Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

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Somatic copy number alterations play a critical role in oncogenesis. Loss of chromosomal regions containing tumor suppressors can lead to collateral deletion of passenger genes. This can be exploited therapeutically if synthetic lethal partners of such passenger genes are known and represent druggable targets. Here, we report that VPS4B gene, encoding an ATPase involved in ESCRT-dependent membrane remodeling, is such a passenger gene frequently deleted in many cancer types, notably in colorectal cancer (CRC). We observed downregulation of VPS4B mRNA and protein levels from CRC patient samples. We identified VPS4A paralog as a synthetic lethal interactor for VPS4B in vitro and in mouse xenografts. Depleting both proteins profoundly altered the cellular transcriptome and induced cell death accompanied by the release of immunomodulatory molecules that mediate inflammatory and anti-tumor responses. Our results identify a pair of novel druggable targets for personalized oncology and provide a rationale to develop VPS4 inhibitors for precision therapy of VPS4B-deficient cancers.

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We found that FccRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P 2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Ia (PIP5-kinase Ia) that colocalized with PI(4,5)P 2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C 374-440 fragment of PIP5-kinase Ia or the pleckstrin homology domain of phospholipase Cd 1 (PLCd 1 -PH), two probes binding PI(4,5)P 2 . These probes reduced the amount of PI(4,5)P 2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P 2 elevation during phagocytosis. Simultaneously, PLCd 1 -PH-GFP reduced the amount of PIP5-kinase Ia associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase Ia-GST bound PI(4,5)P 2 , phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLCd 1 -PH domain, and possibly also the C 374-440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase Ia for PI(4,5)P 2 binding in the plasma membrane leading eventually to PI(4,5)P 2 depletion.

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Molecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 112 endocytosis-related genes. Among them, transcription of the endosomal sorting complex required for transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients, which also showed reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21 (CDKN1A)-mediated inhibition of cell proliferation and sterile inflammatory response driven by the nuclear factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization.

ABSTRACTMolecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 113 endocytosis-related genes. Among them transcription of the Endosomal Sorting Complex Required for Transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients showing also reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21-mediated inhibition of cell proliferation and sterile inflammatory response driven by the Nuclear Factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization, which might occur in CRC patients.SUMMARY STATEMENTEndosomal Sorting Complex Required for Transport (ESCRT)-I destabilization upon concurrent depletion of Vps37 proteins is linked to the activation of sterile inflammatory response and cell growth inhibition.