Epithelial Cells of Deep Infiltrating Endometriosis Harbor Mutations in Cancer Driver Genes.

Endometriosis is an inflammatory condition manifested by the presence of endometrial-like tissue outside of the uterine cavity. The most common clinical presentations of endometriosis are dysmenorrhea, infertility, and severe pelvic pain. Few hypotheses attempt to explain the pathogenesis of endometriosis; however, none of the theories have been fully confirmed or considered universal. We examined somatic mutations in eutopic endometrium samples, deep endometriotic nodules and peripheral blood from 13 women with deep endometriosis of the rectovaginal space. Somatic variants were identified in laser microdissected samples using next-generation sequencing. A custom panel of 1296 cancer-related genes was employed, and selected genes representing cancer drivers and non-drivers for endometrial and ovarian cancer were thoroughly investigated. All 59 detected somatic variants were of low mutated allele frequency (<10%). In deep ectopic lesions, detected variants were significantly more often located in cancer driver genes, whereas in eutopic endometrium, there was no such distribution. Our results converge with other reports, where cancer-related mutations were found in endometriosis without cancer, particularly recurrent KRAS mutations. Genetic alterations located in ectopic endometriotic nodules could contribute to their formation; nevertheless, to better understand the pathogenesis of this disease, more research in this area must be performed.

Clathrin- and dynamin-dependent endocytosis limits canonical NF-κB signaling triggered by lymphotoxin ß receptor.

BACKGROUND: Lymphotoxin ß receptor (LTßR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTßR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTßR internalization to its signaling potential. METHODS: Intracellular localization of LTßR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTßR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTßR stimulation was assessed by qRT-PCR analysis. RESULTS: We demonstrated that LTßR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTßR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTßR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTßR-triggered activation of the non-canonical branch of the NF-κB pathway. CONCLUSIONS: Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTßR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

Cholesterol restricts lymphotoxin ß receptor-triggered NF-κB signaling.

BACKGROUND: Lymphotoxin ß receptor (LTßR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTßR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTßR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTßR-induced NF-κB signaling. METHODS: To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-ß-cyclodextrin and simvastatin were applied. LTßR localization was studied by confocal microscopy. The activity of LTßR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTßR with its protein partners were investigated by immunoprecipitation. RESULTS: We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-ß-cyclodextrin impaired LTßR internalization and potentiated LTßR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTßR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS: Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTßR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTßR-based therapies. Video abstract.

The topology of the lymphotoxin ß receptor that accumulates upon endolysosomal dysfunction dictates the NF-κB signaling outcome.

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

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 ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR 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.

APPL1 endocytic adaptor as a fine tuner of Dvl2-induced transcription.

APPL1 is a multifunctional endocytic adaptor which acts at different steps of various signaling pathways. Here we report that APPL1 interacts with Dvl2, a protein known to activate the canonical and non-canonical Wnt pathways. APPL1 synergizes with Dvl2 and potentiates transcription driven by AP-1 transcription factors, specifically by c-Jun, in non-canonical Wnt signaling. This function of APPL1 requires its endosomal recruitment. Overproduction of APPL1 increases Dvl2-mediated expression of AP-1 target gene encoding metalloproteinase 1 (MMP1) in a JNK-dependent manner. Collectively, we propose a novel role of APPL1 as a positive regulator of Dvl2-dependent transcriptional activity of AP-1.

Multifunctional protein APPL2 contributes to survival of human glioma cells.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long-term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3ß activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis-related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti-apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin-1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction.

Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity.

We have investigated the possible role of Escherichia coli DNA polymerase (Pol) I in chromosomal replication fidelity. This was done by substituting the chromosomal polA gene by the polAexo variant containing an inactivated 3'-->5' exonuclease, which serves as a proofreader for this enzyme's misinsertion errors. Using this strain, activities of Pol I during DNA replication might be detectable as increases in the bacterial mutation rate. Using a series of defined lacZ reversion alleles in two orientations on the chromosome as markers for mutagenesis, 1.5- to 4-fold increases in mutant frequencies were observed. In general, these increases were largest for lac orientations favouring events during lagging strand DNA replication. Further analysis of these effects in strains affected in other E. coli DNA replication functions indicated that this polAexo mutator effect is best explained by an effect that is additive compared with other error-producing events at the replication fork. No evidence was found that Pol I participates in the polymerase switching between Pol II, III and IV at the fork. Instead, our data suggest that the additional errors produced by polAexo are created during the maturation of Okazaki fragments in the lagging strand.

Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex.

Multifunctional adaptor protein APPL1 [adaptor protein containing PH (pleckstrin homology) domain, PTB (phosphotyrosine binding) domain and leucine zipper motif] belongs to a growing group of endocytic proteins which actively participate in various stages of signalling pathways. Owing to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD (nucleosome remodelling and deacetylase), containing both nucleosome remodelling and HDAC (histone deacetylase) activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying HDAC2 as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex containing enzymatically active HDAC2 but not HDAC1 as the only deacetylase. However, the cellular levels of HDAC1 can regulate the extent of APPL1-NuRD interactions, which in turn modulates the nucleocytoplasmic distribution of APPL1. Increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, whereas HDAC1 overexpression exerts an opposite effect. Moreover, we also uncovered a NuRD-independent interaction of APPL1 with HDAC1. APPL1 overexpression affects the composition of the HDAC1-containing NuRD complex and the expression of HDAC1 target p21WAF1/CIP1. Cumulatively, these data reveal a surprising complexity of APPL1 interactions with HDACs, with functional consequences for the modulation of gene expression. In a broader sense, these results contribute to an emerging theme of endocytic proteins playing alternative roles in the cell nucleus.

Nuclear functions of endocytic proteins.

An increasing number of proteins appear to perform multiple, sometimes unrelated functions in the cell. Such moonlighting properties have been recently demonstrated for proteins involved in clathrin-mediated endocytosis. Some clathrin adaptors and endosomal proteins can undergo nucleocytoplasmic shuttling, which is often based on intrinsic sequence motifs and requires active transport mechanisms. Endocytic proteins can associate with nuclear molecules, changing their localization and/or activity and may modulate the levels and specificity of gene transcription. It is not clear how the nuclear and cytoplasmic pools of endocytic proteins are interconnected, or whether these molecules act as nuclear second messengers upon extracellular stimuli, but alike in endocytosis, they seem to form multi-component scaffolding platforms in the nucleus. Added to their endocytic functions, the nuclear roles of Eps15, Epsin1, CALM, HIP1, Dab1/2, beta-arrestins, APPL1/2 and the components of ESCRTs clearly increase the complexity of signaling networks affecting cellular growth, proliferation and homeostasis.

Role of DNA polymerase IV in Escherichia coli SOS mutator activity.

Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli.

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

Mutator phenotype resulting from DNA polymerase IV overproduction in Escherichia coli: preferential mutagenesis on the lagging strand.

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.