E2F1 transcription factor implication in melanoma

Le mélanome est le cancer cutané le plus meurtrier. Il est issu de la transformation maligne des mélanocytes et se dissémine rapidement dans l'organisme sous forme de métastases. A ce stade, ce cancer est réfractaire à pratiquement toutes les thérapies. Ainsi, l'identification de nouvelles cibles thérapeutiques est donc incontournable pour la mise en place de biothérapies spécifiques dans le mélanome. Dans ce contexte, nous nous sommes intéressés au facteur de transcription E2F1 qui joue un rôle prépondérant dans le cycle cellulaire. Plus récemment, il lui a été identifié divers rôles dans les fonctions cellulaires. Ainsi, nous avons cherché à caractériser son implication dans le mélanome. Nous avons observé que E2F1 est faiblement exprimé dans les cellules saines de la peau. En revanche, elle est fortement exprimée dans le mélanome, et est corrélée à un mauvais pronostic clinique. Ainsi, nous avons montré que son inhibition réduisait la viabilité de cellules de mélanomes in vitro et in vivo dû à un arrêt du cycle cellulaire de la sénescence et d'une apoptose. Ces processus semblent être dépendants de la voie p53. Ce travail a permis de caractériser E2F1 comme une potentielle cible thérapeutique dans le mélanome non muté p53. En parallèle, nous avons initié une collaboration avec le Dr Slama-Schwok dont l'étude portait sur un composé appelé NS1, un inhibiteur de la NO-Synthase. Ce composé présente une activité anti-mélanome in vitro. En effet, il induit un stress du réticulum endoplasmique lui même conduisant à une autophagie partielle et une mort des cellules par apoptose. Ce projet ouvre de nouvelles perspectives pour le traitement du mélanome métastatique.Melanoma is the most deadly form of skin cancer. It originates from malignant transformation of melanocytes and quickly disseminates as metastasis through the body. At this stage, this cancer is refractory to almost all therapies. Thus, new therapeutic target identification is needed for setting up specific biotherapies against melanoma. In this context, we focused on E2F1 transcription factor which plays a critical role in cell cycle. Recently, it was also implicated in several cell functions. So we aimed at characterizing its implication in melanoma. We observed that E2F1 is weakly expressed in normal skin cells. On the contrary, it is strongly expressed in melanoma and its expression correlates with a bad clinical prognosis. We also showed that E2F1 inhibition decreased melanoma cell viability in vitro and in vivo, as a result of cell cycle arrest, senescence and apoptosis. These processes seem to depend on p53 pathway. With this work we characterized E2F1 as a potential therapeutic target in non-mutated p53 melanoma. In parallel, we initiated a collaboration with Dr Slama-Schwok for studying NS1 compound, a NO-synthase inhibitor. This compound presents an in vitro anti-melanoma activity. Indeed, it induces endoplasmic reticulum stress, which leads to partial autophagy and cell death by apoptosis. This work opens new perspectives for metastatic melanoma treatment

The ACE2 Receptor for Coronavirus Entry Is Localized at Apical Cell—Cell Junctions of Epithelial Cells

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell—Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry

Cooperative binding of the tandem WW domains of PLEKHA7 to PDZD11 promotes conformation-dependent interaction with tetraspanin 33

Pleckstrin homology domain-containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain-containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin-mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a "hydrophobic hot spot" region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7-PDZD11 interaction modulate the ligand-binding properties of PLEKHA7

Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue

Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite the spectacular achievements of targeted therapies (BRAF inhibitors) or immuno-therapies (anti-CTLA4 or anti-PD1), most patients with melanoma will need additional treatments. Here we used a photoactive NADPH analogue called NS1 to induce cell death by inhibition of NADPH oxidases NOX in melanoma cells, including melanoma cells isolated from patients. In contrast, healthy melanocytes growth was unaffected by NS1 treatment. NS1 established an early Endoplasmic Reticulum stress by the early release of calcium mediated by (a) calcium-dependent redox-sensitive ion channel(s). These events initiated autophagy and apoptosis in all tested melanoma cells independently of their mutational status. The autophagy promoted by NS1 was incomplete. The autophagic flux was blocked at late stage events, consistent with the accumulation of p62, and a close localization of LC3 with NS1 associated with NS1 inhibition of NOX1 in autophagosomes. This hypothesis of a specific incomplete autophagy and apoptosis driven by NS1 was comforted by the use of siRNAs and pharmacological inhibitors blocking different processes. This study highlights the potential therapeutic interest of NS1 inducing cell death by triggering a selective ER stress and incomplete autophagy in melanoma cells harbouring wt and BRAF mutation

The PLEKHA7-PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The Plasma Membrane Calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function in calcium homeostasis and signaling, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is a junctional protein implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adaptor protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane-associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here we test this hypothesis using cultured cell model systems. We showed using immunofluorescence microscopy and a surface biotinylation assay show that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct (mCCD) epithelial cells resulted in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, co-expression of PDZD11 reduced membrane accumulation of overexpressed PMCAx/b and analysis of cytosolic calcium transients showed an effect of PDZD11 on the amplitude of the calcium peaks induced by the expression of PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mCCD) cells increased the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA

Inhibition of Melanogenesis by the Antidiabetic Metformin

International audienceSeveral reports have demonstrated the inhibitory effect of metformin, a widely used drug in the treatment of type 2 diabetes, on the proliferation of many cancers including melanoma. Recently, it has been shown that metformin is able to modulate the cAMP level in the liver. As cAMP has a crucial role in melanin synthesis and skin pigmentation, we investigated the effect of metformin on melanogenesis both in vitro and in vivo. We showed that metformin led to reduced melanin content in melanoma cells and in normal human melanocytes by decreasing cAMP accumulation and cAMP-responsive element-binding protein phosphorylation. This inhibitory effect is correlated with decreased expression of master genes of melanogenesis, microphthalmia-associated transcription factor, tyrosinase, dopachrome tautomerase, and tyrosinase-related protein 1. Furthermore, we demonstrated that the antimelanogenic effect of metformin is independent of the AMPK pathway. Interestingly, topical application of metformin induced tail whitening in mice. Finally, we confirmed the antimelanogenic effect of metformin on reconstituted human epidermis and on human skin biopsies. These data emphasize the depigmenting effect of metformin and suggest a clinical strategy for using metformin in the topical treatment of hyperpigmentation disorders

Increased CD271 expression by the NF-kB pathway promotes melanoma cell survival and drives acquired resistance to BRAF inhibitor vemurafenib

International audienceSpecific BRAFV600E inhibitors (BRAFi) are highly effective in the treatment of melanoma. However, acquired drug resistances invariably develop after the initial response. Therefore, the identification of new mechanisms of acquired resistance gives important clues towards the development of therapies that could elicit long lasting responses. Here we report that CD271 confers resistance to BRAFi in melanoma cells. The expression of CD271 is increased by BRAFi through a stimulation of tumor necrosis factor-alpha (TNFα) secretion that leads to NF-κB signaling pathway activation. CD271 is upregulated in a subset of BRAFi-resistant melanoma cells. The inhibition of TNFα/NF-κB pathway and CD271 silencing restore the BRAFi sensitivity of resistant melanoma cells. Finally, increase of CD271 expression is validated in BRAFi-resistant xenografts tumors and also in tumors from the patients who relapsed under BRAFi. In summary, these results reveal a novel TNFα/NF-κB/CD271 axis whose activation contributes to the acquisition of resistance to BRAFi and therefore may represent a novel therapeutic target to improve the efficacy of therapy in melanoma

A Dock-and-Lock Mechanism Clusters ADAM10 at Cell-Cell Junctions to Promote α-Toxin Cytotoxicity

Summary: We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering. : Shah et al. clarify the mechanism through which adherens junctions render cells more susceptible to death by S. aureus α-toxin. PLEKHA7 and associated proteins create an Achilles heel by clustering the toxin receptor ADAM10 and toxin pores at junctions. Unclustered toxin pores are unstable and endocytosed, promoting cell survival. Keywords: PLEKHA7, PDZD11, Tetraspanin, ADAM10, Afadin, Staphylococcus auereus α-toxin, cell junctions, macropinocytosis, epithelium, cell-deat