Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

[Vitamin D anti-cancer activities: observations, doubts and certainties]. / LES ACTIVITÉS ANTI-CANCÉREUSES DE LA VITAMINE D: les observations, les doutes et les certitudes.

The importance of vitamin D in bone and phosphocalcic status is well recognized by the scientific and medical communities; however, recently identified properties of this cholesterol derived molecule, such as immunomodulator and anticancer activities, are yet discussed. Actually, the debate is not so much about the new vitamin D properties, but rather about the optimal concentration required to reach these properties. The difficulty in determining the norms is rendered even more complex by the existence of a vitamin D receptor gene polymorphism. The body pool of this vitamin depends essentially on its endogenous synthesis, but also on its dietary intakes. Many epidemiological studies interested in Vitamin D serum level and cancer suggest a relation between low Vitamin D level and cancer risk, especially in breast and colon adenocarcinomas. In vitro, many studies showed, in different human and animal malignant cell lines, that this molecule exerts anticancer activities: it induces apoptosis and cell differentiation as well as it inhibits proliferation and angiogenesis. This review tries to update the current knowledge on vitamin D and, more particularly, the potential interest of this molecule in cancer prevention and management.

HDAC5 is required for maintenance of pericentric heterochromatin, and controls cell-cycle progression and survival of human cancer cells.

Histone deacetylases (HDACs) form a family of enzymes, which have fundamental roles in the epigenetic regulation of gene expression and contribute to the growth, differentiation, and apoptosis of cancer cells. In this study, we further investigated the biological function of HDAC5 in cancer cells. We found HDAC5 is associated with actively replicating pericentric heterochromatin during late S phase. We demonstrated that specific depletion of HDAC5 by RNA interference resulted in profound changes in the heterochromatin structure and slowed down ongoing replication forks. This defect in heterochromatin maintenance and assembly are sensed by DNA damage checkpoint pathways, which triggered cancer cells to autophagy and apoptosis, and arrested their growth both in vitro and in vivo. Finally, we also demonstrated that HDAC5 depletion led to enhanced sensitivity of DNA to DNA-damaging agents, suggesting that heterochromatin de-condensation induced by histone HDAC5 silencing may enhance the efficacy of cytotoxic agents that act by targeting DNA in vitro. Together, these results highlighted for the first time an unrecognized link between HDAC5 and the maintenance/assembly of heterochromatin structure, and demonstrated that its specific inhibition might contribute to increase the efficacy of DNA alteration-based cancer therapies in clinic.

Intestinal growth and pathology of Giardia duodenalis assemblage subtype A(I), A(II), B and E in the gerbil model.

This study investigated the significance of the genetic differences between assemblages A, B and E on intestinal growth and virulence. Intestinal growth and virulence were studied in 2 laboratory (A(I): WB and B: GS/M-83-H7) and 6 field isolates of assemblage subtype A(I), A(II), B and E(III). Intestinal trophozoite burdens, body weight and faecal consistency were monitored until day 29 post-infection (p.i.), morphological (mucosal architecture and inflammation) and functional (disaccharidase and alkaline phosphatase enzyme activity) damage to the small intestine were evaluated on days 7 and 18 p.i. The assemblage subtypes A(I) and B were more infectious and produced higher trophozoite loads for a longer period compared to the subtypes A(II) and E(III). The body weight of infected gerbils was significantly reduced compared to uninfected controls, but did not differ between the assemblage subtypes. Consistent softening of the faeces was only observed with assemblage B. Assemblage B next to assemblage subtype A(I) elicited relatively higher pathogenicity, characterized by more extensive damage to mucosal architecture, decreased brush-border enzyme function and infiltration of inflammatory cells. Assemblage E(III) and A(II) isolates showed relatively low virulence. The Giardia assemblage subtypes exhibit different levels of growth and virulence in the gerbil model.

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: benefits over classical vesicles.

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG(750)-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.

[Autologous fat grafting in the breast: oncological implications]. / Transferts graisseux au niveau du sein: implications oncologiques.

Autologous fat grafting for breast is increasing dramatically. This fat injection needs accurate technical conditions, and shows very good and long-lasting clinical results. Nevertheless, in breast conservative treatment sequellae, fat injection could lead to difficulties in breast imaging, but also there is some concerns about the potential oncologic risks of these procedures.

Induction of apoptosis in human promyelocytic leukemia cells by a natural trachylobane diterpene.

BACKGROUND: Trachylobane diterpenes are secondary metabolites, quite rare in nature, and their bioactivities are poorly understood. Recently, we have described the cytotoxic activity of ent-trachyloban-3beta-ol isolated from the leaves of Croton zambesicus, a plant used in African folk medicine. MATERIALS AND METHODS: Cell viability on several cell lines, cell morphology, DNA laddering, annexin Vand caspase-3 activation experiments were undertaken in order to analyse the cytotoxicty of trachylobane diterpene and to determine if this compound is able to induce apoptosis. RESULTS: ent-Trachyloban-3beta-ol exerts a dose-dependent cytotoxic effect, which varies between cell lines. Induction of apoptosis in HL-60 cells could be detected at a concentration of 50 microM after 24-h treatment. CONCLUSION: We show here, for the first time, that a trachylobane diterpene is able to induce apoptosis in human promyelocytic leukemia cells via caspase-3 activation in a concentration-dependent manner.

Spermine induces precocious development of the spleen in mice.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats, it induces maturation of the intestinal tract (liver, pancreas and small intestine). Here we show that this polyamine is able to induce precocious intestinal and splenic development in suckling mice. In fact, in 15-day-old mice which had received spermine orally twice daily for 3 days we observed an increase in the ratio of white pulp surface to total spleen surface in comparison with untreated mice. The two macrophage subsets of the marginal zone and the B-cell population were more developed and reached the development level of 5- or 10-week-old mice. The proliferation rate of B-cells was increased by spermine administration to pups. These observations suggest that spermine might play a role in immune system development; further investigation of its effects are intended, namely the evaluation of its capacity to enhance defence during the neonatal period.

Spermine-Induced alteration of small intestine in suckling rat: involvement of apoptosis or Zn2+ enzymes?

Polyamines are of great importance in several physiological processes, such as cell proliferation and differentiation. The ingestion of spermine by suckling rats induces precocious maturation of their small intestine. Shortly after ingestion, spermine produces cell elimination at the villous top. The origin of this exfoliation was investigated to determine whether it was due to apoptosis. Wistar rats were orally treated with spermine. Apoptosis was analyzed in their small intestine by Tdt-mediated dUTP-fluorescein nick-end labeling reaction, caspase-3-like analysis, and DNA laddering. Polyamine content was measured by HPLC. The intestinal transitory alteration appeared as soon as 2 hr after spermine administration. Apoptosis events increased strongly at the same moment in the small intestine. They were evidenced by Tdt-mediated dUTP-fluorescein nick-end labeling analysis, DNA laddering, and caspase-3-like activity. Changes observed are consistent with apoptosis, but caspase inhibitor did not reduce intestinal alteration, as did Zn2+ chelator.

Dietary polyamines and non-neoplastic growth and disease.

This review presents the data that are now available concerning the effects of dietary polyamines at either postnatal or adult stages in non-neoplastic growth and disease. Polyamines provided by food have a potential role in growth and development of the digestive system in neonatal mammals (and fishes). In humans, this property could be of importance in preventing the appearance of food allergies. Dietary polyamines also seem necessary for the maintenance of normal growth and general properties of adult digestive tract. Their possible therapeutic effects have been investigated in gastric, intestinal, and, more recently, whole-body healing.

Are milk polyamines preventive agents against food allergy?

Insufficient polyamine intake could play a role in the induction of sensitization to dietary allergens. This proposal is based essentially on investigations made in sucking rats and in children. In sucking rats it has been established that oral administration of spermine can induce all the modifications occurring in the digestive tract at weaning. In the intestine events occur in two phases. The early event consists of desquamation of the epithelium resulting from an activation of apoptosis. The late event appears to involve an hormonal cascade in which adrenocorticotropic hormone, cytokines, bombesin and corticosterone are included. Observations in human subjects show that: (1) the spermine and spermidine concentrations are generally lower in infant formulas than in human breast milk. Mothers seem consistently to have relatively high or relatively low concentrations of spermine and spermidine in their milk. These individual variations may be due to diet, lifestyle or genetic background; (2) the probability of developing allergy can reach 80 % if the mean spermine concentration in the milk is lower than 2 nmol/ml milk. It is approximately 0 % if the mean spermine concentration is higher than 13 nmol/ml milk; (3) preliminary results show that the intestinal permeability to macromolecules differs in premature babies when they are fed on breast milk compared with infant formulas (J Senterre, J Rigo, G Forget, G Dandrifosse and N Romain, unpublished results). This difference does not seem to be present when powdered milk is supplemented with polyamines at the concentration found in breast milk; (4) spermine increases proliferation and differentiation of lymphocytes isolated from the tonsils of children.

Cyclosporine A inhibits partially spermine-induced differentiation but not cell loss of suckling rat small intestine.

The polyamines are of great importance in several biological processes, such as cell proliferation, and differentiation. The ingestion of spermine by suckling rats induces the precocious maturation of their small intestine. This phenomenon is preceded by a cell elimination at the villus tip. We hypothesize that these two phenomena could be mediated by the immune system and thus inhibited by an immunosuppressive agent such as cyclosporine A. Cyclosporine A inhibits, at least partially, the spermine-induced increase of the maltase- and sucrase-specific activities in the small intestine but failed to inhibit lactase-specific activity decrease and cell loss. Spermine does not act by the same mechanism in differentiation and in cell loss. Moreover, spermine acts in a different way on lactase-specific activity compared to maltase- or sucrase-specific activity. We hypothesize that spermine acts on differentiation by a T-cell/IL-2-dependent mechanism.

[Statement on polyamines]. / Le point sur les polyamines.

Polyamines are ubiquitous substances. Their intracellular concentration is controlled quickly and rigorously by extremely sophisticated systems. It depends on metabolism and cellular permeability. Polyamines act as structural and functional elements in the cell (nucleic acid conformation, cytoskeleton, radioprotection, apoptosis, proliferation and differentiation of cells...). They also play a role in various diseases (origin of food allergy, cancers...). They present a great therapeutic interest (oncology, molecular transfer to cell nucleus, transfer across the blood-brain barrier, parasitosis, effects on NMDA and GABA receptors in the central nervous system...).

Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine.

In the suckling rats, orally provided spermine induced structural and biochemical changes in the intestine, which are characteristics of the postnatal maturation. This induced maturation was compared to that occurring spontaneously. Eight mumol spermine were administered orally once a day, for one or three days, to suckling rats which were 11 days old at the beginning of the experiment. The animals were killed 0, 2, 4, 6, 8, 10 hours or 3 days after the first treatment. Control rats from the same litter were treated in the same way but received only the vehicle. In order to complete the study of the naturally occurring maturation, another group of rats was killed when they were 12, 13, 15, 16, 17, 18, 19, 20, 21 or 30 days old. Animal and intestine weights were measured. Disaccharidase specific activity, and protein, DNA and RNA contents were estimated in the small intestine. Histological and ultrastructural aspects of the intestinal mucosa were examined. For all these parameters, the maturation induced by spermine ingestion appeared close to that occurring naturally at weaning. Consequently, dietary spermine induces all the morphological and biochemical modifications characterizing the intestinal postnatal maturation in the suckling rat suggesting a role of the polyamines in the naturally occurring processes.

The relationship between spermine content of human milk during the first postnatal month and allergy in children.

DESIGN: Qualitative case study and mathematical model. SETTING: Belgium. OBJECTIVES: To evaluate the correlation between the polyamine mean concentration of the milk drunk during the first postnatal month and the appearance of allergy in children who drank this milk. RESULTS: A model that describes the dependence of the allergy appearance with the spermine mean concentration of milk drunk during the first postnatal month was established. CONCLUSIONS: This model shows that 5.02 nmol ml(-1) of spermine is a critical value to prevent the appearance of allergy.

Role of interleukin-1 beta, interleukin-6, and TNF-alpha in intestinal maturation induced by dietary spermine in rats.

In the present investigation, the authors aimed to evaluate the role of cytokines in intestinal postnatal maturation induced by dietary polyamines. Neonatal rats were administered either saline (8 mumol) orally. Spermine increased interleukin-1 beta (IL-1 beta), IL-6, and TNF-alpha plasma concentration. The maximum concentrations of IL-1 beta, IL-6, and TNF-alpha were, respectively, observed at 4, 4, and 8 h posttreatment. Intraperitoneal (i.p.) injection of IL-1 beta increased the specific activity of sucrase in whole small intestine, whereas the specific activities of maltase and lactase were significantly enhanced only in the jejunum. IL-6 elicited sucrase and increased maltase specific activity in the whole small intestine, but lactase specific activity was not affected. TNF-alpha had no effect on sucrase and maltase specific activity, but a slight augmentation of lactase specific activity was detected in the jejunum. Spermine and spermidine content in the intestine was increased by i.p. injection of IL-1 beta and IL-6. Corticosterone secretion was elevated by single i.p. injection of IL-1 beta, IL-6, or TNF-alpha. These findings suggest that spermine could induce postnatal intestinal development and corticosterone secretion through a cytokine-dependent mechanism.