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SummaryEndosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis in vivo. Indeed, Ptpn23+/− loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23+/−-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23+/− loss increases integrin β1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking.

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat.

SummaryMembrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN) binding to α5β1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5β1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5β1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP) and ubiquitin-associated protein 1 (UBAP1), and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5β1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness.

INTRODUCTION . Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD, and Transient Abnormal Myelopoiesis, TAM) occurs in 10-30% of newborns with Down syndrome (DS). Approx. 20% of infants with TL go on to develop acute myeloid leukemia of DS (ML-DS), typically within the first four years of life. Somatic, clone-specific mutations of GATA1 are found both in the blasts of TL and ML-DS, are concordant within the same individual and thought to function as initiating event in the development of ML-DS. In contrast, additional mutations of cohesin complex and related genes (e.g. RAD21, STAG2, CTCF), epigenetic regulators (e. g. EZH2) and signal transducers (e.g. within RAS, JAK signaling pathways) have been identified only in ML-DS blasts and are thought to cooperate with mutant GATA1 in the progression from TL to ML-DS. It is not known whether these cooperating mutations already mark a minor subclone of TL blasts at birth - allowing, at least in principle, a genetic risk stratification of TL - or are acquired postnatally during the first four years of life. OBJECTIVES . We tested the functional impact of impaired function of cohesin complex genes, CTCF and EZH2 on the progression of TL to ML-DS. We asked if mutations representing putative genetic progression events were already detectable at birth in a minor clone of TL blasts or were acquired postnatally (during the first four years of life). METHODS. The spectrum of GATA1 and cooperating mutations was determined by whole exome sequencing in fractions of TL and ML-DS blasts sorted from blood and bone marrow samples of five patients who had successively developed both disorders including one with a relapse of ML-DS. Corresponding normal T lymphocyte fractions of each patient at the stage of TL and ML-DS served as controls. Numbers of blasts harboring specific mutations were quantified by digital droplet PCR (BioRad, Inc.). Primary TL cells were transduced with lentivirus encoding shRNA (pLVX-shRNA, Clontech, Inc.) to suppress expression of cohesin complex genes, CTCF and EZH2 and intrafemurally injected into 8 week old NSG recipient mice. Engraftment in the bone marrow was assessed 8 weeks later by flow cytometry and GATA1 mutational analysis and compared to TL cells transduced with control vector. RESULTS. TL blasts harbored fewer mutations than those of ML-DS. GATA1 mutations were concordant in TL and ML-DS blasts in the same patient, consistent with development of ML-DS from subclone of TL. Knockdown of RAD21 expression in primary TL blasts, mimicking loss of function mutation of a cohesin complex gene, resulted in significantly increased engraftment of transduced cells in xenograft recipients compared to controls. This finding is consistent with RAD21 loss of function mutations playing the role of a progression event. Mutations of cohesin complex genes (SMC1A, STAG2, RAD21), NRAS and other putative cooperating mutations (with mutant GATA1) were not detectable in any sample of primary TL blasts by either whole exome sequencing or digital droplet PCR. The same result was obtained with control T lymphocytes sorted from TL samples. ML-DS blasts in one case were oligo-clonal with regard to cohesin complex gene mutations. Relapse in this patient arose from a minor clone as defined by cohesin complex gene mutations; mutations of NRAS, KNASL1 and SMC1A were present in ML-DS blasts but absent at relapse. CONCLUSIONS . Increased engraftment of TL cells with suppressed RAD21 expression is consistent with a model in which RAD21 loss of function mutations function as a progression event in the development of ML-DS. Absence of detectable cohesin complex gene mutations and other putative cooperating events in TL blasts suggests these mutations are acquired during the first four years of life and do not mark a minor clone of TL blasts present at birth. Genomic screening of TL blasts at birth therefore is unlikely to predict the risk for development of ML-DS. Disclosures No relevant conflicts of interest to declare.

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis., Author Summary The ability of an organism to adapt to sudden changes in environmental osmolarity is critical to ensure growth, propagation, and survival. The synthesis of organic osmolytes is a common adaptive strategy to survive hyperosmotic stress. However, it was not well understood, which biosynthetic pathways and storage strategies were used by organisms to rapidly generate osmolytes upon acute hyperosmotic stress. Here, we demonstrate that glycogen is an essential reservoir that is used upon acute hyperosmotic stress to generate the organic osmolyte glycerol promoting fast and efficient protection. Importantly, we show that this pathway is regulated by FLCN-1, an ortholog of the human tumor suppressor Folliculin responsible for the Birt-Hogg-Dubé cancer syndrome, and by AMPK, the master regulator of energy homeostasis.

Protein tyrosine phosphatases (PTPs) are key enzymes in the regulation of cellular homeostasis and signaling pathways. Strikingly, not all PTPs bear enzymatic activity. A considerable fraction of PTPs are enzymatically inactive and are known as pseudophosphatases. Despite the lack of activity they execute pivotal roles in development, cell biology and human disease. The present review is focused on the methods used to identify pseudophosphatases, their targets, and physiological roles. We present a strategy for detailed enzymatic analysis of inactive PTPs, regulation of inactive PTP domains and identification of binding partners. Furthermore, we provide a detailed overview of human pseudophosphatases and discuss their regulation of cellular processes and functions in human pathologies.