Clinical value of FAT1 mutations to indicate the immune response in colorectal cancer patients.

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.

Diagnostic Challenges and Emerging Pathogeneses of Selected Glomerulopathies.

Recent progress in glomerular immune complex and complement-mediated diseases have refined diagnostic categories and informed mechanistic understanding of disease development in pediatric patients. Herein, we discuss selected advances in 3 categories. First, membranous nephropathy antigens are increasingly utilized to characterize disease in pediatric patients and include phospholipase A2 receptor (PLA2R), Semaphorin 3B (Sema3B), neural epidermal growth factor-like 1 (NELL1), and protocadherin FAT1, as well as the lupus membranous-associated antigens exostosin 1/2 (EXT1/2), neural cell adhesion molecule 1 (NCAM1), and transforming growth factor beta receptor 3 (TGFBR3). Second, we examine advances in techniques for paraffin and light chain immunofluorescence (IF), including the former's function as a salvage technique and their necessity for diagnosis in adolescent cases of membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) and proliferative glomerulonephritis with monotypic Ig deposits (PGNMID), respectively. Finally, progress in understanding the roles of complement in pediatric glomerular disease is reviewed, with specific attention to overlapping clinical, histologic, and genetic or functional alternative complement pathway (AP) abnormalities among C3 glomerulopathy (C3G), infection-related and post-infectious GN, "atypical" post-infectious GN, immune complex mediated membranoproliferative glomerulonephritis (IC-MPGN), and atypical hemolytic uremic syndrome (aHUS).

Specific antigen-based stratification of membranous nephropathy in patients after haematopoietic stem cell allotransplantation - a case series and literature review.

BACKGROUND: Nephrotic syndrome (NS) is a rare complication that can occur after haematopoietic stem cell transplantation (HSCT). In patients with membranous nephropathy (MN) who have undergone allogeneic HSCT, a new antigen called protocadherin FAT1 has been identified. Our objective is to present a case series of MN patients after HSCT with a novel antigen-based stratification. CASE PRESENTATIONS: Patients who developed full-blown NS due to MN after an HSCT were enrolled in the University Hospital Centre Zagreb study. The first two patients were treated with an HSCT for acute myeloid leukaemia, and both developed NS after cessation of graft versus host disease (GVHD) prophylaxis. The first patient had reduced kidney function, while the second had completely preserved function. Kidney biopsy showed MN with only subepithelial deposits. A thorough examination revealed that there was no secondary cause of the disease. The patients achieved complete remission after undergoing immunosuppression treatment. The third patient underwent HSCT for acute lymphoblastic leukaemia. He developed both acute and chronic GVHD and also experienced avascular hip necrosis. After sixteen years, the patient developed NS with preserved kidney function. The kidney specimen showed membranous nephropathy (MN) with mesangial and subepithelial deposits. Extensive research was conducted, but no secondary cause for the MN was detected. All three cases tested negative for anti-PLA2R antibodies. Biopsy tissue samples were analysed using laser microdissection and tandem mass spectrometry of glomeruli for the detection of different specific antigens. Patients one and two tested positive for FAT1, whereas patient three tested positive for PCSK6. CONCLUSIONS: MN can develop at various time intervals after HSCT. Specific antigen testing can help establish the relationship between MN and HSCT. In the future, serum testing for anti-FAT1 antibodies in HSCT patients could be significant in diagnosing FAT1-associated MN, similar to how anti-PLA2R antibodies are significant in diagnosing PLA2R-associated MN.

[Expression of FAT1 in Lung Adenocarcinoma and Its Relationship 
with Immune Cell Infiltration].

BACKGROUND: Lung cancer is a leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the most common pathological subtype, with adenocarcinoma being the predominant type. FAT atypical cadherin 1 (FAT1) is a receptor-like protein with a high frequency of mutations in lung adenocarcinoma. The protein encoded by FAT1 plays a crucial role in processes such as cell adhesion, proliferation, and differentiation. This study aims to investigate the expression of FAT1 in lung adenocarcinoma and its relationship with immune infiltration. METHODS: Gene expression levels and relevant clinical information of 513 lung adenocarcinoma samples and 397 adjacent lung samples were obtained through The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data. The mRNA expression levels of the FAT1 gene in lung adenocarcinoma tissues were analyzed, along with its association with the prognosis of lung adenocarcinoma patients. Pathway enrichment analysis was conducted to explore the signaling pathways regulated by the FAT1 gene. Immunoblotting was used to detect the differential expression of FAT1 in lung epithelial cells and various lung cancer cell lines, while immunohistochemistry was employed to assess FAT1 expression in lung cancer and adjacent tissues. RESULTS: FAT1 gene mutations were identified in 14% of lung adenocarcinoma patients. TCGA database data revealed significantly higher FAT1 mRNA expression in lung adenocarcinoma tissues compared to adjacent lung tissues. Kaplan-Meier analysis indicated lower survival rates in lung adenocarcinoma patients with higher FAT gene expression. Pathway enrichment analysis suggested the involvement of FAT1 in tumor development pathways, and its expression was closely associated with immune cell infiltration. Immunohistochemical validation demonstrated significantly higher expression of FAT1 in cancer tissues compared to adjacent lung tissues. CONCLUSIONS: FAT1 mRNA is highly expressed in lung adenocarcinoma tissues, and elevated FAT1 mRNA expression is associated with poor prognosis in lung adenocarcinoma patients. FAT1 may serve as a potential biomarker for lung cancer.

The tumour suppressor Fat1 is dispensable for normal murine hematopoiesis.

Loss and overexpression of FAT1 occurs among different cancers with these divergent states equated with tumor suppressor and oncogene activity, respectively. Regarding the latter, FAT1 is highly expressed in a high proportion of human acute leukemias relative to normal blood cells, with evidence pointing to an oncogenic role. We hypothesized that this occurrence represents legacy expression of FAT1 in undefined hematopoietic precursor subsets that is sustained following transformation, predicating a role for FAT1 during normal hematopoiesis. We explored this concept by using the Vav-iCre strain to construct conditional knockout (cKO) mice where Fat1 expression was deleted at the hematopoietic stem cell stage. Extensive analysis of precursor and mature blood populations using multi-panel flow cytometry revealed no ostensible differences between Fat1 cKO mice and normal littermates. Further functional comparisons involving colony forming unit and competitive bone marrow transplantation assays support the conclusion that Fat1 is dispensable for normal murine hematopoiesis.

Pan-cancer analysis predict that FAT1 is a therapeutic target and immunotherapy biomarker for multiple cancer types including non-small cell lung cancer.

FAT1, a substantial transmembrane protein, plays a pivotal role in cellular adhesion and cell signaling. Numerous studies have documented frequent alterations in FAT1 across various cancer types, with its aberrant expression being linked to unfavorable survival rates and tumor progression. In the present investigation, we employed bioinformatic analyses, as well as in vitro and in vivo experiments to elucidate the functional significance of FAT1 in pan-cancer, with a primary focus on lung cancer. Our findings unveiled FAT1 overexpression in diverse cancer types, including lung cancer, concomitant with its association with an unfavorable prognosis. Furthermore, FAT1 is intricately involved in immune-related pathways and demonstrates a strong correlation with the expression of immune checkpoint genes. The suppression of FAT1 in lung cancer cells results in reduced cell proliferation, migration, and invasion. These collective findings suggest that FAT1 has potential utility both as a biomarker and as a therapeutic target for lung cancer.

STAT1 mediated downregulation of the tumor suppressor gene PDCD4, is driven by the atypical cadherin FAT1, in glioblastoma.

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.

FAT1 upregulation is correlated with an immunosuppressive tumor microenvironment and predicts unfavorable outcome of immune checkpoint therapy in non-small cell lung cancer.

Background: Previous studies found that FAT1 was recurrently mutated and aberrantly expressed in multiple cancers, and the loss function of FAT1 promoted the formation of cancer-initiating cells in several cancers. However, in some types of cancer, FAT1 upregulation could lead to epithelial-mesenchymal transition (EMT). The role of FAT1 in cancer progression, which appears to be cancer-type-specific, is largely unknown. Methods: QRT-PCR and immunochemistry were used to verify the expression of FAT1 in non-small cell lung cancer (NSCLC). QRT-PCR and Western blot were used to detect the influence of siFAT1 knockdown on the expression of potential targets of FAT1 in NSCLC cell lines. GEPIA, KM-plotter, CAMOIP, and ROC-Plotter were used to evaluate the association between FAT1 and clinical outcomes based on expression and clinical data from TCGA and immune checkpoint inhibitors (ICI) treated cohorts. Results: We found that FAT1 upregulation was associated with the activation of TGF-ß and EMT signaling pathways in NSCLC. Patients with a high FAT1 expression level tend to have a poor prognosis and hard to benefit from ICI therapy. Genes involved in TGF-ß/EMT signaling pathways (SERPINE1, TGFB1/2, and POSTN) were downregulated upon knockdown of FAT1. Genomic and immunologic analysis showed that high cancer-associated fibroblast (CAF) abundance, decreased CD8+ T cells infiltration, and low TMB/TNB were correlated with the upregulation of FAT1, thus promoting an immunosuppressive tumor microenvironment (TME) which influence the effect of ICI-therapy. Conclusion: Our findings revealed the pattern of FAT1 upregulation in the TME of patients with NSCLC, and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential therapeutic target for NSCLC treatment.

The Planar Cell Polarity Protein Fat1 in Sertoli Cell Function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with ß-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

Half of most frequently mutated genes in breast cancer are expressed differentially between premenopausal and postmenopausal breast cancer patients.

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.

FAT1 inhibits the proliferation of DLBCL cells via increasing the m6A modification of YAP1 mRNA.

Emerging evidence shows that FAT atypical cadherin 1 (FAT1) mutations occur in lymphoma and are associated with poorer overall survival. Considering that diffuse large B cell lymphoma (DLBCL) is the category of lymphoma with the highest incidence rate, this study aims to explore the role of FAT1 in DLBCL. The findings demonstrate that FAT1 inhibits the proliferation of DLBCL cell lines by downregulating the expression of YAP1 rather than by altering its cellular localization. Mechanistic analysis via meRIP-qPCR/luciferase reporter assays showed that FAT1 increases the m6A modification of YAP1 mRNA 3'UTR and the subsequent binding of heterogeneous nuclear ribonucleoprotein D (HNRNPD) to the m6A modified YAP1 mRNA, thus decreasing the stability of YAP1 mRNA. Furthermore, FAT1 increases YAP1 mRNA 3'UTR m6A modification by decreasing the activity of the TGFß-Smad2/3 pathway and the subsequent expression of ALKBH5, which is regulated at the transcriptional level by Smad2/3. Collectively, these results reveal that FAT1 inhibits the proliferation of DLBCL cells by increasing the m6A modification of the YAP1 mRNA 3'UTR via the TGFß-Smad2/3-ALKBH5 pathway. The findings of this study therefore indicate that FAT1 exerts anti-tumor effects in DLBCL and may represent a novel target in the treatment of this form of lymphoma.

FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression.

BACKGROUND: Emerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. METHODS: AML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. RESULTS: In this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-ßR II which decreased the binding of TGF-ßR II to TGF-ßR I and the subsequent phosphorylation of TGF-ßR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. CONCLUSIONS: Collectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFß-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. GENERAL SIGNIFICANCE: Our study suggested that the "FAT1-TGFß-smad2/3-ATG4B-autophagy" pathway may be a novel target for developing new targeted drugs to AML treatment.

Capacidad predictiva de la expresión transcripcional de FAT1 en pacientes con carcinomas escamosos de cabeza y cuello tratados con radioterapia / Predictive capacity of FAT1 transcriptional expression in patients with head and neck squamous cell carcinomas treated with radiotherapy

Objetivo Analizar la capacidad predictiva de la respuesta a nivel local de la expresión transcripcional de FAT1 en pacientes con carcinomas escamosos de cabeza y cuello tratados con radioterapia.Material y métodosLlevamos a cabo un estudio retrospectivo realizado a partir de biopsias de la localización primaria del tumor en 82 pacientes con carcinomas escamosos de cabeza y cuello tratados con radioterapia. Se determinó la expresión transcripcional de FAT1 mediante RT-PCR. Se categorizó el nivel de expresión transcripcional de FAT1 en función del control local tras el tratamiento con radioterapia mediante un análisis de partición recursiva.ResultadosLa expresión transcripcional elevada de FAT1 se relacionó con un incremento en el riesgo de recidiva local tras el tratamiento con radioterapia. Los pacientes con unos niveles de expresión elevada de FAT1 (n=18; 22,0%) tuvieron una supervivencia libre de recidiva local a los 5 años del 42,1% (IC 95%: 18,6-65,6%), en tanto que para los pacientes con una expresión baja (n=64; 78,0%) fue del 72,4% (IC 95%: 61,5-83,3%) (p=0,002). De acuerdo con el resultado de un análisis multivariante, los pacientes con una categoría de expresión elevada de FAT1 tuvieron un riesgo 2,3 veces superior de recidiva local (IC 95%: 1,0-5,2; p=0,043).ConclusionesLa expresión transcripcional elevada de FAT1 se relacionó con un incremento significativo del riesgo de recidiva local en los pacientes con carcinomas escamosos de cabeza y cuello tratados con radioterapia. (AU)

Objective To analyze the predictive capacity at the primary location of the tumor of the FAT1 transcriptional expression in patients with head and neck squamous cell carcinoma treated with radiotherapy.Material and methodsWe conducted a retrospective study from biopsies of the primary location of the tumor in 82 patients with head and neck squamous cell carcinoma treated with radiotherapy. The transcriptional expression of FAT1 was determined by RT-PCR. The level of FAT1 transcriptional expression was categorized according to the local control after radiotherapy using a recursive partitioning analysis.ResultsElevated FAT1 transcriptional expression was associated with an increased risk of local recurrence after radiotherapy. Patients with a high expression level of FAT1 (n=18; 22.0%) had a 5-year local recurrence-free survival of 42.1% (95% CI: 18.6–65.6%), whereas for patients with a low expression (n=64; 78.0%) it was 72.4% (95% CI: 61.5%–83.3%) (P=0.002). According to the result of a multivariate analysis, patients with a high FAT1 expression category had a 2.3-fold increased risk of local recurrence (95% CI: 1.0–5.2; P=0.043).ConclusionsElevated FAT1 transcriptional expression was associated with a significantly increased risk of local recurrence in patients with head and neck squamous cell carcinoma treated with radiotherapy. (AU)