Tumours are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumours in vivo. These cancer stem cells (CSCs) ...represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumour relapse. Despite this realization and great interest in CSCs, it has been difficult to develop CSC-targeted treatments due to our limited understanding of CSC biology. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the CSC phenotype. Utilizing a novel CSC model, we discovered that the HDACs, HDAC1 and HDAC7, are specifically over-expressed in CSCs when compared to non-stem-tumour-cells (nsTCs). Furthermore, we determine that HDAC1 and HDAC7 are necessary to maintain CSCs, and that over-expression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that clinically available HDAC inhibitors (HDACi) targeting HDAC1 and HDAC7 can be used to preferentially target CSCs. These results provide actionable insights that can be rapidly translated into CSC-specific therapies.
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic ...risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to “tune down” the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.
Highlights • We clone and sequence IGF-1 cDNA for two species of Sceloporus lizards. • We validate an RT-qPCR assay for IGF-1 mRNA expression in Sceloporus lizards. • We validate an RIA for ...measurement of circulating IGF-1 levels in Sceloporus lizards. • IGF-1 in Sceloporus is regulated by nutritional status similar to other vertebrates. • Severe nutrient restriction is required to decrease IGF-1 expression.
Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is ...locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.
Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P₂ is ...one of the best characterized PIs; studies in which PtdIns(4,5)P₂ localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P₂ and its derivative Ins(1,4,5)P₃ accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P₂ signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P₃. We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P₂ and Ins(1,4,5)P₃ as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.
Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P ...sub(2) is one of the best characterized PIs; studies in which PtdIns(4,5)P sub(2) localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P sub(2) and its derivative Ins(1,4,5)P sub(3) accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P sub(2) signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P sub(3). We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P sub(2) and Ins(1,4,5)P sub(3) as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.
Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P
2
is ...one of the best characterized PIs; studies in which PtdIns(4,5)P
2
localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P
2
and its derivative Ins(1,4,5)P
3
accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P
2
signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P
3
. We have characterized the Arabidopsis (
Arabidopsis thaliana
)
sac9
mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (
Oryza sativa
) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed
sac9
mutants accumulate elevated levels of PtdIns(4,5)P
2
and Ins(1,4,5)P
3
as compared to wild-type plants. The
sac9
mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.
Growing Holstein heifers n = 6; 104 kg initial body weight (BW) were used to investigate the effects of a daily dose (0, 6.7, 33, 67, 100 and 200 microgram/kg BW) of bovine somatotropin (bST) on ...nutrient utilization. A Latin square design was used, and treatments (daily doses of bST) were administered by intramuscular injection for 14-d periods. Intakes of a total mixed ration were adjusted according to BW at the start of each period. Energy was supplied to allow an estimated daily gain of 0.75 kg, and crude protein intake was increased 15% above recommendations to allow for anticipated increases in protein deposition with bST treatment. Nitrogen balance and nutrient digestibilities were determined on d 7 to 13, and blood samples were obtained on d 14 of treatment. Digestibilities of dry matter, organic matter and N were similar (70 +/- 1%) and not affected by treatments (p > 0.1). Nitrogen retention increased, and plasma urea N and urinary N excretion decreased in a dose-dependent manner. Retention of N in heifers receiving the highest bST dose was 23% greater (p < 0.001) than the zero dose treatment. Serum concentration of insulin-like growth factor I (IGF-I) was also increased with bST treatment in a curvilinear dose-dependent manner. Plasma glucose increased (5 to 8%, p < 0.001) with the three highest doses of bST, but serum concentration of insulin was not altered. The ability of bST to enhance N utilization in a dose-dependent manner was due to postabsorptive changes in nutrient utilization, and the changes in IGF-I are consistent with a possible role for this somatomedin in mediating a portion of the effects of ST.
Two experiments examined whether replacement therapy with recombinantly derived bovine somatotropin (rbST) would induce puberty in heifers that had been actively immunized at 6 mo of age against ...growth hormone-releasing factor (GRF). Heifers received daily i.m. injections of 25 mg of rbST (Exp. 1, n = 6; Exp. 2, n = 4) or vehicle (VEH; Exp. 1, n = 6; Exp. 2, n = 4) for 56 d. Serum concentrations of somatotropin (ST, nanograms/milliliter) were low in all heifers before first injection in Exp. 1 (1.56 +/- 0.04) and 2 (.95 +/- 0.03). During treatment, serum ST was greater (P 0.01) in rbST than in VEH heifers (75.4 +/- 4.8 vs 2.8 +/- 0.1 ng/ml, respectively) in both experiments and remained increased through d 57 (32.2 +/- 6.4 vs 0.90 +/- 0.01 ng/ml). In Exp. 1 and 2, concentrations of serum IGF-I were similar in rbST and VEH heifers before treatment, increased (P 0.01) 12 h after first rbST, and remained increased (P 01) through d 57 in rbST heifers. Concentrations of serum insulin (INS) and plasma glucose (GLU) were similar (P 0.10) in rbST and VEH heifers before first injection (Exp. 1 and 2). Serum INS (micro-units/ milliliter) was greater (P 0.01) in rbST (61.7 +/- 3.7 and 36.0 +/- 2.4) than in VEH (12.4 +/- 1.6 and 8.1 +/- 1.0) heifers on d 1 or 2 only, in Exp. 1 and 2, respectively. In Exp. 1, GLU was increased (P 0.05) by rbST on d 2 through 57, but only on d 1 in Exp. 2. Proportion of heifers pubertal by d 21 tended to be greater (P 0.07) in rbST (3 of 6) than in VEH (0 of 5) heifers in Exp. 1, but not in Exp. 2 (1 of 4 vs 1 of 4, respectively). All heifers in Exp. 1 and 50% of the heifers in Exp. 2 attained puberty by d 56. Daily rbST increased ST, IGF-I, INS, and GLU but did not hasten onset of puberty in heifers immunized against GRF
PDF
