NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

Co-Expression of Podoplanin and CD44 in Proliferative Vitreoretinopathy Epiretinal Membranes.

Epiretinal membranes (ERMs) are sheets of tissue that pathologically develop in the vitreoretinal interface leading to progressive vision loss. They are formed by different cell types and by an exuberant deposition of extracellular matrix proteins. Recently, we reviewed ERMs' extracellular matrix components to better understand molecular dysfunctions that trigger and fuel the onset and development of this disease. The bioinformatics approach we applied delineated a comprehensive overview on this fibrocellular tissue and on critical proteins that could really impact ERM physiopathology. Our interactomic analysis proposed the hyaluronic-acid-receptor cluster of differentiation 44 (CD44) as a central regulator of ERM aberrant dynamics and progression. Interestingly, the interaction between CD44 and podoplanin (PDPN) was shown to promote directional migration in epithelial cells. PDPN is a glycoprotein overexpressed in various cancers and a growing body of evidence indicates its relevant function in several fibrotic and inflammatory pathologies. The binding of PDPN to partner proteins and/or its ligand results in the modulation of signaling pathways regulating proliferation, contractility, migration, epithelial-mesenchymal transition, and extracellular matrix remodeling, all processes that are vital in ERM formation. In this context, the understanding of the PDPN role can help to modulate signaling during fibrosis, hence opening a new line of therapy.

Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models.

Glioblastoma multiforme (GBM) is the most aggressive primary tumor of the central nervous system and the diagnosis is often dismal. GBM pharmacological treatment is strongly limited by its intracranial location beyond the blood-brain barrier (BBB). While Temozolomide (TMZ) exhibits the best clinical performance, still less than 20% crosses the BBB, therefore requiring administration of very high doses with resulting unnecessary systemic side effects. Here, we aimed at designing new negative temperature-responsive gel formulations able to locally release TMZ beyond the BBB. The biocompatibility of a chitosan-ß-glycerophosphate-based thermogel (THG)-containing mesoporous SiO2 nanoparticles (THG@SiO2) or polycaprolactone microparticles (THG@PCL) was ascertained in vitro and in vivo by cell counting and histological examination. Next, we loaded TMZ into such matrices (THG@SiO2-TMZ and THG@PCL-TMZ) and tested their therapeutic potential both in vitro and in vivo, in a glioblastoma resection and recurrence mouse model based on orthotopic growth of human cancer cells. The two newly designed anticancer formulations, consisting in TMZ-silica (SiO2@TMZ) dispersed in the thermogel matrix (THG@SiO2-TMZ) and TMZ, spray-dried on PLC and incorporated into the thermogel (THG@PCL-TMZ), induced cell death in vitro. When applied intracranially to a resected U87-MG-Red-FLuc human GBM model, THG@SiO2-TMZ and THG@PCL-TMZ caused a significant reduction in the growth of tumor recurrences, when compared to untreated controls. THG@SiO2-TMZ and THG@PCL-TMZ are therefore new promising gel-based local therapy candidates for the treatment of GBM.

MAPK15 protects from oxidative stress-dependent cellular senescence by inducing the mitophagic process.

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1-dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15-dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age-associated human diseases.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models.

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis.

In medulloblastomas, genetic alterations resulting in over-activation and/or deregulation of proteins involved in Hedgehog (HH) signaling lead to cellular transformation, which can be prevented by inhibition of primary ciliogenesis. Here, we investigated the role of MAPK15 in HH signaling and, in turn, in HH-mediated cellular transformation. We first demonstrated, in NIH3T3 mouse fibroblasts, the ability of this kinase of controlling primary ciliogenesis and canonical HH signaling. Next, we took advantage of transformed human medulloblastoma cells belonging to the SHH-driven subtype, i.e., DAOY and ONS-76 cells, to ascertain the role for MAPK15 in HH-mediated cellular transformation. Specifically, medullo-spheres derived from these cells, an established in vitro model for evaluating progression and malignancy of putative tumor-initiating medulloblastoma cells, were used to demonstrate that MAPK15 regulates self-renewal of these cancer stem cell-like cells. Interestingly, by using the HH-related oncogenes SMO-M2 and GLI2-DN, we provided evidences that disruption of MAPK15 signaling inhibits oncogenic HH overactivation in a specific cilia-dependent fashion. Ultimately, we show that pharmacological inhibition of MAPK15 prevents cell proliferation of SHH-driven medulloblastoma cells, overall suggesting that oncogenic HH signaling can be counteracted by targeting the ciliary gene MAPK15, which could therefore be considered a promising target for innovative "smart" therapies in medulloblastomas.

RAB7A Regulates Vimentin Phosphorylation through AKT and PAK.

RAB7A is a small GTPase that controls the late endocytic pathway but also cell migration through RAC1 (Ras-related C3 botulinum toxin substrate 1) and vimentin. In fact, RAB7A regulates vimentin phosphorylation at different sites and vimentin assembly, and, in this study, we identified vimentin domains interacting with RAB7A. As several kinases could be responsible for vimentin phosphorylation, we investigated whether modulation of RAB7A expression affects the activity of these kinases. We discovered that RAB7A regulates AKT and PAK1, and we demonstrated that increased vimentin phosphorylation at Ser38 (Serine 38), observed upon RAB7A overexpression, is due to AKT activity. As AKT and PAK1 are key regulators of several cellular events, we investigated if RAB7A could have a role in these processes by modulating AKT and PAK1 activity. We found that RAB7A protein levels affected beta-catenin and caspase 9 expression. We also observed the downregulation of cofilin-1 and decreased matrix metalloproteinase 2 (MMP2) activity upon RAB7A silencing. Altogether these results demonstrate that RAB7A regulates AKT and PAK1 kinases, affecting their downstream effectors and the processes they regulate, suggesting that RAB7A could have a role in a number of cancer hallmarks.

Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats' eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

Chemically stable inhibitors of 14-3-3 protein-protein interactions derived from BV02.

14-3-3 are regulatory proteins that through protein-protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound 1 was able to promote the nuclear translocation of c-Abl at low micromolar concentrations. Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.