Abstract Adaptation to low oxygen tensions is required to survive physiological and pathological stress conditions. We discovered an adenosine monophosphate–activated protein kinase (AMPK)-dependent, hypoxia-specific survival pathway through transcription factors (TFEB, TFE3). This pathway is distinct from nutrient sensing and independent of GAP Activity Towards Rags 1 (GATOR1). Mechanistically, AMPK phosphorylates the tumor suppressor folliculin (FLCN) at a highly conserved serine, inhibiting its GAP activity towards the Rag-GTPases, resulting in mechanistic target of rapamycin complex 1 (mTORC1) inhibition, TFE3 activation, resulting in lysosomal and mitochondrial biogenesis essential for hypoxia adaptation. TFEB/3 activity correlates with the hypoxia signature in hypoxic tumor areas. Cancer patients with elevated TFEB/3 activity show poor survival, specifically in hypoxia. These findings identify the AMPK-FLCN-TFE3 axis as a therapeutic target in tumors.

The 9th Birt-Hogg-Dubé (BHD) International Symposium convened virtually in March 2026. The meeting attracted more than 100 participants internationally and highlighted recent findings in a variety of areas, including genetic insight and molecular understanding of BHD syndrome, also known as the Hornstein-Knickenberg syndrome, structure and function of the tumor suppressor Folliculin (FLCN), therapeutic and clinical advances, as well as patient experiences living with this disease.

Birt-Hogg-Dubé syndrome (BHD) patients are uniquely susceptible to all renal tumor subtypes. However, the underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase the tumorigenic potential of these cells, as evidenced by the formation of larger spheroids under nonadherent conditions. Transcriptomic and proteomic analyses revealed links between the FLCN, cell cycle control and DNA damage response (DDR) machinery. In addition, HK2 cells lacking FLCN had an altered transcriptome profile and enriched cell cycle control genes. G1/S cell cycle checkpoint signaling was compromised by increased protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen revealed that FLCN binds to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked increase in γH2AX and RB1 phosphorylation. The levels of both CCND1 and phosphorylated RB1 remained high during DNA damage, which was associated with defective cell cycle control caused by FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest caused by DNA damage. This work revealed that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

Birt-Hogg-Dubé (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase their tumorigenic potential, forming larger spheroids in non-adherent conditions. Transcriptomic and proteomic analysis uncovered links between FLCN, cell cycle control and the DNA damage response (DDR) machinery. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen uncovered FLCN binding to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked elevation of γH2AX and RB1 phosphorylation. Both CCND1 and RB1 phosphorylation remained raised during DNA damage, showing an association with defective cell cycle control with FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest by DNA damage. This work implicates that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

Les cancers cutanés sont un véritable problème de santé publique, avec une augmentation d'incidence ces cinquante dernières années. Le principal facteur de risque est le rayonnement ultra-violet (UV), dont les indices peuvent être particulièrement importants dans certains départements d'outre-mer. Ainsi, les travailleurs en extérieur sont une population à risque élevé. L'objectif de cette étude était d'estimer la part des cancers cutanés potentiellement attribuables aux expositions solaires professionnelles dans un département d'outre-mer. Cette étude porte sur l'ensemble des mélanomes, carcinomes épidermoïdes cutanés et un échantillon représentatif des carcinomes basocellulaires, diagnostiqués entre le 1er octobre 2020 et le 30 septembre 2021 chez des sujets ayant travaillé au moins 3 ans dans le département. Des entretiens téléphoniques ont été réalisés avec chaque patient afin de recueillir des données sur leur exposition solaire durant l'enfance, les loisirs et le travail. Les cancers ont été classés comme potentiellement attribuables à l'activité professionnelle si le temps d'exposition solaire professionnelle était supérieur au temps d'exposition solaire lors des loisirs, en tenant compte des données de protection solaire et de la localisation du cancer cutané (zones exposées durant l'activité professionnelle). Parmi les 329 patients inclus, 37,7 % présentaient un(des) cancer(s) cutané(s) attribuable(s) à l'exposition professionnelle aux UV. Plusieurs professions à risque étaient mises en évidence dont les agriculteurs. 93 % des patients rapportaient ne jamais avoir eu d'aménagements mis en place par leur employeur en termes de prévention solaire, 73 % n'utilisaient jamais de crème de protection solaire au travail. Le pourcentage de patients ayant un cancer cutané potentiellement attribuable aux expositions solaires professionnelles dans ce département d'outre-mer est considérable. Une relation significative entre les expositions professionnelles aux UV et les carcinomes cutanés a déjà été décrite dans la littérature. Une reconnaissance en tant que maladie professionnelle des cancers cutanés chez les travailleurs exposés devrait être considérée. La prévention et l'éducation à la photoprotection dans les milieux professionnels est primordiale.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

EGFR cell surface density, stability, internalization, and recycling can be measured by cell surface ELISA (cs-ELISA). Performing this experiment on ice impedes receptor internalization; thus the physiological cell surface receptor levels can be measured by cs-ELISA. Cell surface EGFR levels are detected by measuring Amplex Red fluorescence intensity. Although cell surface receptor levels can be measured by flow cytometry, cs-ELISA does not include cell dissociation steps that might affect cell surface receptor levels. For complete details on the use and execution of this protocol, please refer to Kazan et al. (2019).

The 7th Birt-Hogg-Dubé (BHD) International Symposium convened virtually in October 2021. The meeting attracted more than 200 participants internationally and highlighted recent findings in a variety of areas, including genetic insight and molecular understanding of BHD syndrome, structure and function of the tumor suppressor Folliculin (FLCN), therapeutic and clinical advances as well as patients' experiences living with this malady.

Folliculin (FLCN) is a tumor suppressor gene responsible for the inherited Birt-Hogg-Dubé (BHD) syndrome, which affects kidneys, skin and lungs. FLCN is a highly conserved protein that forms a complex with folliculin interacting proteins 1 and 2 (FNIP1/2). Although its sequence does not show homology to known functional domains, structural studies have determined a role of FLCN as a GTPase activating protein (GAP) for small GTPases such as Rag GTPases. FLCN GAP activity on the Rags is required for the recruitment of mTORC1 and the transcriptional factors TFEB and TFE3 on the lysosome, where mTORC1 phosphorylates and inactivates these factors. TFEB/TFE3 are master regulators of lysosomal biogenesis and function, and autophagy. By this mechanism, FLCN/FNIP complex participates in the control of metabolic processes. AMPK, a key regulator of catabolism, interacts with FLCN/FNIP complex. FLCN loss results in constitutive activation of AMPK, which suggests an additional mechanism by which FLCN/FNIP may control metabolism. AMPK regulates the expression and activity of the transcriptional cofactors PGC1α/β, implicated in the control of mitochondrial biogenesis and oxidative metabolism. In this review, we summarize our current knowledge of the interplay between mTORC1, FLCN/FNIP, and AMPK and their implications in the control of cellular homeostasis through the transcriptional activity of TFEB/TFE3 and PGC1α/β. Other pathways and cellular processes regulated by FLCN will be briefly discussed.

Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.

Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.

Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

Increased macroautophagy/autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK (AMP-activated protein kinase) activation and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) inhibition to maintain energy homeostasis and cell survival. TFEB (transcription factor E3) and TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by MTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB and TFE3 transcriptional activity upon nutrient starvation, FLCN (folliculin) depletion and pharmacological manipulation of MTORC1 or AMPK. Collectively, we show that MTORC1 specifically controls TFEB and TFE3 cytosolic retention, whereas AMPK is essential for TFEB and TFE3 transcriptional activity. This dual and opposing regulation of TFEB and TFE3 by MTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1 (unc-51 like autophagy activating kinase 1). Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466, 467 and 469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB and TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.

Non-alcoholic steatohepatitis (NASH) represents a major economic burden and is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat this condition. Emerging data suggests an important role of autophagy in this condition, which serves to degrade intracellular lipid stores, reduce hepatocellular damage, and dampen inflammation. Autophagy is primarily regulated by the transcription factors TFEB and TFE3, which are negatively regulated by mTORC1. Given that FLCN is an mTORC1 activator via its GAP activity towards RagC/D, we generated a liver specific Flcn knockout mouse model to study its role in NASH progression. We demonstrate that loss of FLCN results in reduced triglyceride accumulation, fibrosis, and inflammation in mice exposed to a NASH-inducing diet. Hence, the GAP activity of FLCN could a promising target for small molecule drugs to treat NASH progression by specifically activating autophagy and lysosomal biogenesis while leaving mRNA translation machinery unperturbed. Collectively, these results show an unexpected role for FLCN in NASH progression and highlight new possibilities for treatment strategies through its role in hepatocyte homeostasis.

Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. Research on pseudoenzymes is an emerging area of interest, with new biological functions repurposed from catalytically active relatives. Here, we provide an overview of the pseudophosphatases identified to date in all major phosphatase families. We will highlight the degeneration of the various catalytic sequence motifs and discuss the challenges associated with the experimental determination of catalytic inactivity. We will also summarize the role of pseudophosphatases in various diseases and discuss the major challenges and future directions in this field.

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK conferred pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, while ablation of total AMPK activity abolished this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induced TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages was observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved and pharmacologically actionable mechanism coupling energy status with innate immunity.

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 proximity-dependent biotin identification (BioID) interactome, and confirmed this association using co-immunoprecipitation. HD-PTP loss attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo. HD-PTP depletion abrogates ephrin-B2-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP loss also accelerates ligand-induced EphB2 degradation, contrasting the effects of HD-PTP loss on the relay of signals from other cell surface receptors. Our results link Eph function to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol- 5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a(-/-) Pip4k2b(-/-) mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.