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Breast cancer has distinct causes and molecular characteristics at premenopausal and postmenopausal ages. The age-standardized incidence rate for postmenopausal breast cancer is more than 10 times higher than in premenopausal breast cancer. Here, we showed that the expression of 10 out of 20 most frequently mutated genes in breast cancer (namely, PIK3CA, CDH1, MUC16, PTEN, FAT3, FAT1, SPEN, ARID1A, LRP1B and RUNX1) is higher in premenopausal women with breast cancer than in postmenopausal women with breast cancer. The most significant differences in the expression in terms of menopause status were observed for RUNX1 and FAT1. Furthermore, we found that the majority of these 10 genes also show ER (estrogen receptor) or PR (progesterone receptor) status-dependent expression in both premenopausal and postmenopausal breast cancer patients. Unlike what we observed in the case of ER or PR status, the expression of most of these genes does not change depending on HER2 (human epidermal growth factor receptor 2) status in both premenopausal and postmenopausal breast cancer patients. Combined, our analysis suggests that menopause status might influence the expression of most frequently mutated genes in breast cancer, and that the most of these genes whose expression differ between pre- and post-menopausal women with breast cancer also show ER or PR status-dependent expression in women with breast cancer. [ABSTRACT FROM AUTHOR]

Glomerulocystic kidney disease (GCKD), a rare nephrocystic disorder, was the focus of this study. The FAT1 gene, comprising 27 exons, encoded a single-pass transmembrane protein with multiple biological functions. The extracellular portion of this protein consisted of 33 cadherin repeat sequences, 5 epidermal growth factor-like domains, and a G-layer associated domain. This report detailed a case of glomerular cystic disease caused by a mutation in the FAT1 gene, which was treated with the traditional Chinese medicine formula ‘Shen Jiu Fang Modified’. This formula included Astragalus, Atractylodes, Poria, Angelica, Clematis, Silkworm, Hedyotis diffusa, Hibiscus leaf, among other ingredients, and aimed to invigorate the spleen and promote diuresis, as well as to nourish the kidneys and promote blood circulation. This case demonstrated the potential of traditional Chinese medicine in addressing rare kidney diseases, offering new insights for the diagnosis and treatment of similar complex cases. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) are normally quiescent and non-migratory, regulating the contraction and relaxation of blood vessels to control the vascular tone. In response to arterial injury, these cells become active; they proliferate, secrete matrix proteins, and migrate, and thereby contribute importantly to the progression of several cardiovascular diseases. VSMC migration specifically supports atherosclerosis, restenosis after catheter-based intervention, transplant vasculopathy, and vascular remodeling during the formation of aneurysms. The atypical cadherin FAT1 is expressed robustly in activated VSMCs and promotes their migration. A positive role of FAT1 in the migration of other cell types, including neurons, fibroblasts, podocytes, and astrocyte progenitors, has also been described. In cancer biology, however, the effect of FAT1 on migration depends on the cancer type or context, as FAT1 either suppresses or enhances cancer cell migration and invasion. With this review, we describe what is known about FAT1's effects on cell migration as well as the factors that influence FAT1-dependent migration. In VSMCs, these factors include angiotensin II, which activates FAT1 expression and cell migration, and proteins of the Atrophin family: Atrophin-1 and the short isoform of Atrophin-2, which promote VSMC migration, and the long isoform of Atrophin-2, which exerts negative effects on FAT1-dependent VSMC migration. [ABSTRACT FROM AUTHOR]

The invasion of cancer cells into interstitial tissues in a cohesive unit is termed collective invasion, and it is important for the invasion and metastasis of salivary adenoid cystic carcinoma (SACC). However, the underlying mechanisms regulating SACC collective invasion are still poorly understood. Here, we found that SACC tissues exhibited remarkable FAT1 downregulation and YAP1 upregulation at the invasive front, which was closely associated with collective invasion and distant metastasis. Decreasing FAT1 expression significantly activated the YAP1 signaling pathway and promoted collective invasion. Moreover, miR-183-5p was identified as the candidate regulator of FAT1 by bioinformatic analysis and an online database algorithm. A dual luciferase reporter experiment further confirmed that miR-183-5p directly targeted the FAT1 3′-UTR to reduce FAT1 expression. Increasing or decreasing miR-183-5p expression promoted or attenuated collective invasion, which was reversed by YAP1 siRNA or FAT1 siRNA, respectively. In addition, knocking down miR-183-5p reduced tumor burden and attenuated collective invasion in vivo. Together, these results suggest that the miR-183-5p/FAT1/YAP1 signaling pathway is a critical driver of SACC collective invasion, revealing a novel therapeutic target. [Display omitted] • FAT1 downregulation and YAP1 upregulation are related to metastasis. • Multicellular and single cell methods were used to evaluate collective invasion. • miR-183-5p can directly target the FAT1 3′-UTR to activate the YAP1 signaling pathway. • Knocking down miR-183-5p prevented tumor growth and collective invasion in vivo. [ABSTRACT FROM AUTHOR]

Background: FAT atypical cadherin 1 (FAT1) is a member of the cadherin superfamily whose loss or gain is associated with the initiation and/or progression of different cancers. FAT1 overexpression has been reported in hematological malignancies. This research intended to investigate FAT1 gene expression in adult Iranian acute leukemia patients, compared to normal mobilized peripheral blood CD34+ cells. Materials and Methods: The peripheral blast (peripheral blood mononuclear cells) cells of 22 acute myeloid leukemia (AML), 14 acute lymphoid leukemia (ALL) patients, and mobilized peripheral blood CD34+ cells of 12 healthy volunteer stem cell donors were collected. Then, quantitative real-time polymerase chain reaction (qPCR) was used to compare FAT1 gene expression. Results: Overall, there were no significant differences in FAT1 expression between AML and ALL patients (p>0.2). Nonetheless, the mean expression level of FAT1 was significantly higher in leukemic patients (AML and ALL) than in normal CD34+ cells (p=0.029). Additionally, the FAT1 expression levels were significantly higher in both CD34+ and CD34- leukemic patients than in normal CD34+ cells (p=0.028). Conclusion: No significant differences were found between FAT1 expression in CD34+ and CD34- leukemic samples (p> 0.3). Thus, higher FAT1 expression was evident in ALL and AML leukemia cells but this appeared unrelated to CD34 expression. This suggests in a proportion of adult acute leukemia, FAT1 expression may prove to be a suitable target for therapeutic strategies. [ABSTRACT FROM AUTHOR]

Genetic studies have established a connection between FAT1 (FAT atypical cadherin 1) deletion and variants and autism spectrum disorder (ASD). Here, we describe a 7‐year‐old girl who sought a neurology consultation in order to be evaluated for ASD and was found to have a de novo 4q35.2 duplication containing the FAT1 gene. Similar to other reported cases of FAT1 variants or deletion, this patient exhibits non‐syndromic ASD without facial dysmorphism or brain MRI abnormalities. We suggest also considering FAT1 duplication as a potential ASD cause. [ABSTRACT FROM AUTHOR]

FAT atypical cadherin 1 (FAT1) is among the most frequently mutated genes in many types of cancer. Its highest mutation rate is found in head and neck squamous cell carcinoma (HNSCC), in which FAT1 is the second most frequently mutated gene. Thus, FAT1 has great potential to serve as a target or prognostic biomarker in cancer treatment. FAT1 encodes a member of the cadherin-like protein family. Under normal physiological conditions, FAT1 serves as a molecular "brake" on mitochondrial respiration and acts as a receptor for a signaling pathway regulating cell–cell contact interaction and planar cell polarity. In many cancers, loss of FAT1 function promotes epithelial-mesenchymal transition (EMT) and the formation of cancer initiation/stem-like cells. However, in some types of cancer, overexpression of FAT1 leads to EMT. The roles of FAT1 in cancer progression, which seems to be cancer-type specific, have not been clarified. To further study the function of FAT1 in cancers, this review summarizes recent relevant literature regarding this protein. In addition to phenotypic alterations due to FAT1 mutations, several signaling pathways and tumor immune systems known or proposed to be regulated by this protein are presented. The potential impact of detecting or targeting FAT1 mutations on cancer treatment is also prospectively discussed. [ABSTRACT FROM AUTHOR]

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. It has been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) represents a highly prevalent and deadly malignancy worldwide. The prognosis for locoregionally advanced HNSCC has not appreciably improved over the past 30 years despite advances in surgical, radiation, and targeted therapies and less than 20% of HNSCC patients respond to recently approved immune checkpoint inhibitors. The Hippo signaling pathway, originally discovered as a mechanism regulating tissue growth and organ size, transduces intracellular and extracellular signals to regulate the transcriptional co-activators YAP and TAZ. Alterations in the Hippo pathway resulting in persistent YAP and TAZ activation have emerged as major oncogenic drivers. Our analysis of the human HNSCC oncogenome revealed multiple genomic alterations impairing Hippo signaling and activating YAP and TAZ, which in turn contribute to HNSCC development. This includes mutations and deletions of the FAT1 gene (29%) and amplification of the WWTR1 (encoding TAZ, 14%) and YAP1 genes (8%), together representing one of the most genetically altered signaling mechanisms in this malignancy. Here, we discuss key elements of the mammalian Hippo pathway, detail mechanisms by which perturbations in Hippo signaling promote HNSCC initiation and progression and outline emerging strategies to target Hippo signaling vulnerabilities as part of novel multimodal precision therapies for HNSCC. [ABSTRACT FROM AUTHOR]

Immunotherapy is currently approved for CRC whose tumors have high MSI-H. To find additional biomarkers for immunotherapy in CRC, targeted sequencing was performed on tumor tissues from a discovery cohort of 161 CRC patients. Validation cohorts from the cBioPortal were also used for survival and tumor cell infiltration analyses. The FAT1 -mutated CRC group often co-occurred with MSI events and displayed a higher tumor mutational burden compared to the FAT1 wild-type CRC. Overall survival was higher in patients with FAT1 mutations than in patients with wild type FAT1. The altered PI3K-AKT pathway and immune pathways were enriched in the FAT1 -mutated CRC. A higher infiltration rate of immune cells including CD4+ T cells, CD8+ T cells, macrophages M1 and regulatory T cells were also observed in the colorectal tumors with FAT1 mutation compared to tumors with wild type FAT1. The results showed that CRC patients with FAT1 mutations exhibited an immunotherapy-favorable profile. • The altered PI3K-AKT pathway and immune pathways were enriched in the FAT1 -mutated CRC. • A higher infiltration rate of immune cells including CD4+ T cells, CD8+ T cells, macrophages M1 and regulatory T cells were observed in the colorectal tumors with FAT1 mutation compared to tumors with wild type FAT1. • CRC patients with FAT1 mutations exhibited the cooccurrence of MSI-H, high TMB, longer overall survival, and increased immune cell infiltration, and may benefit from immunotherapy. [ABSTRACT FROM AUTHOR]

Objective To clarify the mechanism of Fat1 on the proliferation of esophageal squamous cell carcinoma (ESCC). Methods KYSE450 cells were transfected with Plko.1-puro-GFP-shRNA-Fat1 plasmid and real time polymerase chain reaction (PCR) was used to verify the efficiency of Fat1 knockdown. The effects of Fat1 and extracellular regulated protein kinase (ERK) pathway inhibitor U0126 on the proliferation of ESCC cells were detected by methyl thiazolyl tetrazolium (MTT). Colony formation assay was used to detect the colony formation ability. Cell cycle was detected by live cell imaging. Western blot was used to observe the level of target protein. Mouse xenograft assay was applied to detect the effect of Fat1 knockdown on KYSE450 cell tumor growth. Immunohistochemistry was used to detect the expressions of related proteins in tumor sections. Results The efficiency of Fat1 knockdown was (77.1±6.9)% in Fat1 sh1 group and (77.7±7.1)% in Fat1sh2 group. Compared with the control group, the cell proliferation and the expression of p-ERK1/2 were significantly increased in Fat1 sh1 and Fat1sh2 group (P<0.05). After U0126 treatment, the effect of Fat1 knockdown on the proliferation of KYSE450 cells disappeared, and the expression of p-ERK1/2 in KYSE450 cells decreased to a level similar to that in the control group. The number of cell clones in the control group was (72±8), lower than (155±28) and (193±9) in the Fat1sh1 and Fat1sh2 groups, respectively (P<0.05). In KYSE450 cell, division time was shortened from 1 622±32 min in control group to 1 408±29 min in Fat1 sh1 group, the difference was statistically significant (P< 0.05). Compared with the control group, the tumor volume of Fat1 knockdown group increased significantly. The tumor weight of control group and Fat1 knockdown group were (0.224±0.028) g and (1.532±0.196) g, respectively, at 4 weeks after inoculation, and the difference was statistically significant ( P < 0. 05). Conclusion Fat1 inhibits cell proliferation via ERK signaling in ESCC. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is one of the most deadly primary malignant brain tumors in adults. R132H mutation of isocitrate dehydrogenase 1 (IDH1) predicts a better prognosis of GBM. IDH1-R132H is associated with increased hypoxia-inducible factor-1α (HIF-1α) expression in GBM tumors. However, the molecular mechanism underlying IDH1-R132H-HIF-1α signaling in GBM is still unclear.Aim: We aimed to investigate the molecular pathway of IDH1-R132H-HIF-1α in the regulation of GBM.Materials and Methods: U87 and U251 GBM cells and xenograft tumor mice were used.Results: We found that overexpression of IDH1-R132H decreased cell proliferation, increased apoptosis, decreased migration and invasion, enhanced temozolomide (TMZ)-induced cytotoxicity, and reduced tumor growth in xenograft mice. Overexpression of IDH1-R132H increased the expression of HIF-1α and downregulation of HIF-1α suppressed IDH1-R132H-induced effect on GBM. Reactive oxygen species (ROS) level was increased by IDH1-R132H over expression and the use of antioxidant inhibited IDH1-R132H-induced increase of HIF-1α expression. FAT Atypical Cadherin 1 (FAT1) expression was increased by IDH1-R132H over expression. Knockdown of FAT1 blocked IDH1-R132H-induced reduction of tumor growth in xenograft mice. Down regulation of FAT1 decreased HIF-1α expression and inhibited IDH1-R132H-induced increase of ROS level.Conclusions: Our findings provide new insights into IDH1-R132H-regulated downstream signaling in GBM and highlight the importance of IDH1-R132H-FAT1-ROS-HIF-1α signaling pathway in potential therapeutic intervention of GBM. [ABSTRACT FROM AUTHOR]

Background: Sporadic medullary thyroid carcinoma (MTC) is a relatively uncommon neuroendocrine malignancy and the molecular tumorigenesis of its sporadic type (sMTC) is only partially understood. In this study, we performed a study focusing on the genomic and transcriptomic characterization of sMTC. Methods: Twenty-nine sMTC patients were included. Whole-exome sequencing (WES) was carried out in 18 patients, including both tumor samples and matched noncancerous tissues. Whole transcriptome sequencing (RNA-Seq) was performed in all 29 tumors. WES, RNA-Seq, and copy number alteration (CNA) data were analyzed. A Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Results: Among the somatic mutations, RET was the only recurrently cancer-related mutated gene (5/18, 27.8%). In the germline, FAT1 and FAT4, two members of the FAT gene family, were identified as the two most common mutated genes. CNA analysis found that FAT1 and FAT4, both located on chromosome 4q, were also two of the genes most commonly affected by somatic chromosomal deletions (4/18, 22.2%). Using TT and MZ-CRC-1 cell lines, the CCK-8 assay showed that FAT1 and FAT4 knockdown could promote MTC cell proliferation. Based on the gene expression profile, patients were clustered into two molecular subtypes: the mesenchymal-like subtype is characterized by epithelial–mesenchymal transition, while the proliferative-like subtype is associated with enrichment of cell cycle pathways. Most events of structural recurrence (80%) occurred in the proliferative-like subtype. Conclusion: In addition to RET, these findings demonstrate that FAT1/FAT4 genomic alterations appear to be frequent in sMTC. Two molecular subtypes of sMTC with distinct biological behavior could be identified. However, these results need to be validated by larger samples and more comprehensive experiments in the future, especially for the frequency and function of FAT1/FAT4 germline variants. [ABSTRACT FROM AUTHOR]

Background: Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1α, stemness genes and EMT markers in glioma cells. Here, we reveal NFкB (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells.Methods: In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0 kb FAT1 promoter (- 3220 bp to + 848 bp w.r.t. TSS + 1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells.Results: Expression levels of NFкB (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n = 16), REMBRANDT GBM-database (n = 214) and TCGA GBM-database (n = 153). In addition to glioma, positive correlation between NFкB (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0 kb FAT1-promoter-construct [- 3220 bp/+ 848 bp, transcription start site (TSS) + 1, having 17 NFкB (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [- 200 bp/+ 848 bp, with 3-NFкB (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NFкB (RelA)-activators [severe-hypoxia/TNF-α/ectopic-NFкB (RelA) + IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NFкB (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NFкB (RelA)-motif at - 90 bp/- 80 bp [pGL3F1δ1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90 bp/- 80 bp and ChIP assay for endogenous-NFкB (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NFкB (RelA).Conclusion: Since FAT1 and NFкB (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1α/EMT/stemness in tumors, targeting the NFкB (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors. [ABSTRACT FROM AUTHOR]

Introduction. Nephrotic syndrome is a heterogeneous disease in children, with nearly 10% categorized as steroid-resistant. In this study we evaluated disease related mutations within NPHS1, NPHS2 and new potential variants in other genes. Methods. In the first phase of study, 25 patients with SRNS were analyzed by Sanger sequencing for NPHS1 (exon 2, 26) and all exons of NPHS2 genes. In the next step, whole exome sequencing was performed on 10 patients with no mutation in NPHS1 and NPHS2. Results. WES analysis revealed a novel mutation in FAT1 (c.10570C > A; Q3524K). We identified 4 pathogenic mutations, located in exon 4 and 5 of NPHS2 gene in 20% of patients (V180M, P118L, R168C and Leu156Phe). Also our study has contributed to the description of previously known pathogenic mutations across WT1 (R205C) and SMARCAL1 (R764Q) and a novel polymorphism in CRB2. Conclusion. It seems that NPHS2, especially exons 4 and 5, should be considered as the first step in genetic evaluation of Iranian patients. We suggest conducting WES after NPHS2 screening to identify the potential genes associated with SRNS, Further studies are required to examine more common genes in the first step and then designing native laboratory panels. [ABSTRACT FROM AUTHOR]

Objective To explore the expression of FAT1 in esophageal squamous cell carcinoma (ESCC) tissues, and its effect on cell proliferation. Methods The expression levels of FAT1 protein in human ESCC tissues and matched adjacent normal tissues were determined by immunohistochemistry (IHC). Lentivirus based knockdown of FAT1 was carried out in YSE2 and Colo680N cell lines and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assays was performed to examine the effect of FAT1 on the proliferation of these ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle and apoptosis. The expression levels of cell cycle markers in FAT1 knock out ESCC cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot. Results The relative expression of FAT1 in ESCC tissues was 66.97±21.53, significantly lower than 78.13±16.76 of adjacent normal tissues(P<0.05). Knockdown of FAT1 promoted cell proliferation and colony formation. In YSE2 cell, the division time in negative control (NC) group was (1 570±51) min, significantly longer than (1 356±31) min in shFAT1 group. In Colo680N cell, division time in NC group was (1 532±53) min, significantly longer than (1 290±30) min in shFAT1 group (P<0.05). Knockdown of FAT1 promoted G1-to S-phase transition and resulted in the upregulation of CDK4/CDK6/CCND1. Conclusion FAT1 inhibits the proliferation and G1-to S-phase transition of ESCC cells through regulating the protein expression of CDK4/CDK6/CCND1 complex. [ABSTRACT FROM AUTHOR]

FAT1 regulates cell-cell adhesion, cell growth, cell migration, and actin dynamics as either oncogene or tumor suppressor in human cancers. We previously identified FAT1 was one of significant mutant genes in esophageal squamous cell carcinoma (ESCC). However, the function and underlying mechanism of FAT1 in ESCC have not been explored. In this study, we report that FAT1 expression was significantly lower in ESCC tumor tissues. Exogenous expression of FAT1 led to inhibition of cell proliferation and colony formation, as well as cell migration and invasion whereas FAT1 knockdown showed the opponent trends in vitro and in vivo. Moreover, FAT1 knockdown led to a statistically decrease of E-cadherin expression and a dramatically increase expression of N-cadherin, Vimentin, and Snail in a MAPK/ERK pathway-dependent manner. Meanwhile, over-expression of FAT1 resulted in the opposite trends. These alterations were abrogated in the presence of U0126, a MEK specific inhibitor. Collectively, our studies identified a novel role for FAT1 in inhibiting tumor growth and EMT occurrence in ESCC. We proposed that disruption of MAPK/ERK pathway by FAT1 contributes the EMT in ESCC and has important implications for understanding ESCC development. [ABSTRACT FROM AUTHOR]

FAT1 is a type 1 transmembrane protein belonging to the cadherin superfamily. Many reports showed either overexpression of FAT1 gene or its loss in solid cancers. Limited studies evaluated its importance in leukemogenesis. Real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) was used to examine the levels of FAT1 mRNA expression in 50 paediatric acute lymphoblastic leukaemia (ALL) and 50 adult cytogenetically normal acute myeloid leukaemia (CN-AML). Each group was compared with 20 age- and gender-matched healthy controls. Peripheral blood (PB) samples were collected at time of diagnosis from all patients and controls. FAT1 mRNA was detected in 66% of the paediatric ALL cases, 50% of the adult CN-AML cases, while not expressed in the PB of the control groups. T cell ALL (T-ALL) cases showed high statistical significant difference in the expression levels of FAT1 when compared to B cell precursor (BCP)-ALL ( P = 0.001). Paediatric FAT1 ALL expressors showed poor response to induction therapy than FAT1 ALL ( P = 0.001) as well as FAT1 expressors in adult CN-AML when compared to FAT1 ( P = 0.01). FAT1 plays an important role in leukemogenesis; its expression levels may be used as an independent prognostic indicator in paediatric ALL and may help in distinguishing an important risk group in adult CN-AML. [ABSTRACT FROM AUTHOR]

Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell contact, actin dynamics, and cell growth suppression. To understand whether the FAT1 gene affects the apoptosis of porcine cumulus cells and to elucidate the mechanism of this potential action, FAT1 was knocked down using RNA interference. The lack of FAT1 resulted in stable expression of CTNNB, enhanced expression of cleaved CASP3, but decreased the BCL2/BAX ratios at both the mRNA and protein levels. These results indicated that FAT1 inhibited porcine cumulus cell apoptosis via different pathways. Taken together, these data provide new insights into the mechanisms of the association between FAT1 and porcine cumulus cell apoptosis. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour. [ABSTRACT FROM AUTHOR]

Intercellular adhesions are vital hubs for signaling pathways during multicellular development and animal morphogenesis. In eukaryotes, under aberrant intracellular conditions, cadherins are abnormally regulated, which can result in cellular pathologies such as carcinoma, kidney disease, and autoimmune diseases. As a member of the Ca2+-dependent adhesion superfamily, Fat proteins were first described in the 1920s as an inheritable lethal mutant phenotype in Drosophila, consisting of four member proteins, FAT1, FAT2, FAT3, and FAT4, all of which are highly conserved in structure. Functionally, FAT1 was found to regulate cell migration and growth control through specific protein-protein interactions of its cytoplasmic tail. FAT2 and FAT3 are relatively less studied and are thought to participate in the development of human cancer through a pathway similar to that of the Ena/VASP proteins. In contrast, FAT4 has been widely studied in the context of biological functions and tumor mechanisms and has been shown to regulate the planar cell polarity pathway, the Hippo signaling pathway, the canonical Wnt signaling cascade, and the expression of YAP1. Overall, Fat cadherins may be useful as emerging disease biomarkers and as novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Synchronous cancers have occasionally been detected at initial diagnosis among patients with breast and ovarian cancer. However, simultaneous coexistence and diagnosis of breast and pancreas cancer has not previously been reported. Case Report: Paternal transmission of a germline BRCA2 mutation to a patient who was diagnosed at age 40 with locally advanced breast and pancreas cancer is presented. Somatic genomic analysis of both cancers with next-generation DNA sequencing confirmed the germline result and reported a variety of variants of unknown significance alterations, of which two were present in both the breast and pancreas cancers. Discussion: The possibility that genomic alterations could have been responsible for modulating the phenotypic or clinical expression of this rare presentation is considered. The authors call attention to the practice of privatizing the clinicogenetic information gained from genetic testing and call for health policy that will facilitate sharing in order to advance the outcomes of patients diagnosed with hereditary cancers. [ABSTRACT FROM AUTHOR]

FAT1 and β-catenin are important tumor regulatory factors. The aim of this study was to detect the possible disparity in their expression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) and to explore its correlation with clinicopathological factors. We used immunohistochemistry to detect protein expression of FAT1 and β-catenin in breast cancer tissues from 113 cases of DCIS and 149 cases of IDC. As compared with DCIS, expression of FAT1 and β-catenin were significantly decreased in IDC ( P < 0.05). In addition, our study also revealed a correlation between their expression and some clinicopathological factors. We found that FAT1 expression was associated with nuclear grade and comedonecrosis ( P < 0.05) in DCIS, whereas FAT1 expression showed significant variation with histological grade and LN status ( P < 0.05) in IDC. Similar associations were observed in the β-catenin subgroup. Furthermore, expressions of FAT1 and β-catenin were correlated with each other in DCIS and IDC ( P < 0.05). FAT1(−), β-catenin(−), or FAT1(−)/β-catenin(−) may indicate worse DFS and OS in breast cancer ( P < 0.05). This study suggests that loss of FAT1 and β-catenin are associated with breast cancer progression, aggressive behavior, and poor prognosis. FAT1 alone or together with β-catenin might be a valuable biomarker in predicting the prognosis of patients with breast cancer. [ABSTRACT FROM AUTHOR]

The n − 3 fatty acid desaturase gene fat1 codes for the n − 3 desaturase enzyme, which can convert n − 6 polyunsaturated fatty acids (PUFAs) to n − 3 PUFAs. The n − 3 PUFAs are essential components required for normal cellular function and have preventive and therapeutic effects on many diseases. Goat is an important domestic animal for human consumption of meat and milk. To elevate the concentrations of n − 3 PUFAs and examine the regulatory mechanism of fat1 in PUFA metabolism in goat cells, we successfully constructed a humanized fat1 expression vector and confirmed the efficient expression of fat1 in goat ear skin-derived fibroblast cells (GEFCs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that fat1 overexpression significantly increased the levels of total n − 3 PUFAs and decreased the levels of total n − 6 PUFAs in GEFCs. In addition, qRT-PCR results indicate that the FADS1 and FADS2 desaturase genes, ELOV2 and ELOV5 elongase genes, ACO and CPT1 oxidation genes, and PPARa and PPARγ transcription factors are up-regulated, and transcription factors of SREBP-1c gene are down-regulated in the fat1 transgenic goat cells. Overall, fat1- overexpression resulted in an increase in the n − 3 fatty acids and altered expression of PUFA synthesis related genes in GEFCs. This work lays a foundation for both the production of fat1 transgenic goats and further study of the mechanism of fat1 function in the PUFAs metabolism. [ABSTRACT FROM AUTHOR]

Background: Herein, we demonstrated the use of a newly generated anti FAT1 antibody (clone mAB198.3) for intracellular delivery of anionic gold NPs, to form active targeting Au nanoparticles with high payload characteristics. Methods: In vitro characterizations were determined by DLS, confocal microscopy, TEM, western blot, MALDI-TOF MS/MS analysis, MTT, ICP-MS and flow cytometry analysis. In vivo targeting efficacy was investigated by in vivo bio-imaging study and ICP-MS. Results: The specificity of the FAT1 recognition in colon cancer was confirmed by pre-adsorbing mAb198.3, adsorption dramatically abolished the antibody reactivity on colon cancer, thus confirming the binding specificity. The DLS size distribution profile of the AuCOOH, AuCOOH(Cy5)_ mAb198.3, AuCOOH(Cy5)_isotype has showed that the modified gold nanoparticles are well dispersed in water, PBS buffer and cell culture medium with 10% FBS. By TEM measurement, the size of Au nanoparticles with spherical morphology is about 10-20 nm. AuCOOH_198.3 NPs were stable in an acidic environment, as well as in PBS buffer, cell culture media and media with 10% serum. MTT results revealed that Au nanoparticles have well biocompatibility. TEM results indicated that conjugation of mAb198.3 on Au nanoparticles can be an effective delivery vehicle for negatively charged gold nanoparticles and increased its intracellular transport. It was also demonstrated by confocal microscopy that AuCOOH(Cy5)_mAb198.3 could attach to the cell membrane in very short time, then gradually delivered into cells. After 4 h incubation, almost all AuCOOH(Cy5)_mAb198.3 have been uptaken into or surrounding the cytoplasm and nucleus. In vivo results showed that only about 20% of AuCOOH accumulated in tumor site due to EPR effect, while nearly 90% of AuCOOH_mAb198.3 was found in tumor, providing sufficient evidence for receptor-specific targeting by mAb198.3. Conclusion: According to in vitro and in vivo research results, the intracellular uptake of negatively charged AuCOOH_ mAB198.3 particles is enhanced to a greater extent. Thus, AuCOOH_mAb198.3 holds significant potential to improve the treatment of cancer. [ABSTRACT FROM AUTHOR]

Dearomatized isoprenylated acylphloroglucinols (DIAPs) are specific natural products mainly distributed in the plants of genus Hypericum. In this study, guided by HPLC-UV screening, 46 DIAPs (approximately 70% of all DIAPs) including 20 new ones and an unprecedented architecture, were discovered from the roots of Hypericum henryi , which were elucidated by comprehensive spectroscopic, X-ray crystallography, and ECD methods. Compounds 1 – 7 , 39 , and 41 – 42 exhibited remarkable cytotoxicities (IC 50 = 0.84–5.63 μM) in human colon cancer HCT116 cells, in which 2 and 6 possessed selective cytotoxicities towards colon cancer cells. The preliminary structure-activity relationships of these tested compounds were discussed. In addition, mechanistic investigations demonstrated that 2 and 6 could significantly suppress the expressions of NFκB, FAT1, and promoted novel tumor suppressor gene PDCD4 in HCT116 cells. Furthermore, in HCT116 colon xenograft-bearing mouse model, treatments with 2 and 6 reduced the growth of xenograft tumors in dose-dependent manner. Expressions of FAT1 in tumors were also decreased in mice treated with 2 and 6 , suggesting their anti-tumor effects were via FAT1 signaling pathway. In conclusion, this is the first report on the mechanistic and in vivo studies of DIAP, indicating that these metabolites can be considered as a new type of anti-colon cancer lead agents for further drug development. [Display omitted] • Diverse DIAPs including 21 new ones were discovered by HPLC-UV screening approach. • These compounds account for about 70% of all reported DIAPs. • 1 – 7 , 39 , and 41 – 42 exhibited remarkable cytotoxicities in HCT116 cells. • Anti-colon cancer activities of 2 and 6 were evaluated in vitro and in vivo. • 2 and 6 reduced the growth of colon tumor via NFκB-FAT1-PDCD4 signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Herein, we demonstrated the use of a newly generated anti FAT1 antibody (clone mAB198.3) for intracellular delivery of anionic gold NPs, to form active targeting Au nanoparticles with high payload characteristics. Methods: In vitro characterizations were determined by DLS, confocal microscopy, TEM, western blot, MALDI-TOF MS/MS analysis, MTT, ICP-MS and flow cytometry analysis. In vivo targeting efficacy was investigated by in vivo bio-imaging study and ICP-MS. Results: The specificity of the FAT1 recognition in colon cancer was confirmed by pre-adsorbing mAb198.3, adsorption dramatically abolished the antibody reactivity on colon cancer, thus confirming the binding specificity. The DLS size distribution profile of the AuCOOH, AuCOOH(Cy5)_ mAb198.3, AuCOOH(Cy5)_isotype has showed that the modified gold nanoparticles are well dispersed in water, PBS buffer and cell culture medium with 10% FBS. By TEM measurement, the size of Au nanoparticles with spherical morphology is about 10-20 nm. AuCOOH_198.3 NPs were stable in an acidic environment, as well as in PBS buffer, cell culture media and media with 10% serum. MTT results revealed that Au nanoparticles have well biocompatibility. TEM results indicated that conjugation of mAb198.3 on Au nanoparticles can be an effective delivery vehicle for negatively charged gold nanoparticles and increased its intracellular transport. It was also demonstrated by confocal microscopy that AuCOOH(Cy5)_mAb198.3 could attach to the cell membrane in very short time, then gradually delivered into cells. After 4 h incubation, almost all AuCOOH(Cy5)_mAb198.3 have been uptaken into or surrounding the cytoplasm and nucleus. In vivo results showed that only about 20% of AuCOOH accumulated in tumor site due to EPR effect, while nearly 90% of AuCOOH_mAb198.3 was found in tumor, providing sufficient evidence for receptor-specific targeting by mAb198.3. Conclusion: According to in vitro and in vivo research results, the intracellular uptake of negatively charged AuCOOH_ mAB198.3 particles is enhanced to a greater extent. Thus, AuCOOH_mAb198.3 holds significant potential to improve the treatment of cancer. [ABSTRACT FROM AUTHOR]

Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica . Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae . While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, Δ Ylpxa1 Δ Ylpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the Δ Ylpxa1 Δ Ylpxa2 Δ Ylant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast. [ABSTRACT FROM AUTHOR]

The interaction between the Drosophila cadherins fat and dachsous is regulated by phosphorylation of their respective ectodomains, a process catalysed by the atypical kinase four-jointed. Given that many signalling functions are conserved between Drosophila and vertebrate Fat cadherins, we sought to determine whether ectodomain phosphorylation is conserved in FAT1 cadherin, and also whether FJX1, the vertebrate orthologue of four-jointed, was involved in such phosphorylation events. Potential Fj consensus phosphorylation motifs were identified in FAT1 and biochemical experiments revealed the presence of phosphoserine and phosphothreonine residues in its extracellular domain. However, silencing FJX1 did not influence the levels of FAT1 ectodomain phosphorylation, indicating that other mechanisms are likely responsible. [ABSTRACT FROM AUTHOR]

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 si RNA 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. [ABSTRACT FROM AUTHOR]

Abstract: Fat1 is an atypical cadherin that controls vascular smooth muscle cell (VSMC) proliferation and migration. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (Ang II) induces the expression and/or activation of both Fat1 and Nox1 proteins. This study tested the hypothesis that Ang II-induced Fat1 activation and VSMC migration are mediated by Nox1-dependent ROS generation and redox signaling. Studies were performed in cultured VSMCs from Sprague–Dawley rats. Cells were treated with Ang II (1μmol/L) for short (5 to 30min) or long term stimulations (3 to 12h) in the absence or presence of the antioxidant apocynin (10μmol/L), extracellular-signal-regulated kinases 1/2 (Erk1/2) inhibitor PD98059 (1μmol/L), or Ang II type 1 receptor (AT1R) valsartan (1μmol/L). siRNA was used to knockdown Nox1 or Fat1. Cell migration was determined by Boyden chamber assay. Ang II increased Fat1 mRNA and protein levels and promoted Fat1 translocation to the cell membrane, responses that were inhibited by AT1R antagonist and antioxidant treatment. Downregulation of Nox1 inhibited the effects of Ang II on Fat1 protein expression. Nox1 protein induction, ROS generation, and p44/p42 MAPK phosphorylation in response to Ang II were prevented by valsartan and apocynin, and Nox1 siRNA inhibited Ang II-induced ROS generation. Knockdown of Fat1 did not affect Ang II-mediated increases in Nox1 expression or ROS. Inhibition of p44/p42 MAPK phosphorylation by PD98059 abrogated the Ang II-induced increase in Fat1 expression and membrane translocation. Knockdown of Fat1 inhibited Ang II-induced VSMC migration, which was also prevented by valsartan, apocynin, PD98059, and Nox1 siRNA. Our findings indicate that Ang II regulates Fat1 expression and activity and induces Fat1-dependent VSMC migration via activation of AT1R, ERK1/2, and Nox1-derived ROS, suggesting a role for Fat1 downstream of Ang II signaling that leads to vascular remodeling. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

In the present study, acyl-CoA synthetase mutants of Saccharomyces cerevisiae were employed to investigate the impact of this activity on certain pools of fatty acids. We identified a genotype responsible for the secretion of free fatty acids into the culture medium. The combined deletion of Faa1p and Faa4p encoding two out of five acyl-CoA synthetases was necessary and sufficient to establish mutant cells that secreted fatty acids in a growth-phase dependent manner. The mutants accomplished fatty acid export during exponential growth-phase followed by fatty acid re-import into the cells during the stationary phase. The data presented suggest that the secretion is driven by an active component. The fatty acid re-import resulted in a severely altered ultrastructure of the mutant cells. Additional strains deficient of any cellular acyl-CoA synthetase activity revealed an almost identical phenotype, thereby proving transfer of fatty acids across the plasma membrane independent of their activation with CoA. Further experiments identified membrane lipids as the origin of the observed free fatty acids. Therefore, we propose the recycling of endogenous fatty acids generated in the course of lipid remodelling as a major task of both acyl-CoA synthetases Faa1p and Faa4p. [ABSTRACT FROM AUTHOR]

We identified 22 polymorphisms in the adipocyte fatty-acid binding protein (FABP4) gene, a strong positional candidate gene for the FAT1 locus in porcine chromosome 4. The most informative polymorphism, an insertion/deletion in intron 1, together with a single nucleotide polymorphism in intron 3, was genotyped in a cross between Iberian and Landrace pigs. After performing QTL, single marker, and haplotype analyses, we showed that there were at least 2 quantitative trait genes in the FAT1 region and that the FABP4 polymorphism was tightly associated to fatness. A comparison of allelic frequencies in a panel of pig breeds suggested that the Del2634C polymorphism was under indirect selection. We also showed that FABP4 is tightly associated to fatness but not growth. Furthermore, a haplotype analysis suggests that there is genetic heterogeneity at the FAT1 locus within the Landrace breed. [ABSTRACT FROM AUTHOR]

Role of Fat1 in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney. Background. Fat1 is a member of the cadherin superfamily characterized by its 34 cadherin repeats in the extracellular domain. Fat1 was originally found as a component of the slit diaphragm of podocytes, but its function in podocytes remains obscure. To gain insight into its role in podocytes, we expanded our study of Fat1 expression to puromycin aminonucleoside (PAN) nephrosis, the neonatal kidney, and the primary podocyte culture, where slit diaphragms are absent or disappear. Methods. Expression of Fat1 was examined in isolated glomeruli of PAN nephrosis by the ribonuclease protection assay and Western blot analysis and in the neonatal kidney by in situ hybridization. Fat1 localization in glomeruli and in the primary culture was confirmed by immunofluorescence or immunoelectron microscopy. Results. In PAN nephrotic rats, glomerular expression of Fat1 increased rather than decreased at both transcript and protein levels in comparison with normal rats. Immunofluorescence microscopy revealed distinct staining for Fat1 along the glomerular capillary wall, where nephrin staining was weakened or disappeared. Immunoelectron microscopy demonstrated significant accumulation of immunogold particles for Fat1 at intercellular junctions newly formed between podocytes in the nephrosis. In the primary culture of podocytes, Fat1 was mainly localized at cell-cell contact sites and in tips of cellular processes. In the neonatal kidney, immature podocytes expressed Fat1 more intensely than mature podocytes as shown by in situ hybridization. Double-labeled immunostaining using anti-pan cadherin antibody revealed that Fat1 in podocytes colocalized with cadherin in immature glomeruli, indicating that junctional complexes of developing podocytes contain Fat1. Conclusion. These findings suggest that Fat1 may be a fundamental component of intercellular junctions of podocytes, and may be involved in the initial step of cell contacts of podocytes. [ABSTRACT FROM AUTHOR]

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue. [ABSTRACT FROM AUTHOR]

We report on two siblings suffering from different pathogenic conditions, born to consanguineous parents. A multigene panel for brain malformations and microcephaly identified the homozygous splicing variant NM_005886.3:c.1416+1del in the KATNB1 gene in the older sister. On the other hand, exome sequencing revealed the homozygous frameshift variant NM_005245.4:c.9729del in the FAT1 gene in the younger sister, who had a more complex phenotype: in addition to bilateral anophthalmia and heart defects, she showed a right split foot with 4 toes, 5 metacarpals, second toe duplication and preaxial polydactyly on the right hand. These features have been never reported before in patients with pathogenic FAT1 variants and support the role of this gene in the development of limb buds. Notably, each parent was heterozygous for both of these variants, which were ultra-rare and rare, respectively. This study raises awareness about the value of using whole exome/genome sequencing rather than targeted gene panels when testing affected offspring born to consanguineous couples. In this way, exomic data from the parents are also made available for carrier screening, to identify heterozygous pathogenetic and likely pathogenetic variants in genes responsible for other recessive conditions, which may pose a risk for subsequent pregnancies. [ABSTRACT FROM AUTHOR]

Simple Summary: With the application of immunotherapy in patients with non-small-cell lung cancer (NSCLC), we found that immunotherapy for some patients cannot achieve long-term effects. Therefore, the purpose of this study is to explore the characteristics of these patients and make a model that can effectively predict the prognosis of immunotherapy patients. The results of this study will make it easier for clinicians to screen out NSCLC patients for immunotherapy. (1) Background: The immune checkpoint blockade (ICB) has shown promising efficacy in non-small-cell lung cancer (NSCLC) patients with significant clinical benefits and durable responses, but the overall response rate to ICBs is only 20%. The lack of responsiveness to ICBs is currently a central problem in cancer immunotherapy. (2) Methods: Four public cohorts comprising 2986 patients with NSCLC were included in the study. We screened 158 patients with NSCLC with no durable clinical benefit (NDB) to ICBs in the Rizvi cohort and identified NDB-related gene mutations in these patients using univariate and multivariate Cox regression analyses. Programmed death-ligand 1 (PD-L1) expression, tumor mutation burden (TMB), neoantigen load, tumor-infiltrating lymphocytes, and immune-related gene expression were analyzed for identifying gene mutations. A comprehensive predictive classifier model was also built to evaluate the efficacy of ICB therapy. (3) Results: Mutations in FAT1 and KEAP1 were found to correlate with NDB in patients with NSCLC to ICBs; however, the analysis suggested that only mutation in FAT1 was valuable in predicting the efficacy of ICB therapy, and that mutation in KEAP1 acted as a prognostic but not a predictive biomarker for NSCLC. Mutations in FAT1 were associated with a higher TMB and lower multiple lymphocyte infiltration, including CD8 (T-Cell Surface Glycoprotein CD8)+ T cells. We established a prognostic model according to PD-L1 expression, TMB, smoking status, treatment regimen, treatment type, and FAT1 mutation, which indicated good accuracy by receiver operating characteristic (ROC) analysis (area under the curve (AUC) for 6-months survival: 0.763; AUC for 12-months survival: 0.871). (4) Conclusions: Mutation in FAT1 may be a predictive biomarker in patients with NSCLC who exhibit NDB to ICBs. We proposed an FAT1 mutation-based model for screening more suitable NSCLC patients to receive ICBs that may contribute to individualized immunotherapy. [ABSTRACT FROM AUTHOR]

FAT atypical cadherin 1 (FAT1) regulates cell-cell adhesion and extracellular matrix architecture, while acting as tumor suppressor or oncogene, context-dependently. Despite implication of FAT1 in several malignancies, its role in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we document the driver-oncogene role of FAT1, and its mediation of cell-death evasion, proliferation, oncogenicity, and chemoresistance in OSCC. In-silica analyses indicate FAT1 mutations are frequent and drive head-neck SCC, with enhanced expression defining high-risk population and poor prognosis. We demonstrated aberrant FAT1 mRNA and protein expression in OSCC compared with non-cancer tissues, whereas loss-of-FAT1-function attenuates human primary SAS and metastatic HSC-3 OSCC cell viability, without affecting normal primary human gingival fibroblast cells. shFAT1 suppressed PCNA and upregulated BAX/BCL2 ratio in SAS and HSC-3 cells. Moreover, compared with wild-type cells, shFAT1 concomitantly impaired HSC-3 cell migration, invasion, and clonogenicity. Interestingly, while over-expressed FAT1 characterized cisplatin-resistance (CispR), shFAT1 synchronously re-sensitized CispR cells to cisplatin, enhanced glutathione (GSH)/GSH synthetase (GSS)-mediated oxidative stress and deregulated LRP5/WNT2 signaling. Concisely, FAT1 is an actionable driver-oncogene in OSCC and targeting FAT1 in patients with erstwhile cisplatin-resistant OSCC is therapeutically promising. [ABSTRACT FROM AUTHOR]

Background: FAT1 is a transmembrane protein and function as an adhesion molecule. FAT1 gene is located on chromosome 4q35.2 encoding a 506KDa protein. The role of FAT1 in tumors is not fully characterized. Few reports suggest it behaves as a tumor suppressor and few reported it as oncogenic. Here in our study we are characterizing the role of FAT1 in primary brain tumors. There are studies showing altered expression of FAT1 in human gliomas with ~30% of them showing LOH at the FAT1 locus. TP53 is mutated in 50% of primary brain tumors. P53 being a transcription factor, regulates the expression of a number of target genes including regulating biological functions such as cell cycle arrest, apoptosis, senescence, differentiation and antiangiogenesis. Glioma is known to have hypoxic environment in its tumour core and hypoxic tumour micro environment and HIF1A is believed to play role in tumour progression . So, in this study we are analysing the interaction between FAT1, P53 and HIF1A in glioma progression. Methodology: In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies in glioma cell lines followed by expression analysis by Q-PCR, Immunofluorescence and Western Blot. Migratory potential of cells was assessed by Migration assay and invasion potential were assessed using Matrigel invasion assay and images were captured using Nikon fluorescence microscopy and confocal microscopy. For cell proliferation and viability assay MTT/ XTT were used. Cell cycle analysis was carried out using PI staining in Flow cytometry (BD canto FACS) Sample Collection: - Glial tumor samples were collected from Department of Neurosurgery/Pathology of All India Institute of Medical Sciences, India. Tissue were collected in 1 ml of RNA-Later solution and incubated for 24 hours at 4°C. After removing RNA-Later solution the tumor tubes were immediately stored at -70°C till further use. For the control, normal human brain RNA was purchased commercially. Transfection of cell lines: - 1) For Knockdown: Selection of target siRNA and corresponding control- We have been using commercially available siRNA (Invitrogen) for FAT1 knockdown in our lab. FAT1 and control siRNA Oligos are designed by Genscript siRNA construct builder software(http://www.genscript. com/siRNA_construct_builder.html). 2) FAT1_trunc (Truncated FAT1) transfection: plasmid containing truncated FAT1 gene in pcDNA vector 3.1 was used (gifted by Dr Timothy A Chan). Truncated FAT1 plasmid contains first two repeats of cadherins at N terminals and around 22 repeats of cadherins are removed retaining last few cadherin repeats along with intercellular region of FAT1 gene. The vectors is tested for its expression and also known to retain all its functions. Transfection is carried out for 48 hr. Empty vector PCDNA is used as control. Transfection was carried out after plating 40000 to 50000 cells in 6 well plates for U87MG cells line and 1.2-1.5 lakhs cells for GOS and U373 Cell lines. The total duration of transfection was 72 hr (excluding plating) for knockdown and 48 hr for overexpression experiments. Hypoxia exposure was given with 0.2% oxygen for 48 hr After transfection following experimental techniques were done • MTT/XTT assay - For Cell survival • Flow cytometry- For Apoptosis assay/cell cycle analysis • RNA isolation and lysate preparation- For pathway(s) analysis by Real-Time PCR and Western blotting. • Migration and invasion assay - BD Matrigel • ICC Results Hypoxic treatment exhibited a significant increase in expression levels (7 to 9-fold) of FAT1 in U87MG, U373MG and GOS3 glioma cell lines. On FAT1 Knockdown under severe hypoxia, P53 and HIF1A expression was decreased and on Fat1 Knockdown under normoxia the expression of p53 and HIF1A was increased. The downstream targets of P53 (Noxa, Puma, p21 and MDM2) and downstream targets of HIF1A (VEGF, Ca9, GLUT1 and PGK3) also showed similar trend as p53 and HIF1A. On western blot analysis, protein levels of HIF1A and p53 showed increased levels after FAT1 knockdown under normoxia and no significant change was observed under FAT1 Knockdown in hypoxia. Our array data showed differential gene expression on FAT1 after knockdown. We observed reduction in HIF1A expression after FAT1 Knockdown under hypoxia and increased p53 and HIF1A on FAT1 Knockdown under normoxia. Increased in FAT1 expression under hypoxia and reduction of HIF1A after FAT1 Knockdown seems to suggest a direct link of FAT1 and HIF1A. Further, on subjecting U87 MG (grade IV GBM cell line) to hypoxia, we observed the shift in subcellular localization of FAT1 from perinuclear and plasma membrane location to inside the nucleus on ICC experiments suggesting FAT1 itself act as a transcription factors or help other transcription factors to increase or decrease gene expression involved in hypoxia and tumour progression. On over expressing FAT1 trunc we observed increased p53 [removed]1.49 fold) in normoxia and 1.87 fold increase under hypoxia (0.2%O2). MDM2 (downstream target gene of p53) also increased after FAT1 over expression by 1.629 fold in normoxia and 1.42 in hypoxia. We also observed elevated HIF1A in U87 and GOS3 along with their downstream molecules. RNA was isolated from histopath positive glioma grade IV tumors samples and gene expression analysis was performed in 18 tumour samples. We observed in GBM patients samples a positive correlation between FAT1 expression and P53 (r2 0.723 p=0.0029) and positive correlation of p53 with HIF1A (r2 =0.620, p=0.0106) and correlation of FAT1 expression with HIF1A showed r2 0.44, p=0.063. The expressions correlating analysis for patient samples and qPCR analysis of glioma cell lines suggests an existence of an intermediary molecule connecting the HIF1A and p53 regulation by FAT1. We examined p300 a co transcription factor necessary for both p53 and HIF1A transcription activity. P300 is upregulated when FAT1 is knockdown under normoxic condition. Absence of FAT1 (knockdown) under normoxia in U87MG cell lines increased p53 (1.7fold) and HIF1A (1.5 fold) and also p300 (1.8 fold). Increase in P53 in FAT1 Knockdown under normoxia is due to C/EBP beta. C/EBP is a known transcription factor and has two binding site in p53 promotors and C/EBP beta expressions is increased on FAT1 knockdown under normoxia to 1.56 fold. HIF1A being onocogene should promotes proliferation/migration and invasion but in our study we observed decreased in migration and invasion (Matrigel assay) and decreased in proliferation (MTT/XTT cell proliferation assay) and increased in apoptosis (Annexin FITC and PI flowcytometry assay) and increased Sub G0 population (PI cell cycle assay) on Knock down of FAT1 in normoxia. Increased p53 and its downstream genes after FAT1 Knockdown activated p300/p53 combination over p300/HIF1A resulting in decreased oncogenicity in normoxia in absences of FAT1. On other hand after FAT1 over expressions we observed decreased p300 and decreased p53 and its downstream and increased migration/ invasion and viability. The cell viability was 74.61 % after FAT1 over expression compared to control under normoxia. In hypoxia, the cell viability was increased after FAT1 over [removed]91% compared to control). Migratory properties also showed significant increase in over expression sample (4.19 times) then mock control under normoxia. This observation points to the critical and important role of FAT1 in cell migration. In conclusion FAT1 plays an oncogenic role in glioma and HIF1A and p53 plays and important role. The exact mechanism involved in the oncogenesis by FAT1 is under study. [ABSTRACT FROM AUTHOR]