothelial progenitor cells (EPCs) play a fundamental role in postnatal vascular repair and CAD. The role of microRNAs in CAD pathogenesis and their potential as biomarkers remain to be elucidated. MicroRNA-31 (miR-31) level in both the plasma and EPCs of patients with CAD is found lower. miR-31 regulates EPC activities by targeting FAT atypical cadherin 4 and thromboxane A2 receptor, which show increased expression in CAD EPCs. Overexpressing miR-31 in CAD EPCs rescued their angiogenic and vasculogenic abilities both in vitro and in vivo. When exploring approaches to restore endogenous miR-31, we found that far-infrared treatment enhanced the expression of not only miR-31, but also miR-720 in CAD EPCs. miR-720, which was also decreased in EPCs and the plasma of patients with CAD, stimulated EPC activity by targeting vasohibin 1. The miR720-vasohibin 1 pair was shown to be downstream of FAT atypical cadherin 4, but not of thromboxane A2 receptor. FAT atypical cadherin 4 inhibited miR-720 expression via repression of the planar cell polarity signaling gene four-jointed box 1 (FJX1), which was required for miR-720 expression through a hypoxia-inducible factor 1, α subunit-dependent mechanism. Restoring miR-720 level strengthened activity of CAD EPCs. The miR-31-miR-720 pathway is shown critical to EPC activation and that downregulation of this pathway contributes to CAD pathogenesis. Circulating levels of miR-31, miR-720, and vasohibin 1 have the potential to allow early diagnosis of CAD and to act as prognosis biomarkers for CAD and other EPC-related diseases. Manipulating the expression of the miR-31-miR-720 pathway in malfunction EPCs should help develop novel therapeutic modalities.

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

It's not over until the FAT lady sings

The death receptors FAS, TRAIL-Rs and TNFR1 play critical roles in programmed cell death, particularly in the immune system. Upon ligation of death receptors, caspase-8 is activated within the so-called ‘Death Induced Signalling Complex’ (DISC) but the mechanisms that mediate and modulate the activation of caspase-8 are still not fully understood. This is an important issue because caspase-8 is essential for apoptosis induced by death receptors. In this issue of The EMBO Journal, Kranz and Boutros (2014) describe their findings from a whole genome siRNA screen for the identification of novel regulators of death receptor induced apoptosis signalling. They identified the atypical cadherin FAT1 as a negative regulator of TRAIL-R-mediated caspase-8 activation and consequent induction of apoptosis, although it had no impact on NF-κB activation. The authors also show that FAT1 depletion substantially increased TRAIL-induced killing of glioblastoma-derived cell lines, suggesting a potential novel approach for treatment of this highly aggressive cancer.

Regulation and Function of the atypical cadherin FAT1 in hepatocellular carcinoma

Regulation and Function of the atypical cadherin FAT1 in hepatocellular carcinoma

Expression and function of the atypical cadherin FAT1 in chronic liver disease

Hepatic fibrosis can be considered as wound healing process in response to hepatocellular injury. Activation of hepatic stellate cells (HSCs) is a key event of hepatic fibrosis since activated HSCs are the cellular source of enhanced extracellular matrix deposition, and reversion of liver fibrosis is accompanied by clearance of activated HSCs by apoptosis. The atypical cadherin FAT1 has been shown to regulate diverse biological functions as cell proliferation and planar cell polarity, and also to affect wound healing. Here, we found increased FAT1 expression in different murine models of chronic liver injury and in cirrhotic livers of patients with different liver disease. Also in hepatic tissue of patients with non-alcoholic steatohepatitis FAT1 expression was significantly enhanced and correlated with collagen alpha I(1) expression. Immunohistochemistry revealed no significant differences in staining intensity between hepatocytes in normal and cirrhotic liver tissue but myofibroblast like cells in fibrotic septa of cirrhotic livers showed a prominent immunosignal. Furthermore, FAT1 mRNA and protein expression markedly increased during in vitro activation of primary human and murine HSCs. Together, these data indicated activated HSCs as cellular source of enhanced FAT1 expression in diseased livers. To gain insight into the functional role of FAT1 in activated HSCs we suppressed FAT1 in these cells by siRNA. We newly found that FAT1 suppression in activated HSCs caused a downregulation of NFκB activity. This transcription factor is critical for apoptosis resistance of HSCs, and consequently, we detected a higher apoptosis rate in FAT1 suppressed HSCs compared to control cells. Our findings suggest FAT1 as new therapeutic target for the prevention and treatment of hepatic fibrosis in chronic liver disease.

Expression of the atypical cadherin Fat1 in hepatic fibrosis

Expression of the atypical cadherin Fat1 in hepatic fibrosis

Expression and function of the atypical cadherin Fat1 in liver fibrosis

The activation of hepatic stellate cells (HSC) is a key event of hepatic fibrosis, and previous studies found that cadherin expression is altered in activated hepatic stellate cells. Fat1 is an atypical cadherin of more than 500kDa and is characterized by large extracellular domains.

Abstract 436: The Fat1 Cadherin Intracellular Domain Interacts with Atrophin Proteins and Recruits Robust Transcription Activity

Background: We recently reported that the atypical cadherin Fat1 is strongly induced after arterial injury, inhibits growth and promotes migration of vascular smooth muscle cells (VSMCs), and can undergo cleavage, with subsequent translocation of its intracellular domain (Fat1IC) to the cell nucleus. In Drosophila, dFat and Atrophin, a nuclear receptor transcriptional corepressor implicated in the control of cell polarity and migration, have been linked by both physical and genetic interaction. Together, these findings suggest an important integrative role for Fat1 in active vascular remodeling, and raise the possibility that some Fat1 effects are mediated from the nucleus via interaction with transcriptional regulators. Objectives, methods, findings: We hypothesized that the interaction of mammalian Fat1IC with nuclear proteins, such as the mammalian Atrophin homologues atro-1 and atro-2, might contribute to the phenotypic effects of Fat1 on VSMC activation. To test for such interactions, we performed co-immunoprecipitation and Western analysis, identified a physical interaction of Fat1 with atro-1 and atro-2, and mapped necessary Fat1IC sequences to a discrete domain. Immunocytochemical staining showed partial co-localization of Fat1 and atro-1 and -2 in the nucleoplasm. In mammalian two-hybrid assays, surprisingly, tethering of the Fat1IC to DNA yielded very robust transcriptional activation, not repression. This activity was augmented by COUP-TFs, orphan nuclear receptor transcription factors known to associate with atrophins and implicated in both angiogenesis and cardiac development, but not previously linked to Fat1. The biological relevance of these interactions is further supported by quantitative PCR analysis that showed induction of both atro-1 and atro-2 mRNAs after vascular injury, similar to the pattern we identified for Fat1. Conclusion: The biological significance and molecular functions of the multiple cadherin superfamily proteins expressed in VSMCs are not well understood. Our studies demonstrate the physical and functional interaction of the Fat1IC with multiple nuclear proteins involved in transcriptional regulation, and point to novel pathways by which Fat1 may affect VSMC growth and migration.