Hyperglycemia triggers HIPK2 protein degradation

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies

Reduced chemotherapeutic sensitivity in high glucose condition: implication of antioxidant response

Resistance to chemotherapy represents a major obstacle to successful treatment. The generation of reactive oxygen species (ROS) has been directly linked to the cytotoxic effects of several antitumor agents, including Adriamycin (ADR), and modulation of the oxidative balance has been implicated in the development and/or regulation of resistance to chemotherapeutic drugs. We recently showed that high glucose (HG) markedly diminished the cancer cell death induced by anticancer agents such as ADR. In the present study we attempted to evaluate the mechanism that impaired the cytotoxic effect of ADR in HG. We found that, in colon cancer cells, HG attenuated ADR-induced ROS production that consequently diminished ADR-induced H2AX phosphorylation and micronuclei (MN) formation. Mechanistically, HG attenuation of ADR-induced ROS production correlated with increased antioxidant response promoted by NRF2 activity. Thus, pharmacologic inhibition of NRF2 pathway by brusatol re-established the ADR cytotoxic effect impaired by HG. Together, the data provide new insights into chemotherapeutic-resistance mechanisms in HG condition dictated by increased NRF2-induced antioxidant response and how they may be overcome in order to restore chemosensitivity and ADR-induced cell death

Divergent Modulation of Neuronal Differentiation by Caspase-2 and -9

Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells

p53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions

BACKGROUND: As an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA. METHODS AND RESULTS: Here we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression. CONCLUSIONS: Altogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo

Innovative Therapeutic Strategies for Cystic Fibrosis: Moving Forward to CRISPR Technique

One of the most revolutionary technologies in recent years in the field of molecular biology is CRISPR-Cas9. CRISPR technology is a promising tool for gene editing that provides researchers the opportunity to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases. Cystic fibrosis (CF) is one of the most common lethal genetic diseases caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Although CF is an old acquaintance, there is still no effective/resolutive cure. Life expectancy has improved thanks to the combination of various treatments, but it is generally below average. Recently, a significant number of additional key medications have become licensed in Europe for the CF treatment including CFTR modulators. But innovative genomically-guided therapies have begun for CF and it is predictable that this will lead to rapid improvements in CF clinical disease and survival in the next decades. In this way, CRISPR-Cas9 approach may represent a valid tool to repair the CFTR mutation and hopeful results were obtained in tissue and animal models of CF disease

TERAPIE AVANZATE: LE SPERANZE DEL DOMANI

La mappatura del genoma umano e lo sviluppo di tecnologie per il sequenziamento dei geni hanno portato rapidamente ad una maggiore comprensione delle basi genetiche di molte malattie. I notevoli progressi tecnologici nel campo delle manipolazioni in vitro di cellule (isolamento, coltivazione, espansione, diffe-renziamento) e del trasferimento di materiale genetico da una cellula ad un’altra hanno consentito di sviluppare tecniche di ingegneria cellulare tramite le quali manipolare sia cellule staminali che cellule ad uno stadio di differenziamento terminale. Queste metodiche, applicate a cellule umane, lasciano presagire nuove opzioni terapeutiche per un’ampia gamma di patologie, trasformando e talora mettendo in discussione la nozione convenzionale di “medicina”. Il termine “terapie avanzate” fa riferimento a nuovi prodotti medicinali (Advanced Therapeutic Medicinal Products/ATMPs) che utilizzano geni (terapia genica), cellule (terapia cellulare) e tessuti (ingegneria tissutale) quali preparazioni farmaceutiche a scopo terapeutico o preventivo (ad esempio, se un difetto genetico evidenziato dall’analisi molecolare viene corretto prima che la patologia si manifesti in modo conclamato). Le distinzioni fra le categorie sono sottili ed occasionalmente alcuni prodotti potrebbero rientrare in più di una categoria. La produzione e sperimentazione di questi prodotti è strettamente regolata a livello internazionale. Tale regolamentazione impone criteri specifici atti a garantire in primo luogo la sicurezza del prodotto finale, poiché destinato alla somministrazione a pazienti, ma non secondariamente anche l’efficacia, poiché non è ritenuto etico sottoporre dei pazienti ad un trattamento che non abbia dato sufficiente prova di efficacia terapeutica

Compliance o aderenza: uno strumento essenziale per l’efficacia terapeutica

Drugs don’t work in patients who don’t take them. — C. Everett Koop L’efficacia e la tollerabilità di un trattamento farmacologico vengono valutate attraverso studi sperimentali (trial clinici), disegnati per analizzare la relazione tra la somministrazione di un farmaco e gli esiti clinici associati. Questi studi sono anche organizzati in modo che siano attuate le condizioni di trattamento più adeguate perché si possa mettere in evidenza l’effetto terapeutico desiderato. Nel trattamento dell'ipertensione arteriosa, ad esempio, queste condizioni sono rappresentate dal dosaggio, dalla durata e dalla continuità del trattamento considerato. Stimare il grado di osservanza al trattamento da parte del paziente non è stato sempre considerato di primaria importanza al momento dell’impostazione di un trial clinico. L’accertamento della compliance terapeutica, intesa come aderenza del paziente alla terapia nella durata e nel dosaggio prescritto dal medico curante, rappresenta oggi una condizione necessaria per la verifica del raggiungimento degli esiti previsti. La rilevanza clinica dei risultati di uno studio dovrebbe essere giudicata sulla base dell’effettiva influenza che essi avranno nel modificare la pratica clinica reale. L’efficacia sperimentale attesa (efficacy) è la capacità di un trattamento di modificare in maniera positiva il decorso di una malattia, in condizioni organizzative migliori di quelle della pratica quotidiana. L’efficacia nella pratica (effectiveness) è il beneficio che un trattamento mostra durante la sua applicazione nelle condizioni di pratica clinica corrente, di solito con minore controllo dell’aderenza e peggiori condizioni organizzative di quelle proprie di uno studio sperimentale. Negli ultimi anni, è emerso che il grado di compliance al trattamento rappresenta una variabile molto importante nel determinare differenze tra gli esiti clinici riscontrati durante i trials clinici e la pratica clinica quotidiana. Ciò ha incentivato lo sviluppo di studi volti ad una valutazione quantitativa dei processi che realmente avvengono nella pratica clinica e dei risultati di efficacia terapeutica che da essi ne derivano. Rilevare e quantificare l’aderenza dei pazienti alla terapia negli studi clinici è di grande valore al fine di ottenere risultati di efficacia altamente generalizzabili, in quanto si riferiscono ai pazienti che hanno effettivamente portato a termine la sperimentazione in rapporto a quanti hanno abbandonato la terapia nel corso dello studio. L’analisi di questo fenomeno ha assunto una rilevante importanza perché può ridimensionare l'efficacia complessiva finale di uno schema terapeutico e, anche, metterne in dubbio l’applicabilità futura

HIPK2 as a Novel Regulator of Fibrosis

Fibrosis is an unmet medical problem due to a lack of evident biomarkers to help develop efficient targeted therapies. Fibrosis can affect almost every organ and eventually induce organ failure. Homeodomain-interacting protein kinase 2 (HIPK2) is a protein kinase that controls several molecular pathways involved in cell death and development and it has been extensively studied, mainly in the cancer biology field. Recently, a role for HIPK2 has been highlighted in tissue fibrosis. Thus, HIPK2 regulates several pro-fibrotic pathways such as Wnt/β-catenin, TGF-β and Notch involved in renal, pulmonary, liver and cardiac fibrosis. These findings suggest a wider role for HIPK2 in tissue physiopathology and highlight HIPK2 as a promising target for therapeutic purposes in fibrosis. Here, we will summarize the recent studies showing the involvement of HIPK2 as a novel regulator of fibrosis

Reactivation of mutant p53 by capsaicin, the major constituent of peppers

Mutations in the p53 oncosuppressor gene are highly frequent in human cancers. These alterations are mainly point mutations in the DNA binding domain of p53 and disable p53 from transactivating target genes devoted to anticancer activity. Mutant p53 proteins are usually more stable than wild-type p53 and may not only impair wild-type p53 activity but also acquire pro-oncogenic functions. Therefore, targeting mutant p53 to clear the hyperstable proteins or change p53 conformation to reactivate wild-type p53 protein functions is a powerful anticancer strategy. Several small molecules have been tested for p53 reactivation in mutant p53-carrying cells while studies exploiting the effect of natural compounds are limited. Capsaicin (CPS) is the major constituent of peppers and show antitumor activity by targeting several molecular pathway, however, its effect on mutant p53 reactivation has not been assessed yet. In this study we aimed at investigating whether mutant p53 could be a new target of capsaicin-induced cell death and the underlying mechanisms