Glioblastoma (GB) is a highly malignant primary brain tumor with limited treatment options and poor prognosis. Despite current treatment approaches, including surgical resection, radiation therapy, ...and chemotherapy with temozolomide (TMZ), GB remains mostly incurable due to its invasive growth pattern, limited drug penetration beyond the blood-brain barrier (BBB), and resistance to conventional therapies. One of the main challenges in GB treatment is effectively eliminating infiltrating cancer cells that remain in the brain parenchyma after primary tumor resection. We've reviewed the most recent challenges and surveyed the potential strategies aimed at enhancing local treatment outcomes.
Macroautophagy/autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. ...Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to 'autophagy addiction'. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors.
Abbreviations: AAK1: AP2 associated kinase 1; AMPK: 5ʹ-prime-AMP-activated protein kinase; AKT1: AKT serine/threonine kinase 1; BAF: bafilomycin A
1
; CA: constitutively activated; CDK17: cyclin dependent kinase 17; CDK18: cyclin dependent kinase 18; CHUK: conserved helix-loop-helix ubiquitous kinase; EGF: epidermal growth factor; ERBB2: erb-b2 receptor tyrosine kinase 2; FGF: fibroblast growth factor; FM: full medium; GALK2: galactokinase 2; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKE: inhibitor of nuclear factor kappa B kinase subunit epsilon; IKK: IκB kinase complex; KD: kinase dead; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1: mitogen-activated protein kinase 1; MAPK15: mitogen-activated protein kinase 15; MTORC1: mammalian target of rapamycin kinase complex 1; myr: myristoylation/myristoylated; NFKBIA: NFKB inhibitor alpha; PDGF: platelet derived growth factor; PFKL: phosphofructokinase, liver type; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKCD: protein kinase C delta; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; WB: western blot; WT: wild-type.
The cargo receptor NCOA4 mediates autophagic ferritin degradation. Here we show that NCOA4 deficiency in a knockout mouse model causes iron accumulation in the liver and spleen, increased levels of ...transferrin saturation, serum ferritin, and liver hepcidin, and decreased levels of duodenal ferroportin. Despite signs of iron overload, NCOA4-null mice had mild microcytic hypochromic anemia. Under an iron-deprived diet (2–3 mg/kg), mice failed to release iron from ferritin storage and developed severe microcytic hypochromic anemia and ineffective erythropoiesis associated with increased erythropoietin levels. When fed an iron-enriched diet (2 g/kg), mice died prematurely and showed signs of liver damage. Ferritin accumulated in primary embryonic fibroblasts from NCOA4-null mice consequent to impaired autophagic targeting. Adoptive expression of the NCOA4 COOH terminus (aa 239–614) restored this function. In conclusion, NCOA4 prevents iron accumulation and ensures efficient erythropoiesis, playing a central role in balancing iron levels in vivo.
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•Genetic disruption of NCOA4 causes ferritin accumulation in tissues•NCOA4 deficiency blocks iron mobilization from ferritin storage and induces anemia•NCOA4-null mice are hypersensitive to iron overload
NCOA4 is crucial for autophagic ferritin degradation. Bellelli et al. find that genetic disruption of NCOA4 causes ferritin accumulation in tissues and defective iron mobilization from storage. NCOA4-null mice display impaired erythropoiesis associated with microcytic and hypochromic anemia, which is more pronounced in mice fed an iron-depleted diet.
Autophagy, a pathway for bulk protein degradation and removal of damaged organelles, represents one of the major responses of cells to stress, thereby exerting a strict control on their correct ...functioning. Consequently, this process has been involved in the pathogenesis and therapeutic responses of several human diseases. Mitogen-activated protein (MAP) kinase 15 (MAPK15) is an atypical member of the MAP kinase family that recently emerged as a key modulator of autophagy and, through this, of cell transformation. Still, no information is available about signaling pathways mediating the effect of MAPK15 on this process, nor is it known which phase of autophagosome biogenesis is affected by this MAP kinase. Here, we demonstrate that MAPK15 stimulated 5′-AMP–activated protein kinase–dependent activity of UNC-51-like kinase 1 (ULK1), the only protein kinase among the ATG-related proteins, toward downstream substrates and signaling intermediates. Importantly, MAPK15 directly interacted with the ULK1 complex and mediated ULK1 activation induced by starvation, a classical stimulus for the autophagic process. In turn, ULK1 and its highly homologous protein ULK2 are able to transduce MAPK15 signals stimulating early phases of autophagosomal biogenesis in a multikinase cascade that offers numerous potential targets for future therapeutic intervention in cancer and other autophagy-related human diseases.
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.
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-β-glycerophosphate-based thermogel (THG)-containing mesoporous SiO
nanoparticles (THG@SiO
) 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@SiO
-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 (SiO
@TMZ) dispersed in the thermogel matrix (THG@SiO
-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@SiO
-TMZ and THG@PCL-TMZ caused a significant reduction in the growth of tumor recurrences, when compared to untreated controls. THG@SiO
-TMZ and THG@PCL-TMZ are therefore new promising gel-based local therapy candidates for the treatment of GBM.
A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a ...myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel "druggable" link between BCR-ABL1 and human CML.
Aberrant production of reactive oxygen species (ROS) from dysfunctional mitochondria leads to oxidative stress and DNA damage, which induces the cellular senescence stress response pathway. This, ...while exerting strong beneficial suppressive effects on the development of cancer, also contributes to aging and different age-related disorders. Mitophagy is a key mechanism to constantly eliminate old and damaged mitochondria, strongly contributing to keep low levels of intracellular ROS. Here, we discuss our recent findings showing the involvement of the atypical MAP kinase family member MAPK15 in controlling the mitophagic process, thereby preventing ROS accumulation, extensive DNA damage and activation of the cellular senescence phenotype.
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.
Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. ...In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7A
WT
, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7A
T22N
mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7A
V162M
mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.
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