A short story of 3AB-OS cancer stem cells, a possible model for studying cancer stemness

Cancer Stem Cells (CSCs) are thought to be the cause of cancer initiation, growth and development. Thus, a challenge in cancer research is their identification and eradication. In our laboratory, by chemical treatment of the human osteosarcoma (OS) MG63 cell line, we have isolated and characterized 3AB-OS cells, a human OS CSC line. 3AB-OS cells transdifferentiate in vitro into cells of the three derivatives germ layers and, when xenografted in athymic mice they are highly tumorigenic and recapitulate in vivo crucial features of human OS. They even express a reprogrammed energy metabolism, with a dependence on glycolytic metabolism more strong than parental MG63 cells. 3AB-OS cells have chromosomes showing a great number of abnormalities which are very similar to abnormalities found in both pediatric and adult osteosarcomas. In comparison with parental MG63 cells (where TP53 gene is hypermethylated, rearranged and in single copy), 3AB-OS cells have TP53 gene unmethylated, rearranged and in multiple copies. Moreover, the mutp53 (p53-R248W/P72R) is post-translationally stabilized, has nuclear localization and a gain of function. A great number of results obtained in our laboratories suggested that p53 mutation could be the “driver mutation” at the origin of the transformation of MG63 cells into 3AB-OS CSCs

Inhibition of hydrogen sulfide biosynthesis sensitizes lung adenocarcinoma to chemotherapeutic drugs by inhibiting mitochondrial DNA repair and suppressing cellular bioenergetics

Therapeutic manipulation of the gasotransmitter hydrogen sulfide (H(2)S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H(2)S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H(2)S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H(2)S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H(2)S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance

<i>SNHG5</i> promotes colorectal cancer cell survival by counteracting STAU1-mediated mRNA destabilization

We currently have limited knowledge of the involvement of long non-coding RNAs (lncRNAs) in normal cellular processes and pathologies. Here, we identify and characterize SNHG5 as a stable cytoplasmic lncRNA with up-regulated expression in colorectal cancer. Depletion of SNHG5 induces cell cycle arrest and apoptosis in vitro and limits tumour outgrowth in vivo, whereas SNHG5 overexpression counteracts oxaliplatin-induced apoptosis. Using an unbiased approach, we identify 121 transcript sites interacting with SNHG5 in the cytoplasm. Importantly, knockdown of key SNHG5 target transcripts, including SPATS2, induces apoptosis and thus mimics the effect seen following SNHG5 depletion. Mechanistically, we suggest that SNHG5 stabilizes the target transcripts by blocking their degradation by STAU1. Accordingly, depletion of STAU1 rescues the apoptosis induced after SNHG5 knockdown. Hence, we characterize SNHG5 as a lncRNA promoting tumour cell survival in colorectal cancer and delineate a novel mechanism in which a cytoplasmic lncRNA functions through blocking the action of STAU1

Cellule staminali di osteosarcoma umano 3AB-OS: un modello per analizzare le proprietà oncogeniche di p53 mutata.

Alterazioni del gene TP53 si riscontrano in circa il 50% dei tumori umani (1). Negli ultimi anni è stato dimostrato che la proteina mutata p53 (mp53) acquisisce nuove proprietà oncogeniche, definite “gain of function” (GOF), che contribuiscono alla progressione tumorale (2). In questo studio è stato analizzato lo stato del gene TP53, della proteina da esso espressa e il loro ruolo nella promozione della proliferazione, invasività, resistenza all’apoptosi e staminalità delle cellule 3AB-OS, una linea tumorale staminale precedentemente isolata dalle cellule di osteosarcoma umano MG63 (3). Analisi comparative di RT-PCR, Methylation-Specific-PCR (MSP), Fluorescent-in situ-hybridization (FISH) e sequenziamento hanno evidenziato che, nelle cellule 3AB-OS, il gene TP53 è mutato, presenta un promotore non metilato, è espresso ad elevati livelli ed è presente in copie multiple che non sempre co-localizzano con il cromosoma 17 (dove normalmente è localizzato il gene TP53). Nelle cellule parentali MG63, invece, non è possibile apprezzare livelli misurabili di espressione del gene TP53, che è presente in singola copia, non co-localizza con il cromosoma 17 ed è caratterizzato da un promotore metilato. Inoltre, analisi di western-blot e immunofluorescenza per mp53, hanno dimostrato che nelle cellule 3AB-OS essa risulta espressa, stabilizzata da modifiche post-traduzionali (fosforilazione/acetilazione) e localizzata esclusivamente nel nucleo. Per valutare l’acquisizione di proprietà oncogeniche da parte di mp53, sono stati condotti studi di silenziamento genico transiente, mediante l’utilizzo di small-interfering-RNA. Il knockdown di mp53 induce un’evidente riduzione della proliferazione cellulare e dell’invasività assieme a un incremento della sensibilità all’induttore di morte TRAIL. Analisi di Real-Time-PCR e di Western-Blot condotte su geni e relative proteine implicati nella cancerogenesi e nella staminalità, mostrano risultati congrui con i dati precedenti. Complessivamente, questi studi dimostrano che nelle cellule 3AB-OS la proteina endogena mp53 svolge un ruolo oncogenico, suggerendo che tali cellule possano rappresentare un valido modello sperimentale per la comprensione sia dei meccanismi molecolari attraverso cui mp53 esplica le sue attività oncogeniche che del suo ruolo nell’iniziazione del cancro

Clivaggio e shuttling nucleo-citoplasmatico della proteina Sirt1, in cellule di carcinoma mammario MDA-MB231.

Sirt1 è una proteina nota per il suo ruolo di istone deacetilasi NAD+ dipendente che sembra essere coinvolta in una ampia gamma di processi cellulari, quali regolazione genica, controllo dello stato metabolico, meccanismi di sopravvivenza allo stress. Dalla letteratura emergono dati contrastanti concernenti la funzione di Sirt1 nei tumori, le vengono infatti attribuiti ruoli sia di oncogene che di soppressore tumorale, argomento fortemente dibattuto. A conferma di ciò, la localizzazione subcellulare e la funzione di Sirt1 variano nei differenti tipi cellulari (1). E’ anche noto che Sirt1 risulta frequentemente clivata in varie linee cellulari grazie ad attività proteolitiche nucleari (2). Questo studio ha lo scopo di identificare lo stato della proteina Sirt1 in cellule di carcinoma mammario MDA-MB231. I nostri risultati dimostrano che Sirt1 risulta espressa a bassi livelli nella forma “full lenght”, mentre si riscontrano alti livelli di sue forme clivate sia nucleari che citoplasmatiche. Sulla base di analisi bioinformatiche sui possibili siti di clivaggio di Sirt1 (Peptide Cutter Prediction-ExPASy), abbiamo ipotizzato che alcune delle forme di clivaggio da noi osservate, possano essere attribuite all’azione della caspasi-8. Abbiamo evidenziato all’interno del nucleo di cellule MDA-MB231, la forma procaspasica (55 kDa) accompagnata dalla presenza di frammenti attivi (48-18 kDa). Abbiamo, anche evidenziato una ulteriore forma della caspasi-8 a maggior peso molecolare (75 kDa) che, secondo dati bibliografici, potrebbe essere attribuita ad una forma di sumoilazione, da alcuni indicata come segnale di import nucleare (3). Abbiamo pertanto effettuato analisi su immunoprecipitati della caspasi-8 sviluppati contro anticorpo anti-SUMO1, dimostrando che nel nucleo di cellule MDA-MB231, la caspasi-8 è anche in forma sumoilata. Allo scopo di dimostrare che i frammenti di Sirt1 da noi osservati, sono effettivamente frutto dell’azione della caspasi-8, abbiamo trattato le cellule MDA-MB231 con un inibitore generico delle caspasi (Z-VAD-FMK). I risultati dimostrano il decremento nella frazione nucleare dei livelli di una delle forme di clivaggio di Sirt1, un frammento c-terminale di 65 kDa (p65-Sirt1). Dati preliminari sembrano indicare che il frammento p65-Sirt1 possa essere esportato nel citosol. In cellule MDA-MB231 trattate con Leptomicina B (inibitore dell’export nucleare), analisi di immunofluorescenza e analisi di western blotting effettuate su frazioni citosolico-nucleari, hanno evidenziato che il frammento p65-Sirt1 decrementa nella frazione citosolica e si accumula nel nucleo. E’ stato suggerito che frammenti della proteina Sirt1 possano sequestrare il citocromo c svolgendo così un ruolo anti-apoptotico (2). Nostri studi futuri avranno l’obiettivo di verificare tale aspetto

TREM2-induced activation of microglia contributes to synaptic integrity in cognitively intact aged individuals with Alzheimer's neuropathology

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Nondemented with AD neuropathology" (NDAN), suggests that some mechanisms are triggered to resist cognitive impairment. Exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible mediator of this process is TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, we performed a comparative study of human post-mortem frontal cortices of aged-matched, AD and NDAN individuals. We studied the distribution of activated microglia (IBA1, IBA1(+)/CD68(+) cells) and phagocytic microglia-related proteins (TREM2, DAP12), demonstrating higher microglial activation and TREM2 expression in NDAN versus AD. A study of the preservation of synapses around plaques, assessed using MAP2 and beta III tubulin as dendritic and axonal markers, respectively, and PSD95 as a postsynaptic marker, revealed preserved axonal/dendritic structure around plaques in NDAN versus AD. Moreover, high levels of PSD95 around NDAN plaques and the colocalization of PSD95 with CD68 indicated a prompt removal of damaged synapses by phagocytic microglia. Furthermore, Annexin V assay on aged-matched, AD and NDAN individuals synaptosomes revealed increased levels of ePS in NDAN, confirming damaged synapses engulfment. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals

Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

Abstract This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response in pulmonary epithelial cells. Extrusion of mtDNA into the bronchoalveolar lavage fluid occurs as an early event in mice subjected to cigarette smoke injury, concomitantly with the depletion of mtDNA in the lung tissue. In cultured lung epithelial cells, prolonged, low-level oxidative stress damages the mtDNA, without any detectable damage to the nuclear DNA. In turn, cellular depletion of the mtDNA occurs, together with a transient remodeling of cellular bioenergetics and morphology - all without any detectable impairment in overall cell viability. Damaged mtDNA first enters the cytoplasm, where it binds to Z-DNA binding protein 1 (ZBP1) and triggers inflammation via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Fragments of the mtDNA are subsequently released into the extracellular space via exosomes. MtDNA-containing exosomes are capable of inducing an inflammatory response in naïve (non-oxidatively stressed) epithelial cells. In vivo, administration of isolated mtDNA into the in lungs of naïve mice induces the production of pro-inflammatory mediators, without histopathologic evidence of tissue injury. We propose that mtDNA-specific damage, and subsequent activation of the ZBP1 pathway, is a mechanism that links prolonged, low-level oxidative stress to autocrine and paracrine inflammation during the early stages of inflammatory lung disease

Inhibition of Mitochondrial Bioenergetics by Esterase-Triggered COS/H₂S Donors

Hydrogen sulfide (H₂S) is an important biological mediator, and synthetic H₂S donating molecules provide an important class of investigative tools for H₂S research. Here, we report esterase-activated H₂S donors that function by first releasing carbonyl sulfide (COS), which is rapidly converted to H₂S by the ubiquitous enzyme carbonic anhydrase (CA). We report the synthesis, self-immolative decomposition, and H₂S release profiles of the developed scaffolds. In addition, the developed esterase-triggered COS/H₂S donors exhibit higher levels of cytotoxicity than equivalent levels of Na₂S or the common H₂S donors GYY4137 and AP39. Using cellular bioenergetics measurements, we establish that the developed donors reduce cellular respiration and ATP synthesis in BEAS 2B human lung epithelial cells, which is consistent with COS/H₂S inhibition of cytochrome c oxidase in the mitochondrial respiratory chain although not observed with common H₂S donors at the same concentrations. Taken together, these results may suggest that COS functions differently than H₂S in certain biological contexts or that the developed donors are more efficient at delivering H₂S than other common H₂S-releasing motifs