Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies.

Extracellular vesicles (EVs) have emerged as potential vehicles for targeted drug delivery and diagnostic applications. However, achieving consistent and reliable functionalization of EV membranes remains a challenge. Copper-catalyzed click chemistry, commonly used for EV surface modification, poses limitations due to cytotoxicity and interference with biological systems. To overcome these limitations, we developed a standardized method for functionalizing an EV membrane via copper-free click chemistry. EVs derived from plasma hold immense potential as diagnostic and therapeutic agents. However, the isolation and functionalization of EVs from such a complex biofluid represent considerable challenges. We compared three different EV isolation methods to obtain an EV suspension with an optimal purity/yield ratio, and we identified sucrose cushion ultracentrifugation (sUC) as the ideal protocol. We then optimized the reaction conditions to successfully functionalize the plasma-EV surface through a copper-free click chemistry strategy with a fluorescently labeled azide, used as a proof-of-principle molecule. Click-EVs maintained their identity, size, and, more importantly, capacity to be efficiently taken up by responder tumor cells. Moreover, once internalized, click EVs partially followed the endosomal recycling route. The optimized reaction conditions and characterization techniques presented in this study offer a foundation for future investigations and applications of functionalized EVs in drug delivery, diagnostics, and therapeutics.

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells.

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature.

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-ß-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-ß structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-ß structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

A quantitative biology approach correlates neuronal toxicity with the largest inclusions of TDP-43.

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Ultrastructural imaging reveals vascular remodeling in migraine patients.

Migraine is a neurological disorder and one of the most common pain conditions worldwide. Despite its prevalence, the basic biology and underlying mechanisms contributing to the development of migraine are still poorly understood. It is still unclear, for instance, whether the vasculature, both extra and intracranial, plays a significant role in the generation of migraine pain. Neuroimaging data, indeed, have reported conflicting results on blood vessels abnormalities like vasodilation, while functional studies suggest that vessels dysfunction may extend beyond vasodilation. Here we combined light and electron microscopy imaging to investigate the fine structure of superficial temporal (STA) and occipital arteries (OA) from patients that underwent minimally invasive surgery for migraine. Using optical microscopy, we observed that both STA and OA vessels showed marked endothelial thickening and internal elastic lamina fragmentation. In the muscular layer, we found profound shape changes of vascular smooth muscle cells (VSMCs), abundant extracellular matrix, and the presence of clear extracellular vacuoles. The electron microscopy analysis confirmed putative VSMCs infiltrated within the intima layer and revealed a consistent shifting of VSMCs from contractile to a synthetically active phenotype. We also report the presence of (i) abundant extracellular vacuoles filled with fine granular material and membranes, (ii) multilamellar structures, (iii) endosome-like organelles, and (iv) bona fide extracellular vesicles in the matrix space surrounding synthetically active cells. As both the endothelial layer and VSMCs coordinate a variety of vascular functions, these results suggest that a significant vascular remodeling is occurring in STA and OA of migraine patients. Thus, this phenomenon may represent an important target for future investigation designed toward the development of new therapeutic approaches.

Role of extracellular vesicles from adipose tissue- and bone marrow-mesenchymal stromal cells in endothelial proliferation and chondrogenesis.

The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle- and small-sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC-EVs overexpressed pro-angiogenic factors in comparison to the BMSC-counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications.

Simulated Microgravity Effects on Human Adenocarcinoma Alveolar Epithelial Cells: Characterization of Morphological, Functional, and Epigenetic Parameters.

BACKGROUND: In space, the reduction or loss of the gravity vector greatly affects the interaction between cells. Since the beginning of the space age, microgravity has been identified as an informative tool in biomedicine, including cancer research. The A549 cell line is a hypotriploid human alveolar basal epithelial cell line widely used as a model for lung adenocarcinoma. Microgravity has been reported to interfere with mitochondrial activity, energy metabolism, cell vitality and proliferation, chemosensitivity, invasion and morphology of cells and organelles in various biological systems. Concerning lung cancer, several studies have reported the ability of microgravity to modulate the carcinogenic and metastatic process. To investigate these processes, A549 cells were exposed to simulated microgravity (µG) for different time points. METHODS: We performed cell cycle and proliferation assays, ultrastructural analysis of mitochondria architecture, as well as a global analysis of miRNA modulated under µG conditions. RESULTS: The exposure of A549 cells to microgravity is accompanied by the generation of polynucleated cells, cell cycle imbalance, growth inhibition, and gross morphological abnormalities, the most evident are highly damaged mitochondria. Global miRNA analysis defined a pool of miRNAs associated with µG solicitation mainly involved in cell cycle regulation, apoptosis, and stress response. To our knowledge, this is the first global miRNA analysis of A549 exposed to microgravity reported. Despite these results, it is not possible to draw any conclusion concerning the ability of µG to interfere with the cancerogenic or the metastatic processes in A549 cells. CONCLUSIONS: Our results provide evidence that mitochondria are strongly sensitive to µG. We suggest that mitochondria damage might in turn trigger miRNA modulation related to cell cycle imbalance.

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells.

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration-approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30-100 nm) ERBB2- EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs.

Trastuzumab Modulates the Protein Cargo of Extracellular Vesicles Released by ERBB2+ Breast Cancer Cells.

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (Tz) induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. Because these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

Liposomes Loaded with the Proteasome Inhibitor Z-Leucinyl-Leucinyl-Norleucinal are Effective in Inducing Apoptosis in Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is one of the main causes of cancer-related death in developed countries. Targeted therapies and conventional chemotherapeutics have been developed to help treat this type of aggressive cancer. Among these, the monoclonal antibodies cetuximab (Cxm) and panitumumab specifically target and inactivate the signaling of ERBB1 (EGF receptor), a key player in the development and progression of this cancer. Unfortunately, these antibodies are effective only on a small fraction of patients due to primary or secondary/acquired resistance. However, as ERBB1 cell surface expression is often maintained in resistant tumors, ERBB1 can be exploited as a target to deliver other drugs. Liposomes and immunoliposomes are under intensive investigation as pharmaceutical nanocarriers and can be functionalized with specific antibodies. In this study, we first investigated the anti-cancer activity of a cell permeable tripeptide, leucine-leucin-norleucinal (LLNle), an inhibitor of gamma-secretase and proteasome, in three different CRC cell lines that express ERBB1. We formulated LLNle-liposomes and Cxm-conjugated LLNle-loaded liposomes (LLNle-immunoliposomes) and evaluated their efficacy in inhibiting cell survival. Despite similar pro-apoptotic effects of free LLNle and LLNle-liposomes, immunoliposomes-LLNle were significantly less effective than their unconjugated counterparts. Indeed, immunoliposomes-LLNle were readily internalized and trafficked to lysosomes, where LLNle was likely trapped and/or inactivated. In conclusion, we demonstrated that LLNle was readily delivered to CRC cell lines by liposomes, but immunoliposomes-LLNle failed to show significant anti-cancer activity.

The chromodomain helicase CHD4 regulates ERBB2 signaling pathway and autophagy in ERBB2+ breast cancer cells.

The chromodomain helicase DNA-binding 4 (CHD4), a member of the nucleosome remodeling and deacetylases (NuRD) complex, has been identified as an oncogene that modulates proliferation and migration of breast cancers (BC). ERBB2 is an oncogenic driver in 20-30% of BC in which its overexpression leads to increased chemoresistance. Here we investigated whether CHD4 depletion affects the ERBB2 cascade and autophagy, which represents a mechanism of resistance against Trastuzumab (Tz), a therapeutic anti-ERBB2 antibody. We show that CHD4 depletion in two ERBB2+ BC cell lines strongly inhibits cell proliferation, induces p27KIP1 upregulation, Tyr1248 ERBB2 phosphorylation, ERK1/2 and AKT dephosphorylation, and downregulation of both ERBB2 and PI3K levels. Moreover, CHD4 silencing impairs late stages of autophagy, resulting in increased levels of LC3 II and SQSTM1/p62, lysosomal enlargement and accumulation of autolysosomes (ALs). Importantly, we show that CHD4 depletion and concomitant treatment with Tz prevent cell proliferation in vitro Our results suggest that CHD4 plays a critical role in modulating cell proliferation, ERBB2 signaling cascade and autophagy and provide new insights on CHD4 as a potential target for the treatment of ERBB2+ BC.

Pharmacological activation of autophagy favors the clearing of intracellular aggregates of misfolded prion protein peptide to prevent neuronal death.

According to the "gain-of-toxicity mechanism", neuronal loss during cerebral proteinopathies is caused by accumulation of aggregation-prone conformers of misfolded cellular proteins, although it is still debated which aggregation state actually corresponds to the neurotoxic entity. Autophagy, originally described as a variant of programmed cell death, is now emerging as a crucial mechanism for cell survival in response to a variety of cell stressors, including nutrient deprivation, damage of cytoplasmic organelles, or accumulation of misfolded proteins. Impairment of autophagic flux in neurons often associates with neurodegeneration during cerebral amyloidosis, suggesting a role in clearing neurons from aggregation-prone misfolded proteins. Thus, autophagy may represent a target for innovative therapies. In this work, we show that alterations of autophagy progression occur in neurons following in vitro exposure to the amyloidogenic and neurotoxic prion protein-derived peptide PrP90-231. We report that the increase of autophagic flux represents a strategy adopted by neurons to survive the intracellular accumulation of misfolded PrP90-231. In particular, PrP90-231 internalization in A1 murine mesencephalic neurons occurs in acidic structures, showing electron microscopy hallmarks of autophagosomes and autophagolysosomes. However, these structures do not undergo resolution and accumulate in cytosol, suggesting that, in the presence of PrP90-231, autophagy is activated but its progression is impaired; the inability to clear PrP90-231 via autophagy induces cytotoxicity, causing impairment of lysosomal integrity and cytosolic diffusion of hydrolytic enzymes. Conversely, the induction of autophagy by pharmacological  blockade of mTOR kinase or trophic factor deprivation restored autophagy resolution, reducing intracellular PrP90-231 accumulation and neuronal death. Taken together, these data indicate that PrP90-231 internalization induces an autophagic defensive response in A1 neurons, although incomplete and insufficient to grant survival; the pharmacological enhancement of this process exerts neuroprotection favoring the clearing of the internalized peptide and could represents a promising neuroprotective tool for neurodegenerative proteinopathies.

Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells.

BACKGROUND: ERBB2 is overexpressed in up to 20-30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2+) cells and whether CA might restore Tz sensitivity in Tz-resistant cells. METHODS: We have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy. RESULTS: We report that, in ERBB2+ BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21WAF1 and CDKN1B/p27KIP1. Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line. CONCLUSIONS: Our data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2+ BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2+ cancers.

Identification of an HSP90 modulated multi-step process for ERBB2 degradation in breast cancer cells.

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes.

Neuroblastoma-targeted nanocarriers improve drug delivery and penetration, delay tumor growth and abrogate metastatic diffusion.

Selective tumor targeting is expected to enhance drug delivery and to decrease toxicity, resulting in an improved therapeutic index. We have recently identified the HSYWLRS peptide sequence as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current therapies. Here we validated the specific binding of HSYWLRS to neuroblastoma cell suspensions obtained either from cell lines, animal models, or Schwannian-stroma poor, stage IV neuroblastoma patients. Binding of the biotinylated peptide and of HSYWLRS-functionalized fluorescent quantum dots or liposomal nanoparticles was dose-dependent and inhibited by an excess of free peptide. In animal models obtained by the orthotopic implant of either MYCN-amplified or MYCN single copy human neuroblastoma cell lines, treatment with HSYWLRS-targeted, doxorubicin-loaded Stealth Liposomes increased tumor vascular permeability and perfusion, enhancing tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy, as evaluated by bioluminescence imaging and micro-PET, leading to (i) delay of tumor growth paralleled by decreased tumor glucose consumption, and (ii) abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span. Our findings are functional to the design of targeted nanocarriers with potentiated therapeutic efficacy towards the clinical translation.

Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH) in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein alpha(s)-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments.

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793-804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C-gamma, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.

A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans.

Redundant gene function frequently hampers investigations of the physiological roles of mammalian proteins. This is the case for Eps8, a receptor tyrosine kinase (RTK) substrate that participates in the activation of the Rac-specific guanine nucleotide-exchange function of Sos1 (refs 2-5), thereby regulating actin remodelling by RTKs. EPS8-knockout mice, however, exhibit no evident phenotype, owing to the redundant function of three other EPS8-related genes. Here we show that in the nematode Caenorhabditis elegans, only one orthologue of the EPS8 gene exists, which gives rise to two alternatively spliced isoforms, EPS-8A and EPS-8B, differing at their carboxyl termini. In the nematode, eps-8 is essential for embryonic development. Furthermore, EPS-8A, but not EPS-8B, is specifically required for proper apical morphogenesis in the intestinal cells. This latter phenotype could be precisely correlated with a previously unknown actin barbed-end-capping activity, which is present in the C terminus of the EPS-8A isoform. Therefore, nematode genetics allowed not only the unmasking of distinct EPS-8-linked phenotypes, but also the definition of a novel function for this molecule in actin dynamics.