Simvastatin reduces melanoma progression in a murine model.

Statins are a class of drugs that inhibit the rate-limiting step in the cholesterol biosynthetic pathway and show an anticancer effect, probably through the inhibition of cell proliferation. To date, the exact mechanism of cancer cell growth arrest induced by statins is not known. We report that simvastatin is able to induce apoptosis in melanoma cells but not in normal cells and also able to contrast the growth of tumor in an experimental melanoma murine model. We observed a delay in the tumor development in almost the 50% of the simvastatin administered animals and a strong reduction of the tumor volume with a differences of ~150% compared to the controls. Also the survival rate was significantly higher in mice that received the drug with a survival increase of ~130% compared to the controls. The tumor growth reduction in mice was supported by the results of cell migration assay, confirming that simvastatin clearly reduced cell migration. Moreover, simvastatin induced a strong downregulation of NonO gene expression, an important growth factor involved in the splicing regulation. This result could explain the decrease of melanoma cells proliferation, suggesting a possible action mechanism. The results derived from our experiments may sustain the many reports on the anticancer inhibitory property of statins and encourage new studies on this drug for a possible use in therapy, probably in combination with conventional chemotherapy.

Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease.

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB-mediated cellular clearance.

A highly secreted sulphamidase engineered to cross the blood-brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA.

Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs.

Simvastatin inhibits cancer cell growth by inducing apoptosis correlated to activation of Bax and down-regulation of BCL-2 gene expression.

The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been proven to be effective in lowering cholesterol and as anti-lipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation. Probably, these effects are due to suppression of the mevalonate pathway leading to the depletion of various downstream products that play an essential role in cell cycle progression, cell signaling and membrane integrity. To date, although many hypotheses have been proposed, the exact mechanism at the basis of cancer cell growth arrest induced by statins is not known. In this study, we have demonstrated that simvastatin, at a dose of 20 µM for 24-72 h, induced in cancer cells but not in normal cells precise features of apoptosis including increased DNA fragmentation while, at the molecular level simvastatin induced overexpression of the pro-apoptotic gene Bax together with an inhibition of BCL-2, the gene that has the well-known function of protecting cells from apoptosis. The simvastatin-mediated induction of apoptosis in similar cancer cells but not in normal cells is very interesting and may be at the basis of cancer therapy using statins, usually in combination with chemotherapy or to be used as a cancer protective drug. Simvastatin may, thus, play a dual prophylactic role as a lipid-lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain types of cancers.

Efficacy of a combined intracerebral and systemic gene delivery approach for the treatment of a severe lysosomal storage disorder.

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.

Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44.

Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.

Mutations in splicing factor PRPF3, causing retinal degeneration, form detrimental aggregates in photoreceptor cells.

PRPF3 is an element of the splicing machinery ubiquitously expressed, yet mutations in this gene are associated with a tissue-specific phenotype: autosomal dominant retinitis pigmentosa (RP). Here, we studied the subcellular localization of endogenous- and mutant-transfected PRPF3. We found that (i) subcellular distribution of the endogenous wild-type protein co-localizes with small nuclear ribonucleoproteins, partially with a nucleolar marker and accumulates in speckles labeled by SC35; (ii) in human retinas, PRPF3 does not show a distinctive abundance in photoreceptors, the cells affected in RP and (iii) the RP causing mutant PRPF3, differently from the wild-type protein, forms abnormally big aggregates in transfected photoreceptor cells. Aggregation of T494M mutant PRPF3 inside the nucleus triggers apoptosis only in photoreceptor cells. On the basis of the observation that mutant PRPF3 accumulates in the nucleolus and that transcriptional, translational and proteasome inhibition can induce this phenomenon in non-photoreceptor cells, we hypothesize that mutation affects splicing factor recycling. Noteworthy, accumulation of the mutant protein in big aggregates also affects distribution of some other splicing factors. Our data suggest that the mutant protein has a cell-specific dominant effect in rod photoreceptors while appears not to be harmful to epithelial and fibroblast cells.

Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency.

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1(-/-) mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients, Sumf1(-/-) mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1(-/-) mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.