Multi-State Gene Cluster Switches Determine the Adaptive Mitochondrial And Metabolic Landscape of Breast Cancer.

Adaptive metabolic switches are proposed to underlie conversions between cellular states during normal development as well as in cancer evolution. Metabolic adaptations represent important therapeutic targets in tumors, highlighting the need to characterize the full spectrum, characteristics, and regulation of the metabolic switches. To investigate the hypothesis that metabolic switches associated with specific metabolic states can be recognized by locating large alternating gene expression patterns, we developed a method to identify interspersed gene sets by massive correlated biclustering (MCbiclust) and to predict their metabolic wiring. Testing the method on breast cancer transcriptome datasets revealed a series of gene sets with switch-like behavior that could be used to predict mitochondrial content, metabolic activity, and central carbon flux in tumors. The predictions were experimentally validated by bioenergetic profiling and metabolic flux analysis of 13C-labelled substrates. The metabolic switch positions also distinguished between cellular states, correlating with tumor pathology, prognosis, and chemosensitivity. The method is applicable to any large and heterogeneous transcriptome dataset to discover metabolic and associated pathophysiological states.

Inositol trisphosphate receptor-mediated Ca2+ signalling stimulates mitochondrial function and gene expression in core myopathy patients.

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.

Targeting RORs nuclear receptors by novel synthetic steroidal inverse agonists for autoimmune disorders.

Designing novel inverse agonists of NR RORγt still represents a challenge for the pharmaceutical community to develop therapeutics for treating immune diseases. By exploring the structure of NRs natural ligands, the representative arotenoid ligands and RORs specific ligands share some chemical homologies which can be exploited to design a novel molecular structure characterized by a polycyclic core bearing a polar head and a hydrophobic tail. Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was identified as lead compound which was chemically modified at position 2 in order to obtain a small library for preliminary SARs. Cell viability and estrogenic activity of compounds 7, 8, 19a, 30, 31 and 32 were evaluated to attest selectivity. The selected 7, 8, 19a and 31 compounds were assayed in a Gal4 UAS-Luc co-transfection system in order to determine their ability to modulate RORγt activity in a cellular environment. They were evaluated as inverse agonists taken ursolic acid as reference compound. The potency of compounds was lower than that of ursolic acid, but their efficacy was similar. Compound 19a was the most active, significantly reducing RORγt activity at low micromolar concentrations.

Sexually Diergic Trophic Effects of Estradiol Exposure on Developing Bovine Cerebellar Granule Cells.

In the mammalian brain, the differentiation of neural cells and the developmental organization of the underlying circuitry are influenced by steroid hormones. The estrogen 17-ß estradiol (E2) is one of the most potent regulators of neural growth during prenatal life, synthetized locally from steroid precursors including prenatal testicular testosterone. Estradiol promotes brain differentiation counting sexually dimorphic neural circuits by binding to the estrogen receptors, ER-α and ER-ß. The cerebellum has been described as a site of estrogen action and a potentially sexually dimorphic area. The goal of this study was to analyze the capacity of E2 to affect the growth of male and female fetal bovine cerebellar granule. We performed primary cultures of fetal cerebellar granules, and verified the mRNA expression of the ER-α and ER-ß in both sexes. Moreover, the distribution of ERs in the male and female cerebellar granules of the second fetal stage was characterized by immunohistochemistry. We measured morphological parameters in presence (or absence) of estradiol administration, focusing on the variations of the dendritic branching pattern of granule neurons. By using the nonparametric combination and permutation testing approach, we proposed a sophisticated multivariate statistical analysis to demonstrate that E2 induces multifarious and dimorphic changes in the granule cells. E2 exerts trophic effects in both female and male granules and this effect is stronger in female. Male granules treated with E2 became similar to female control granule. Bos taurus species has a long gestation and a large brain that offers an interesting alternative in comparative neuroscience.

Altered Ca2+ homeostasis and endoplasmic reticulum stress in myotonic dystrophy type 1 muscle cells.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.

Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells.

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.