A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review.

BACKGROUND: TFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics. CASE PRESENTATION: We report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient's molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-ß, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed. CONCLUSION: We clarified the patient's challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

TFEB regulates dendritic cell antigen presentation to modulate immune balance in asthma.

OBJECTIVE: Asthma stands as one of the most prevalent chronic respiratory conditions in children, with its pathogenesis tied to the actived antigen presentation by dendritic cells (DCs) and the imbalance within T cell subgroups. This study seeks to investigate the role of the transcription factor EB (TFEB) in modulating the antigen presentation process of DCs and its impact on the differentiation of T cell subgroups. METHODS: Bone marrow dendritic cells (BMDCs) were activated using house dust mites (HDM) and underwent RNA sequencing (RNA-seq) to pinpoint differentially expressed genes. TFEB mRNA expression levels were assessed in the peripheral blood mononuclear cells (PBMCs) of both healthy children and those diagnosed with asthma. In an asthma mouse model induced by HDM, the TFEB expression in lung tissue DCs was evaluated. Further experiments involved LV-shTFEB BMDCs co-cultured with T cells to explore the influence of TFEB on DCs' antigen presentation, T cell subset differentiation, and cytokine production. RESULTS: Transcriptomic sequencing identified TFEB as a significantly differentially expressed gene associated with immune system pathways and antigen presentation. Notably, TFEB expression showed a significant increase in the PBMCs of children diagnosed with asthma compared to healthy counterparts. Moreover, TFEB exhibited heightened expression in lung tissue DCs of HDM-induced asthmatic mice and HDM-stimulated BMDCs. Silencing TFEB resulted in the downregulation of MHC II, CD80, CD86, and CD40 on DCs. This action reinstated the equilibrium among Th1/Th2 and Th17/Treg cell subgroups, suppressed the expression of pro-inflammatory cytokines like IL-4, IL-5, IL-13, and IL-17, while augmenting the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: TFEB might have a vital role in asthma's development by impacting the antigen presentation of DCs, regulating T cell subgroup differentiation, and influencing cytokine secretion. Its involvement could be pivotal in rebalancing the immune system in asthma. These research findings could potentially unveil novel therapeutic avenues for treating asthma.

Genome-wide identification of ZmMYC2 binding sites and target genes in maize.

BACKGROUND: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified. RESULTS: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA. CONCLUSIONS: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

An unusual case of primary splenic soft part alveolar sarcoma: case report and review of the literature with emphasis on the spectrum of TFE3-associated neoplasms.

BACKGROUND: Alveolar soft part sarcoma is a rare tumour of soft tissues, mostly localized in muscles or deep soft tissues of the extremities. In rare occasions, this tumour develops in deep tissues of the abdomen or pelvis. CASE PRESENTATION: In this case report, we described the case of a 46 year old man who developed a primary splenic alveolar soft part sarcoma. The tumour displayed typical morphological alveolar aspect, as well as immunohistochemical profile notably TFE3 nuclear staining. Detection of ASPSCR1 Exon 7::TFE3 Exon 6 fusion transcript in molecular biology and TFE3 rearrangement in FISH confirmed the diagnosis. CONCLUSION: We described the first case of primary splenic alveolar soft part sarcoma, which questions once again the cell of origin of this rare tumour.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation.

BACKGROUND: Muscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported. METHODS: Wild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux. RESULTS: WT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner. CONCLUSIONS: Females have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells.

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

A genome-first approach to variants in MLXIPL and their association with hepatic steatosis and plasma lipids.

BACKGROUND: Common variants of the max-like protein X (MLX)-interacting protein-like (MLXIPL) gene, encoding the transcription factor carbohydrate-responsive element-binding protein, have been shown to be associated with plasma triglyceride levels. However, the role of these variants in steatotic liver disease (SLD) is unclear. METHODS: We used a genome-first approach to analyze a variety of metabolic phenotypes and clinical outcomes associated with a common missense variant in MLXIPL, Gln241His, in 2 large biobanks: the UK Biobank and the Penn Medicine Biobank. RESULTS: Carriers of MLXIPL Gln241His were associated with significantly lower serum levels of triglycerides, apolipoprotein-B, gamma-glutamyl transferase, and alkaline phosphatase. Additionally, MLXIPL Gln241His carriers were associated with significantly higher serum levels of HDL cholesterol and alanine aminotransferase. Carriers homozygous for MLXIPL Gln241His showed a higher risk of SLD in 2 unrelated cohorts. Carriers of MLXIPL Gln241His were especially more likely to be diagnosed with SLD if they were female, obese, and/or also carried the PNPLA3 I148M variant. Furthermore, the heterozygous carriage of MLXIPL Gln241His was associated with significantly higher all-cause, liver-related, and cardiovascular mortality rates. Nuclear magnetic resonance metabolomics data indicated that carriage of MLXIPL Gln241His was significantly associated with lower serum levels of VLDL and increased serum levels of HDL cholesterol. CONCLUSIONS: Analyses of the MLXIPL Gln241His polymorphism showed a significant association with a higher risk of SLD diagnosis and elevated serum alanine aminotransferase as well as significantly lower serum triglycerides and apolipoprotein-B levels. MLXIPL might, therefore, be a potential pharmacological target for the treatment of SLD and hyperlipidemia, notably for patients at risk. More mechanistic studies are needed to better understand the role of MLXIPL Gln241His on lipid metabolism and steatosis development.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases.

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

[Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway].

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

TXNIP deficiency attenuates renal fibrosis by modulating mTORC1/TFEB-mediated autophagy in diabetic kidney disease.

Thioredoxin-interacting protein (TXNIP) is an important regulatory protein for thioredoxin (TRX) that elicits the generation of reactive oxygen species (ROS) by inhibiting the redox function of TRX. Abundant evidence suggests that TXNIP is involved in the fibrotic process of diabetic kidney disease (DKD). However, the potential mechanism of TXNIP in DKD is not yet well understood. In this study, we found that TXNIP knockout suppressed renal fibrosis and activation of mammalian target of rapamycin complex 1 (mTORC1) and restored transcription factor EB (TFEB) and autophagy activation in diabetic kidneys. Simultaneously, TXNIP interference inhibited epithelial-to-mesenchymal transformation (EMT), collagen I and fibronectin expression, and mTORC1 activation, increased TFEB nuclear translocation, and promoted autophagy restoration in HK-2 cells exposed to high glucose (HG). Rapamycin, an inhibitor of mTORC1, increased TFEB nuclear translocation and autophagy in HK-2 cells under HG conditions. Moreover, the TFEB activators, curcumin analog C1 and trehalose, effectively restored HG-induced autophagy, and abrogated HG-induced EMT and collagen I and fibronectin expression in HK-2 cells. Taken together, these findings suggest that TXNIP deficiency ameliorates renal fibrosis by regulating mTORC1/TFEB-mediated autophagy in diabetic kidney diseases.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1.

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Liver ChREBP deficiency inhibits fructose-induced insulin resistance in pregnant mice and female offspring.

High fructose intake during pregnancy increases insulin resistance (IR) and gestational diabetes mellitus (GDM) risk. IR during pregnancy primarily results from elevated hormone levels. We aim to determine the role of liver carbohydrate response element binding protein (ChREBP) in insulin sensitivity and lipid metabolism in pregnant mice and their offspring. Pregnant C57BL/6J wild-type mice and hepatocyte-specific ChREBP-deficient mice were fed with a high-fructose diet (HFrD) or normal chow diet (NC) pre-delivery. We found that the combination of HFrD with pregnancy excessively activates hepatic ChREBP, stimulating progesterone synthesis by increasing MTTP expression, which exacerbates IR. Increased progesterone levels upregulated hepatic ChREBP via the progesterone-PPARγ axis. Placental progesterone activated the progesterone-ChREBP loop in female offspring, contributing to IR and lipid accumulation. In normal dietary conditions, hepatic ChREBP modestly affected progesterone production and influenced IR during pregnancy. Our findings reveal the role of hepatic ChREBP in regulating insulin sensitivity and lipid homeostasis in both pregnant mice consuming an HFrD and female offspring, and suggest it as a potential target for managing gestational metabolic disorders, including GDM.

Stress-induced microautophagy is coordinated with lysosome biogenesis and regulated by PIKfyve.

Lysosome turnover and biogenesis are induced in response to treatment of cells with agents that cause membrane rupture, but whether other stress conditions engage similar homeostatic mechanisms is not well understood. Recently we described a form of selective turnover of lysosomes that is induced by metabolic stress or by treatment of cells with ionophores or lysosomotropic agents, involving the formation of intraluminal vesicles within intact organelles through microautophagy. Selective turnover involves noncanonical autophagy and the lipidation of LC3 onto lysosomal membranes, as well as the autophagy gene-dependent formation of intraluminal vesicles. Here, we find a form of microautophagy induction that requires activity of the lipid kinase PIKfyve and is associated with the nuclear translocation of TFEB, a known mediator of lysosome biogenesis. We show that LC3 undergoes turnover during this process, and that PIKfyve is required for the formation of intraluminal vesicles and LC3 turnover, but not for LC3 lipidation onto lysosomal membranes, demonstrating that microautophagy is regulated by PIKfyve downstream of noncanonical autophagy. We further show that TFEB activation requires noncanonical autophagy but not PIKfyve, distinguishing the regulation of biogenesis from microautophagy occurring in response to agents that induce lysosomal stress.

Establishment and characterization of NCC-ASPS2-C1: a novel patient-derived cell line of alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal tumor characterized by rearrangement of the ASPSCR1 and TFE3 genes and a histologically distinctive pseudoalveolar pattern. ASPS progresses slowly, but is prone to late metastasis. As ASPS is refractory to conventional chemotherapy, the only curative treatment is complete surgical resection. The prognosis of advanced and metastatic cases is poor, highlighting the need for preclinical research to develop appropriate treatment options. However, ASPS is extremely rare, accounting for < 1% of all soft tissue sarcomas, and only one patient-derived ASPS cell line is available from public cell banks worldwide for research. This study reports the establishment of a novel ASPS cell line derived from the primary tumor tissue of an ASPS patient, named NCC-ASPS2-C1. This cell line retains the ASPSCR1-TFE3 fusion gene, which is characteristic of ASPS. The characterization of this cell line revealed stable growth, spheroid formation, and invasive properties. By screening a drug library using NCC-ASPS2-C1, we identified several drugs that inhibited the proliferation of ASPS cells. In conclusion, the establishment of NCC-ASPS2-C1 provides a valuable resource for advancing ASPS research and developing novel treatments for this challenging disease.

A nomogram based on TFE3 IHC results and clinical factors as a preliminary screening scheme for TFE3-rearranged renal cell carcinoma.

BACKGROUND: TFE3 immunohistochemistry (TFE3-IHC) is controversial in the diagnosis of TFE3-rearranged renal cell carcinoma (TFE3-rearranged RCC). This study is to investigate the accuracy and sensitivity of IHC and establish a predictive model to diagnose TFE3-rearranged RCC. METHODS: Retrospective analysis was performed by collecting IHC and fluorescence in situ hybridization (FISH) results from 228 patients. IHC results were evaluated using three scoring systems. Scoring system 1 is graded based on nuclear staining intensity, scoring system 2 is graded based on the percentage of stained tumor cell nuclei, and scoring system 3 is graded based on both the nuclear staining intensity and the percentage. We collected patients' IHC results and clinical information. Important variables were screened based on univariate logistic regression analysis. Then, independent risk factors were established through multivariate logistic regression, and a nomogram model was constructed. The model was validated in internal test set and external validation set. The receiver operating characteristic curve (ROC curve), calibration curve, and decision curve analysis (DCA) were generated to assess discriminative ability of the model. RESULTS: The accuracy of IHC based on three scoring systems were 0.829, 0.772, and 0.807, respectively. The model included four factors including age, gender, lymph node metastasis and IHC results. Area under the curve (AUC) values were 0.935 for the training set, 0.934 for the internal test set, 0.933 for all 228 patients, and 0.916 for the external validation set. CONCLUSIONS: TFE3 IHC has high accuracy in the diagnosis of TFE3-rearranged RCC. Clinical information such as age and lymph node metastasis are independent risk factors, which can be used as a supplement to the results of TFE3 IHC. This study confirms the value of IHC in the diagnosis of TFE3-rearranged RCC. The accuracy of the diagnosis can be improved by incorporating IHC with other clinical risk factors.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Autophagy inhibition suppresses hormone production and cell growth in pituitary tumor cells: A potential approach to pituitary tumors.

Pituitary tumors (PTs) represent about 10% of all intracranial tumors, and most are benign. However, some PTs exhibit continued growth despite multimodal therapies. Although temozolomide (TMZ), an alkylating chemotherapeutic agent, is a first-line medical treatment for aggressive PTs, some PTs are resistant to TMZ. Existing literature indicated the involvement of autophagy in cell growth in several types of tumors, including PTs, and autophagy inhibitors have anti-tumor effects. In this study, the expression of several autophagy-inducible genes, including Atg3, Beclin1, Map1lc3A, Map1lc3b, Ulk1, Wipi2, and Tfe3 in two PT cell lines, the mouse corticotroph AtT-20 cells and the rat mammosomatotroph GH4 cells were identified. Down regulation of Tfe3, a master switch of basal autophagy, using RNA interference, suppressed cell proliferation in AtT-20 cells, suggesting basal autophagy contributes to the maintenance of cellular functions in PT cells. Expectedly, treatment with bafilomycin A1, an autophagy inhibitor, suppressed cell proliferation, increased the cleavage of PARP1, and reduced ACTH production in AtT-20 cells. Treatment with two additional autophagy inhibitors, chloroquine (CQ) and monensin, demonstrated similar effects on cell proliferation, apoptosis, and ACTH production in AtT-20 cells. Also, treatment with CQ suppressed cell proliferation and growth hormone production in GH4 cells. Moreover, the combination of CQ and TMZ had an additive effect on the inhibition of cell proliferation in AtT-20 and GH4 cells. The additive effect of anti-cancer drugs such as CQ alone or in combination with TMZ may represent a novel therapeutic approach for PTs, in particular tumors with resistance to TMZ.